Virtual Library

Background
Malignant mesothelioma (MM) is a highly aggressive cancer with a very poor prognosis. Debulking surgery is often used as the principal therapy but is seldom curative. Adjuvant chemotherapy or radiotherapy can be used as to target residual disease, but these too are largely ineffective, while some early post-surgery immunotherapy strategies had limited clinical success. However, there is renewed interest in the use of immunotherapy to treat MM as new modalities have been developed. Recent work form our laboratory and others, has demonstrated that specific immunotherapies can alert the immune system to the presence of tumour. These therapies are particularly useful when used in conjunction with standard treatment protocols such as surgery or chemotherapy. Here we describe the development of a Prime Boost (P/B) anti-tumour vaccination protocol that when combined with debulking surgery and removal of CD4 T cells improved survival outcome of AB1-HA tumour bearing mice.

Methods
Using our established mouse model of mesothelioma, AB1-HA tumour bearing BALB/c mice received influenza A PR/8/34/H1N1 (PR8; Prime) and HA expressing recombinant modified Vaccinia Ankara (rMVA‑HA; Boost) anti‑tumour vaccinations before (neoadjuvant) or after (adjuvant) 75% debulking surgery. Diphtheria toxin (DTX) was administered to tumour bearing BALB/c FoxP3.dtr mice to specifically deplete CD4+ FoxP3+ regulatory T cells (Treg). In both models, tumour growth and overall survival was monitored and immunological parameters assessed by multicolour FACS.

Results
Neoadjuvant P/B vaccination alone or in combination with 75% debulking surgery induced a significant increase in splenic tumour‑specific CD8 T cells as well as significant increases in the proportion, activation and proliferation status of peripheral CD8 T cells relative to other treatment groups. However, a significant delay in tumour growth was only observed when neoadjuvant P/B vaccination was combined with debulking surgery. Specific depletion of CD8 T cells demonstrated that they were essential for the delay in tumour growth, although their presence was not sufficient to eliminate the tumour outright. Depletion of CD4 T cells during P/B vaccination enhanced the survival outcome of the surgery + vaccination group with 60% of these mice remaining tumour free for > 60 days post-surgery. Data from preliminary experiments in which Treg in tumour bearing FoxP3.dtr mice resulted in complete tumour regression in 20% of DTX treated mice. Tumour specific immunological memory was confirmed as all surviving mice remained tumour free for at least 60 days post rechallenge with the parental AB1 tumour.

Conclusion
Anti-tumour P/B vaccination induced tumour‑specific immunity resulting in delayed tumour growth when combined with debulking surgery. Depletion of CD4 T cells during neoadjuvant P/B vaccination enhanced P/B vaccine efficacy leading to cures in 60% of treated mice. Transient depletion of CD4+ FoxP3+ Treg suggesting that vaccine induced anti‑tumour immunity is “restrained”, possibly by regulatory T cells. Based on these findings we are investigating whether combining novel immunotherapies with conventional treatments in the absence of “immunological restrainers” may generate effective therapy for MM. Financial disclosure: This research was funded by a research grant from the Workers’ Compensation Dust Diseases Board, an agency of the New South Wales Government.

Background
BIM (BCL2L11) is a BH3-only pro-apoptotic member of the Bcl-2 protein family. BIM upregulation is required for apoptosis induction by EGFR tyrosine kinase inhibitors (EGFR-TKIs) in EGFR-mutant forms of non-small cell lung cancer (NSCLC). Notably, a BIM deletion polymorphism occurs naturally in 12.9% of East Asian individuals, impairing the generation of the pro-apoptotic isoform required for the EGFR-TKIs gefitinib and erlotinib and therefore conferring an inherent drug resistant phenotype. Indeed, NSCLC patients who harbored this host BIM polymorphism exhibited significantly inferior responses to EGFR-TKI treatment than individuals lacking this polymorphism. In attempt to correct this response defect in the resistant group, we investigated whether the histone deacetylase (HDAC) inhibitor vorinostat could circumvent EGFR-TKI resistance in EGFR mutant NSCLC cell lines that also harbored the BIM polymorphism.

Methods
not applicable

Results
We found that such cells with BIM polymorphism were much less sensitive to gefitinib-induced apoptosis than EGFR mutant cells which did not harbor the polymorphism. Notably, vorinostat increased expression in a dose-dependent manner of the pro-apoptotic BH3 domain-containing isoform of BIM, which was sufficient to restore gefitinib death sensitivity in the EGFR mutant, EGFR-TKI resistant cells. In xenograft models, while gefitinib induced marked regression, via apoptosis, of tumors without the BIM polymorphism, its combination with vorinostat was needed to induce marked regression of tumors with the BIM polymorphism in the same manner.

Conclusion
Our results show how HDAC inhibition can epigenetically restore BIM function and death sensitivity of EGFR-TKI, in cases of EGFR mutant NSCLC where resistance to EGFR-TKI is associated with a common BIM polymorphism.

Background
Analysis for EGFR mutation became new standard in management of lung adenocarcinoma. Mutations in EGFR tyrosine kinase domain such as L858R or small deletions in exon 19 result in sustained phosphorylation of EGFR and become driver oncogenic mutation. EGFR TKI, such as gefitinib, induces dramatic response in lung adenocarcinoma with sensitive mutations. Unfortunately, this dramatic response can not last long and resistance to EGFR TKI emerges and induces treatment failure. More than 50% of resistant mutations are EGFR T790M mutation. In this study, we investigated the role of siRNA specific to EGFR T790M and its clinical significance.

Methods
We designed three sequences (siRNA1, 2, 3) specific to EGFR T790M according to siRNA design guideline. Lung cancer cells were used: A549, NCI H460 (EGFR; wild type), NCI H1975 (EGFR L858R + T790M), PC9 (EGFR small deletion in exon 19), PC9-G (EGFR small deletion in exon 19 + T790M). We investigated the effect of three siRNAs on suppression of EGFR T790M and reversal of resistance to gefitinib.

Results
Transfection of siRNA 1 and 3 showed marked suppression of EGFR expression in NCI H1975 and PC9-G, however, siRNA 2 failed to suppress. All siRNA don't affect EGFR expression in A549, NCI H460 and PC-9. This finding suggested that suppressions of EGFR by siRNA 1 and 3 were specific to EGFR T790M. EGFR T790M siRNA 1 and 3 not 2, markedly suppressed the growth of NCI H1975 and PC9-G via increased apoptotic cell death and also suppressed in vitro tumorigenicity. No significant effect was found in other cell lines. This finding strongly supports that EGFR T790M is another oncogenic driver mutation. Cotreatment of EGFR siRNA 1 and 3 with gefitinib induced marked increase in sensitivity of NCI H1975 and PC-9 to gefitinib (synergistic interaction), however, no effects were found in A549 and NCI H460.

Conclusion
Application of EGFR T790M specific siRNA can reverse the resistance of lung adenocarcinoma and shows its potential to be a breakthrough in EGFR TKI. Further study will focus on preclinical application with efficient delivery system, such as, nanotechnology or viral vectors. (This study was supported by a grant from the National Research Foundation of Korea, 2011-0002169).

Background
The PDGFR is among the significantly mutated pathways and the PDGFR and downstream Src family protein kinases are often aberrantly activated and play important roles in mediating oncogenic signaling and modulating sensitivity to the molecularly-targeted therapy in lung cancer. We have previously shown that the novel tumor suppressor TUSC2 (FUS1) functions as a key mediator in the apoptosis signaling pathway and down-regulates activities of multiple oncogenic protein kinases such as EGFR, PDGFR, Src, and c-Abl in lung cancer cells. A systemic treatment with FUS1-DOTAP:Cholesterol nanoparticles demonstrated a potent antitumor efficacy in preclinical lung cancer animal models and showed promising clinical benefits in advanced lung cancer patients.

Methods
In this study, we evaluated a rationalized therapeutic strategy using a combined systemic treatment with the multifunctional FUS1-nanoparticles and the PDGFR kinase inhibitor imatinib (gleevec) or the Src inhibitor Dasatinib or KX2-391 to simultaneously target the dysregulated PDGFR-Src-PI3K-Akt signaling pathways and suppress tumor cell growth by facilitating apoptosis in human lung cancer cells in vitro and in vivo.

Results
We have compared the effectiveness of the orally-available Src inhibitor dasatinib (a ATP competitive inhibitor) or KX2-391 (a novel non-competitive inhibitor that interrupts binding of the Src kinase to its substrates) as an single agent or in combination with FUS1-nanopaticles for potentiating their anticancer efficacy in NSCL and SCLC cells. We found that the dasatinib treatment alone showed a moderate level of tumor cell growth arrest and cell viability reduction but a low degree of apoptosis induction in selected NSCLC cells and exhibited a very low degree of tumor cell killing in SCLC cells. In comparison, the KX2 demonstrated a 10-100 fold higher tumor cell killing and apoptosis induction than Dasatinib in more than 20 NSCLC and SCLC cells tested. The ectopic expression by FUS1- nanoparticle-mediated gene transfer in these lung cancer cells markedly enhanced the dasatinib- or KX2-mediated tumor cell killing. The combination treatment with FUS1-nanoparticles and Src inhibitors dramatically reduced their IC50s in NSCLC cells and suppressed NSCLC cells growth through a mechanism of action by a significant induction of apoptosis and the down-regulation of activated EGFR, PI3K, Akt, and Src kinases. Furthermore, the ectopic expression of wt-FUS1 in the PDGFRß-expressing SCLC H128, N417. and NSCLC H358 cell lines inactivated PDGFR oncogenic signaling, as evidenced by a significant reduction in levels of phospho-PDGFRß and downstream phospho-PI3-K and phospho-AKT protein expression, relative to untransfected or lacZ-transfected controls. A combined treatment with FUS1-nanoparticles and the PTK inhibitor imatinib synergistically inhibited growth and induced apoptosis in SCLC and NSCLC cell lines and in preclinical mouse models with N417 SCLC orthotopic lung tumor xenografts.

Conclusion
Our findings suggest that a combination of the pro-apoptotic FUS1-nanoparticle with novel PDGFR or Src inhibitors targeting the PDGFR-Src-PI3K-Akt signaling pathway that is significantly mutated and predominantly activated in lung cancer cells could sensitize their response to PDGFR and Src inhibitors by more efficiently inhibiting tumor cell proliferation and survival, facilitating apoptosis, and overcoming drug resistance. (This abstract is supported by NIH/NCI Grants SPORE P50CA70907 and RO1CA116322).

Background
Somatic activating mutations in the tyrosine kinase (TK) domain of EGFR are harbored by 10-20% of Caucasian NSCLC patients (pts). Reversible TK inhibitors (TKIs), including erlotinib or gefitinib, have demonstrated significantly longer progression-free survival compared to chemotherapy alone in EGFR-mutated pts. Nevertheless, the majority of these tumors develop drug resistance due to an acquired mutation (T790M) in EGFR that determines disease progression. Recent clinical trials have demonstrated interesting activity of the irreversible EGFR-TKI afatinib (BIBW-2992) in advanced NSCLC carrying EGFR mutations and in unselected pts failing previous treatments with reversible TKIs. The aim of this study was to clarify the mechanisms of acquired resistance to afatinib using in vitro models of resistant cell lines.

Methods
A dose-escalation study was performed to establish afatinib-resistant (R) clones in NSCLC cell lines harboring different EGFR mutations: H-1650 (exon 19 delE746-A750) and H-1975 (exon 21 L858R/exon 20 T790M). The entire genomes of parental (P) and R cells were screened by array comparative genomic hybridization (aCGH) using a 105 k oligonucleotide microarray. All EGFR and KRAS exons and 10 known hot spots (5 in genes involved in the EGFR signaling cascade and 5 in genes frequently altered in NSCLC) were deep sequenced using an Ion PGM™ Sequencer in P and R cells. The relative expression of 92 genes belonging to the EGF pathway was studied by quantitative polymerase chain reaction (qPCR). The expression of proteins related to the EGF pathway, including EGFR, AKT and ERK (total and activated forms), was investigated by Western blot.

Results
Genomic analysis indicated that both R cell lines had genomic profiles similar to the P cells. However, H-1975-R showed 3p and 12p loss and gains at 4q and 10q compared to the P cell line. Sequence analyses identified a novel frame-shift mutation within exon 14 of MET and confirmed EGFR mutation status in 100% of H1975-R cells. In contrast, H-1650-R cells showed a single-base deletion 12 bp upstream of exon 8 of PIK3CA within a sequence of nine repeated Ts. Furthermore, a novel missense variant (exon 8 K368E) was found in FGFR2 in both R cell lines compared to the P cell line. Gene expression profiles identified an increase in the FGFR2 and PIK3 regulatory subunits and EGFR ligand silencing in H-1975-R. Notably, H-1975-R cells maintained in afatinib-free medium for over 6 months showed higher EGFR and AKT phosphorylation compared to the P cell lines.

Conclusion
The lack of novel EGFR mutations suggests the involvement of other mechanisms implicated in afatinib resistance. In particular, the identification of mutations involving MET and FGFR2 in H-1975 and PI3KCA in H-1650 suggests their contribution to resistance against irreversible TKIs, sparing EGFR activation. Furthermore, the different mutation status of the two cell lines indicates that the T790M mutation may be partially responsible for the mechanism of resistance. Validation studies are ongoing to confirm the genomic results. In conclusion, these preliminary data may help identify novel therapeutic strategies to delay or reverse resistance to irreversible TKIs in EGFR-mutated NSCLC patients.

Background
The outcome for lung cancer patients remains poor and new therapeutic approaches are urgently needed. Cediranib is an orally available inhibitor of all 3 VEGFR tyrosine kinases. We evaluated the therapeutic efficacy and radiosensitizing effects of cediranib and paclitaxel, alone or in combination, in orthotopic models of human lung adenocarcinoma that mimic clinical patterns of malignant progression.

Methods
PC14PE6 or NCI-H441 human lung adenocarcinoma cells (1 x 10[6]) were injected into the left lungs of nude mice. Mice were randomized (8/group) to treatment with vehicle control, cediranib (3 mg/kg/day po), paclitaxel (200 µg/week ip), radiation to the left lung and mediastinum (20 Gy in 5 fractions over 2 weeks), or radiation with cediranib and/or paclitaxel. When controls became moribund, all mice were sacrificed and assessed for lung tumor burden and mediastinal nodal metastasis. Lung tumors and adjacent tissues were analyzed immunohistochemically.

Results
All treatments were well tolerated without significant differences in body weight between groups. In both models, cediranib or radiation therapy alone inhibited tumor growth and lymph node metastasis with efficacy superior to paclitaxel. Cediranib markedly enhanced the antitumor and antimetastatic effects of radiation with 99.3% and 92.1% reductions in primary lung tumor volume in the PC14PE6 and NCI-H441 models, respectively, while paclitaxel only modestly improved the effects of radiation therapy. Trimodality therapy resulted in a near-complete suppression of tumor growth and metastasis, with 99.8% and 98.3% reductions in tumor volume compared to control in the PC14PE6 and NCI-H441 models, respectively, without evidence of lymph node metastasis. Immunohistochemical analyses of lung tumors revealed that cediranib inhibited angiogenesis and tumor cell proliferation and increased tumor and endothelial cell apoptosis. The antiangiogenic and apoptotic effects of cediranib were substantially enhanced when combined with radiation and paclitaxel. Cediranib alone or in combination with radiation and/or paclitaxel increased VEGFR2 expression, but VEGF expression was not significantly impacted by treatment. VEGFR2/3 activation was blocked by cediranib alone or in combination therapy.

	PC14PE6			NCI-H441
Treatment	Left Lung Weight (mg)	Left Lung Tumor Volume (mm[3])	Mediastinal Lymph Node Metastasis	Left Lung Weight (mg)	Left Lung Tumor Volume (mm[3])	Mediastinal Lymph Node Metastasis
Vehicle	710 (490-1210)	753 (254-1089)	7/8	935 (800-1230)	1146 (860-1601)	8/8
Paclitaxel 200ug/week	545 (150-860)	506 (37-817)	6/8	785 (485-820)	820 (576-1208)	7/8
Radiation 20Gy/5fractions	220** (50-360)	154* (34-270)	4/8	485** (330-820)	501* (333-879)	6/8
Cediranib 3mg/kg/day	215* (70-540)	137* (13-316)	4/8	395** (230-570)	414** (261-698)	5/8
Radiation +Paclitaxel	185** (60-260)	87** (21-268)	2/8	360** (260-650)	327** (236-651)	5/8
Cediranib + Paclitaxel	125** (60-260)	41** (0-150)	1/8[†]	225** (160-630)	241** (79-651)	4/8[†]
Radiation + Cediranib	50* (40-60)	0** (0-28)	0/8[†]	120** (70-190)	88** (1-182)	2/8[†]
Radiation + Cediranib + Paclitaxel	40** (40-60)	0** (0-1)	0/8[†]	100** (60-120)	9** (1-64)	0/8[†]
Data are presented as medians and ranges or as incidence. [†]p<0.05 versus vehicle (lymph nodes), *p<0.01, **p<0.001 versus vehicle (others)

Conclusion
Trimodality therapy with cediranib, paclitaxel, and radiation resulted in the near complete suppression of lung tumor growth and metastasis with markedly enhanced antiangiogenic and apoptotic effects. The radiosensitizing effects of cediranib upon lung tumors and their vasculature was superior to those of paclitaxel with markedly enhanced apoptosis. The combination of cediranib with radiotherapy or chemoradiotherapy is a potentially promising therapy for cancer and our data provides a strong basis for the design of clinical trials in lung adenocarcinoma patients.

Background
Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy with poor prognosis. MPM is typically recalcitrant to treatment and new therapies are urgently needed. Multiple genes involved in proliferation and metabolic activity are upregulated in MPM and these represent attractive targets for an siRNA-based therapeutic intervention.

Methods
We carried out an RNAi-based screen of 40 target genes previously shown to be upregulated in MPM to identify candidate genes with roles in cell growth and survival in MPM cell lines. Effects of target gene silencing were measured using standard in vitro proliferation assays. Lead candidates were further assessed with siRNA dose response experiments. The specificity of siRNA-mediated growth inhibition was confirmed by assessing gene knockdown by real-time qPCR and Western blotting. The effects of the most potent siRNAs on xenograft tumour growth were assessed in vivo by delivery using EGFR-targeted, siRNA-loaded, minicells.

Results
All 40 genes were effectively silenced, and for 6 genes (PLK1, CDK1, NDC80, RRM1, RRM2 and BIRC5) knockdown with 2 independent siRNAs resulted in significant growth inhibition over time in multiple cell lines. Dose response experiments revealed that siRNAs specific for RRM1 and RRM2 were the most effective at inhibiting growth with IC50 values in the low nanomolar range. Intravenous administration of RRM1 siRNA packaged in minicells targeted with EGFR-specific antibodies (2x10[9] minicells per dose, 4 times per week for 3 weeks) led to consistent and dose-dependent inhibition of MPM tumor growth compared with treatment with an inactive siRNA. Reducing the dose and number of administrations did not reduce growth inhibition; as little as 1x10[9] minicells administered once a week were sufficient to completely inhibit MPM tumour growth.

Conclusion
RRM1 is an attractive target for siRNA-based inhibition, and siRNA delivery with EGFR-targeted minicells represents a novel therapeutic approach for MPM.

Background
The fidelity of established NSCLC cell line models to reflect patient tumors has been challenged. Patient-derived primary tumor xenografts (PTXGs) established directly from patient tumors in immunodeficient mice reproduce closely the histology of the primary tumors, thus are potentially better preclinical models to investigate novel therapies. We previously reported that early stage NSCLC patients whose tumors form PTXGs have significantly greater risk of relapse after surgery (Clin Cancer Res 2011; 17: 134-141). We report here a more extended analysis of clinical-molecular-pathological features of early stage NSCLC that are associated with engraftment and its impact on patient outcome.

Methods
Resected NSCLC tumors were harvested within 30 minutes after surgery and were implanted into severely immunodeficient mice to establish PTXGs. Tumors that grew were propagated for up to 3 passages. The mutational profiles of the primary tumors were assessed by the MassARRAY platform that included 133 mutations with ‘putative’ driver function, which have been reported in COSMIC database as recurrent in NSCLC. All identified mutations were verified by direct sequencing in both the primary and PTXG tumors. Engraftment rate among clinical factors were tested using the Fisher’s exact or Mann-Whitney tests. The Kaplan-Meier method was used to estimate 3-year overall (OS) and disease-free survival (DFS) probabilities. The effect of engraftment on OS and DFS adjusting for clinical variables was assessed using a Cox proportional hazards model.

Results
From April 2005 to December 2010, 261 rigorously verified resected primary non-carcinoid NSCLCs were engrafted; 38 xenografts that were lymphoma were excluded from further analysis. For the remaining 223 primaries, 101 (45.3%) successfully engrafted and formed PTXG lines. Engraftment rates were 33.8% (48/142) for adenocarcinoma (AdC), 67.7% (42/62) for squamous cell carcinoma (SqCC), 66.7% (4/6) for large cell neuroendocrine carcinoma, and 53.8% (7/13) for others. The tumors forming PTXGs were more likely to be poorly differentiated (p=0.00012) and of larger tumor size and higher pT stage (p<0.0001), but were not correlated with the pN stage. Among 95/101 (94.1%) PTXG cases profiled for mutations, 6 had mutations in the EGFR tyrosine kinase domain, 18 in KRAS/HRAS, 5 in PIK3CA, 2 in paxillin and 1 in STK11/LKB gene; 56 (62.2%) were negative for mutations. The median follow-up time was 2.7 years (range 0.04 – 7.5 years). Patients whose tumors engrafted had decreased DFS (HR 2.68, 95% CI 1.16-4.60, Wald p<0.0001) and OS (HR 3.14, 95% CI 1.56-6.33, Wald p=0.0014). Significantly poorer survival was maintained in AdC. Among 33 patients with EGFR mutation, only 6 (18.2%) engrafted. Engraftment was associated with significantly poorer DFS (HR 4.76; 1.43-15.86, log-rank p=0.005) and OS (HR 8.55, 95% CI 0.77-94.3, log-rank p=0.035) in this population.

Conclusion
The ability to form PTXGs of early stage NSCLC is confirmed as a very strong poor prognostic marker. Although EGFR mutant tumors usually do not engraft, engraftment of EGFR mutant tumors is associated with poor patient survival. PTXGs appear to represent biologically aggressive NSCLC.

Background
Epithelial to mesenchymal transition (EMT) is related with reduced sensitivity to many chemotherapeutic drugs including EGFR tyrosine kinase inhibitors. We investigated whether EMT also could contribute to the resistance to crizotinib and there are other therapeutic options overcoming EMT-mediated resistance.

Methods
We established a crizotinib-resistant subline (H2228/CR), which was derived from the parental H2228 cell line by long-term exposure to increasing concentration of crizotinib. Characteristics related with EMT including morphology, EMT marker proteins and cellular mobility were analyzed. We examined whether the induction of EMT affect sensitivity to Hsp90 inhibitors.

Results
Compared with the H2228 cell, the growth of H2228/CR cells was independent on EML4-ALK, and they showed cross-resistance to TAE-684 (a 2[nd]-generation ALK inhibitor). Phenotypic change of a spindle-cell shape was found in H2228/CR, which was accompanied by a decrease of E-cadherin and an increase of vimentin and AXL. In addition, they showed the increased secretion and expression of TGF-β1. The capability of invasion and migration was dramatically increased in H2228/CR cells. TGF-b1 treatment for 72 h in parental H2228 cells induced reversible EMT leading to crizotinib-resistance while this was reversed through the removal of TGF-β1. Suppression of vimentin by siRNA treatment in H2228/CR cells restored the sensitivity to crizotinib. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitors similar to parental cells, H2228. HSP90 inhibition resulted in downregulation of TGF-β receptor II in addition to ALK.

Conclusion
EMT should be considered as one of possible acquired resistant mechanisms to crizotinib and HSP90 inhibitors can be a promising therapeutic option for EMT-mediated resistance.

Background
Lung cancer has the highest cancer incidence and mortality worldwide. Small cell lung cancer (SCLC) accounts for 15% of all cases. Platinum-based chemotherapy induces responses in up to 70%. However, treatment-resistant recurrence is near universal, and 5-year survival remains poor at 1-2%. Therefore, there is urgent need for pre-clinical models that accurately recapitulate the parent tumour and allow testing for predictive biomarkers of response and resistance to drugs, and also screening of novel anticancer agents. Furthermore, as the vast majority of SCLC are inoperable, it is crucial that the mode of tumour tissue acquisition be minimally invasive and repeatable in cases of recurrence. Here we describe a novel pre-clinical model using samples obtained by the minimally invasive technique of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to develop primary xenografts of SCLC.

Methods
Cell suspensions from samples of SCLC obtained by EBUS-TBNA were implanted directly into the flanks of NSG (Non-Obese Diabetic, Severe Combined Immune Deficient, IL2Rγ knockout) mice to generate primary xenografts. The mice were monitored for tumour growth, and if engraftment was successful, pre-graft and post-graft tumours were compared in terms of morphology, immunohistochemistry and molecular characteristics.

Results
Thus far, 14 SCLC specimens have been implanted, with 7 cases completing 6 months of tumour monitoring. Of these, 6 have undergone successful engraftment (86%). Samples typically contained over 1 million tumour cells with minimal stromal contamination. Mean engraftment lag time was 96 days. In all cases of engraftment, histological and molecular fidelity to the original tumour was demonstrated.

Conclusion
This is the first report of the generation of a primary xenograft model of lung cancer using a new method of tissue acquisition by EBUS-TBNA. Furthermore, it is the largest reported group of primary xenografts of SCLC. The primary xenograft lines from these specimens may provide the much-needed basis for more accurate pre-clinical modeling of SCLC, and hold great translational promise for novel therapeutic agents.

Background
Lung cancer remains the most common cancer diagnosed and has one of the lowest survival rates of all cancers, with less than a third of all patients undergoing a curative resection. Electrochemotherapy (ECT) has emerged as a novel treatment in treating various kinds of malignancies. ECT is the local potentiation, by means of permeabilising electric pulses, of the anti-tumour activity of a non-permeant anticancer drug possessing a high intrinsic cytotoxicity. We have devised a study to demonstrate the safety of using a needle based electrode device (ThoraVe) in delivering electrochemotherapy to lung tissues. A comparison of ECT with other modalities such as radiofrequency ablation (RFA) and Irreversible Electroporation (IRE) was carried out to explore the safety of electrochemotherapy as a novel treatment in lung tumours.

Methods
Healthy female pigs were randomised into the following treatment groups: ECT, Electroporation only (EP), RFA, IRE and No procedure (Sham). Each animal underwent a pre-treatment CT scan as a baseline. The scans were repeated at Days 1, 3, 7, 10 and 21 following treatment. The area of trauma/opacification seen radiologically was calculated using volume-analysis software. Histological samples were taken from each group at the same time points. Secondary outcomes analysed included airleak and drainage volume following each procedure

Results
A total of 65 pigs were used, 3 in each treatment group and 2 for each histological time point. Following each treatment, the area of trauma to the lung represented by an area of opacification on the CT scan, was identified and calculated. The ECT and EP groups were similar in terms of volume of opacification over the 3-week period, and demonstrated less radiological changes than RFA and IRE. The volume of opacification was minimal at the end of the 3-week period compared to the RFA and IRE groups. H&E staining demonstrated evidence of alveolar edema and hyperaemia in all groups but more marked in the RFA and IRE groups at day 1 and 3. Area of necrosis was evident in the RFA and IRE groups, which persisted until Day 21. ECT and EP groups did not show any evidence of persisting necrosis with normal lung parenchyma seen on Day 10 and 21. There was minimal to no air leak measured for the Sham, EP and ECT groups at the end of the surgical procedure with no air leak observed by day 1 postoperatively for the 3 groups. RFA and IRE groups showed significant air leakage immediately following treatment. The drainage volume was minimal and comparable in the Sham, EP and ECT groups. Both RFA and IRE groups had significantly higher drainage volume on Day 1. Drainage persisted until Day 3 in the IRE group.

Conclusion
We have successfully demonstrated the feasibility and safety of using ECT as compared to other established and experimental treatment modalities. Our data show radiological and histological evidence of preservation of lung parenchyma post ECT treatment, which was well tolerated, with minimal complications such as air leaks or bleeding.

Background
Lung cancer is the leading cause of cancer deaths in the world, and is classified into two major groups, non-small cell lung cancer (NSCLC, ~85%) and small-cell lung cancer (SCLC, ~15%). EGFR mutation is a validated predictive marker for response and progression-free survival with EGFR tyrosine kinase inhibitors in advanced lung adenocarcinoma. However, secondary EGFR mutation may cause drug resistant and cancer relapse. Further investigation and drug development is necessary for lung cancer therapy. KNG-I-484C is an analog of Desmosdumotin B compound, isolated and modified from the roots of Desmos dumosus. Previous studies showed that KNG-I-484C can inhibit cell proliferation of multidrug resistant (MDR) cancer cell line, KB-V, as well as multiple non-MDR cancer cell lines. Therefore, KNG-I-484C may act as a potential antitumor agent to inhibit drug-resistant cancer cells.

Methods
KNG-I-484C anti-tumorigenesis activity is estimated in non-small cell lung cancer cell lines by SRB assay and by the soft agar colony formation assay. Flow cytometry is used for cell cycle progression and cell apoptosis evaluation. The centrosomes observation is by the IF staining. The gene expression affected by the compound is by DNA microarray. Nude mice are subcutaneously injected with non-small cell lung cancer cell lines. When the tumor volume reaches about 2 mm[3], KNG-I-484C is administered by intra-peritoneal injection. The body weight of mice will be monitored. Before tumor volume reaches 1 cm[3], the mice will be sacrificed for the measurement of the tumor volume and blood.

Results
KNG-I-484C can inhibit cell proliferation and colonies formation in the soft agar in NSCLC cell lines. The compound induces G2/M arrest by flow cytometry and the G2/M markers, cyclin B1 and phospho-histone H3, are upregulated at the early stage. And it then causes cell apoptosis by annexin-V staining assay, and the apoptotic markers, caspase 3 and cleaved PARP increases by the treatment. KNG-I-484C treatment causes abnormal formation of centrosomes in NSCLC cell lines. The microarray results showed that EGR1 (early growth response protein 1) may be one of the target candidate. In the animal model, KNG-I-484C tends to inhibit the tumor growth.

Conclusion
KNG-I-484C can inhibit cell proliferation and induce cell apoptosis in lung cancer cell lines by directly inhibiting tubulin polymerization. Additional mechanisms of action may go through the centrosome abnormality. Therefore, KNG-I-484C may serve as a new and potential antitumor agent against NSCLC.

Background
Epidermal growth factor receptor (EGFR) is activated in subsets of non-small cell lung cancer (NSCLC) by mutations such as L858R and exon19 deletions. EGFR-tyrosine kinase inhibitors such as gefitinib and erlotinib have been successfully used to treat tumors with these mutations, but responses tend to be limited by the development of resistance, often through further mutations in EGFR such as T790M. EGFR and its mutated forms are clients of HSP90 and so dependent on this chaperone for their stability. HSP90 inhibition is therefore an alternative mechanism for targeting EGFR-driven disease, which should be effective on EGFR inhibitor-sensitive or -resistant disease alike. AT13387 is a novel, potent, fragment-derived HSP90 inhibitor and is the subject of a number of Phase II clinical trials, including one in NSCLC.

Methods
The activity of AT13387 was investigated in vitro and in vivo in erlotinib-sensitive and -resistant EGFR-activated NSCLC cell lines and mouse xenograft models (see Table). The HCC827R cell line was generated by prolonged incubation of HCC827 cells with erlotinib. Cell proliferation was measured by Alamar blue assay. Protein levels were determined by western blotting.

Results
AT13387 was tested in a panel of EGFR-driven NSCLC cell lines and potently inhibited proliferation of both erlotinib-sensitive and -resistant cells including a cell line with acquired erlotinib resistance (HCC827R) (see Table).

Inhibition of proliferation of EGFR-dependent NSCLC cell lines

Cell line	EGFR mutation status	Erlotinib inhibition of proliferation IC50 (nM)	AT13387 inhibition of proliferation IC50 (nM)
HCC827	Exon19 Del	57	33
NCI-H1650	Exon19 Del	> 10 000	54
NCI-H1975	L858R/T790M	> 10 000	30
H820	Exon19 Del/T790M	> 10 000	70
HCC827R	N/D	> 10 000	26

Treatment of both erlotinib-sensitive and -resistant cell lines with AT13387 resulted in depletion of EGFR and its phospho-form, irrespective of its mutation status (L858R, T790M, Exon19 deletion). Other clients such as AKT were also depleted. A decrease in the levels of phospho-ERK and phospho-S6 indicated that EGFR signalling was also being inhibited in both erlotinib-sensitive and -resistant cells. In vivo, AT13387 significantly inhibited tumor growth in EGFR-driven tumor xenograft models (HCC827, NCI-H1975) when administered at 70 mg/kg ip once weekly. As expected, erlotinib dosed at 12.5 mg/kg once daily caused regression in HCC827 xenografts, whilst 75 mg/kg once daily had no effect on tumor growth in the resistant NCI-H1975 model. Levels of EGFR and phospho-EGFR were depleted for up to 72 hours in xenograft tumors treated with a single dose of 70 mg/kg AT13387, whilst a reduction in phospho-ERK and phospho-S6 again demonstrated an inhibition of signalling.

Conclusion
AT13387 was shown to be effective in erlotinib-sensitive and -resistant NSCLC models, depleting levels of EGFR regardless of its mutation status. These data suggest that AT13387 treatment may also be a potential approach for combating EGFR inhibitor resistance in the clinic.

Background
When tumor cells undergo apoptosis in response to chemotherapy, the levels of apoptotic biomarkers such as phosphatidylserine and histone H1 are increased at the cell surface. Here we hypothesized that the chemotherapy-induced apoptosis would amplify in situ apoptotic biomarkers (homing signals) for apoptosis-targeted drug carriers and enhance drug delivery to lung cancer.

Methods
To examine this possibility, we employed a phage display-identified CQRPPR peptide (ApoPep-1) as a targeting moiety, which was able to recognize apoptotic cells by binding to histone H1 on the surface of apoptotic cells.

Results
When injected into lung cancer-bearing mice, ApoPep-1-labeled, fluorescent liposomes containing doxorubicin inhibited tumor growth more efficiently than untargeted or folate-labeled liposomes. Moreover, in vivo fluorescence imaging could enable monitoring of tumor response during the chemotherapy. The imaging signals at tumor were increased by the homing of apoptosis-targeted liposomes, which was correlated with the increase of apoptosis and the amount of doxorubicin (payloads) at the tumor and, conversely, with the decrease of tumor volume. Next, we harnessed the chemotherapy-induced apoptosis of tumor cells as a homing signal for the delivery of apoptosis-targeted T cells to lung cancer. When labeled with ApoPep-1 using an oleyl acid-derived membrane anchor, targeted T cells preferentially bound to apoptotic tumor cells over living cells. In vivo imaging showed higher levels of tumor homing of targeted, fluorescent T cells in mice treated with chemotherapy more than those of untargeted T cells.

Conclusion
These results demonstrate that the apoptosis-targeted delivery can efficiently enhance the delivery of cells or drugs to lung cancer and, when combined with imaging of apoptosis, provides a real-time monitoring of tumor response for lung cancer theragnosis.

Background
Lung cancer is the leading cause of death worldwide with a high metastasis and recurrence rate. Non-small cell lung cancer (NSCLC) accounts for 75-85% of lung cancers. Growing evidences show that some, if not all, tumors derive from a minor subpopulation of cancer cells, also known as cancer stem-like cells (CSCs), either retain or acquire the capacity for self-renewal and drug resistance. By the virtue of altered cell signaling pathways related to cell survival and/or apoptosis, CSCs are able to survive radiation or chemotherapeutic insults. Thus, the targeting of key signaling pathway(s) that is active in CSCs is very attractive therapeutic strategy to treating cancers. However, research has been hampered due to the lack of distinct molecular makers on CSCs. We take advantage of a rare subset of cells that can efflux the DNA binding dye out of the cell. These cells, called side population (SP) cells, are proved to be enriched with CSCs and have stem cell characteristics.

Methods
The SPs in PC-9 cells ware detected by staining them with Hoechst 33342. CSC population in PC-9 cells, the phosphorylation of EGFR at Tyr1068, AKT at Ser473 and ERK at Thr202/Tyr204 were investigated by FCM after treatment with EGFR/PI3K/AKT signaling inhibitors including Gefitinib, LY294002, U0126 and Erlotinib. significantly reduced the stem-like cancer cells. The effects of over-expression and silencing of β-catenin on the CSC population in PC-9 cells were detected by FCM. The PC-9 transplanted tumor model was used to detect the effects of Gefitinib and Cisplantin on CSC population in PC-9 cells. The Boyden chamber was used to determine the effects of Gefitinib and Cisplntin on the vitro invasion of PC-9 cells.

Results
EGFR-TKIs (Gefitinib or Erlotinib) regulate CSC, constitutive activation of EGFR increased the subpopulation almost 4.5-fold to 4.0%. EGFR-TKI almost completely ablated it resulting in only 0.2% or 0.3% of the total cells. EGF promote CSC population, the phosphorylation of EGFR at Tyr1068, AKT at Ser473 and ERK at Thr202/Tyr204 were investigated and they are all positive. EGFR/PI3K/AKT signaling inhibitors including Gefitinib, LY294002, U0126 and Erlotinib, significantly reduced the stem-like cancer cells. A significant decrease in cancer stem-like cells was observed following β-catenin suppression. The treatment with Gefitinib dramatically reduced the tumor numbers and size in vivo xenograft model with PC9 cells. Although there were few SP cells (1.3% as detected) in Gefitinib-treated mice in the primary tumors, more discernible numbers of SP cells were detected in Cisplatin-treated (13.6%) or control-treated tumors (8.3%). Tthe reduction of SP cells by Gefitinib treatment significantly reduced the migration capability of PC-9 cells. As a comparison, those primary culture cells derived from Cisplatin-treated tumors had an increased migration rate.

Conclusion
EGFR-TKI can dramatically decrease the CSC population and invasion ability in PC-9 cells in vitro and in vivo. The molecular mechanisms of EGFR-TKI decrease CSCs of lung cancer might be related to that EGFR-TKIs can suppress the Wnt/β-cateninsignal pathway.

Background
Malignant pleural mesothelioma (MPM), an asbestos exposure related disease, is a highly aggressive tumor. Pemetrexed is a third-generation multitargeted antifolate approved as single agent or in combination with cisplatin as standard of care in first/second line treatment of unresectable MPM. Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is the main target of Pemetrexed and its overexpression has been related to Pemetrexed-resistance. Experimental data suggest that c-SRC tyrosine kinase hyperactivation has a key role in MPM. The effect of c-SRC pharmacological inhibition in correlation with TS expression levels and Pemetrexed resistance, has been investigated in three MPM cell lines.

Methods
Cell growth inhibitory effects of both Pemetrexed and Dasatinib (10nM-100μM) were evaluated by MTS proliferation assay in epithelial (MPP89, REN) and biphasic (MSTO-211) MPM cell lines. Apoptosis was detected by AnnexinV-propidium iodide method using a FACScan, while drug-mediated changes in invasive ability were tested using “wound healing” scratch assay. Real-Time PCR and Western blot were assessed to identify drugs-associated genes and/or proteins modulation.

Results
The cell lines assayed displayed different sensitivity to both Pemetrexed and Dasatinib treatments. Among the three cell lines, MSTO-211 was the most sensitive to Pemetrexed (IC~50 ~0.5 μM); on the contrary REN was the most resistant (IC~50 ~5 μM). A similar trend was observed upon Dasatinib treatment with IC50 values ranging from 1 to 5 μM. The synergistic effect of Dasatinib and Pemetrexed was also evaluated, being significantly relevant in MPP89 and REN after 72h treatment while, in MSTO-211, was already detectable after 48h. Early and late apoptosis assessment confirmed, for both drugs, the ability to induce apoptosis, being MPP89 the most Dasatinib-sensitive and MSTO-211 the most Pemetrexed-sensitive cell line. In MPP89 and REN cells co-administration of Pemetrexed/Dasatinib significantly increased the apoptotic rate of 16 folds and this behaviour was enhanced in MSTO-211 (up to 27 folds). Both TS, gene and protein levels were higher in REN compared to MPP89 and MSTO-211 cells. Pemetrexed administration increased TS levels over time, in those cells most sensitive to the drug. Interestingly, in REN cells Pemetrexed treatment did not affect the high baseline TS levels but Dasatinib administration suppressed TS protein and, to a lesser extent, mRNA expression, thus increasing sensitivity to Pemetrexed. In addition, in REN cells the pretreatment with Dasatinib (5 μM) enhanced Pemetrexed sensitivity leading to a strong cell viability reduction. In all 3 cell lines, SRC was expressed (mRNA and protein), decreasing its levels from MSTO and MPP89 to REN, and activated. Dasatinib impaired also cell migration, as observed by wound-healing assay.

Conclusion
In vitro data suggest that inhibition of both TS and SRC might represent a potential therapeutic strategy in MPM. The evidence indicates that Dasatinib plays a role by inhibiting cell motility and, more surprisingly, by down-regulating TS. Dasatinib-mediated TS expression impairment suggests a cross-talk between SRC and TS pathways thus leading to hypothesize a therapeutic use of Dasatinib to sensitize those Pemetrexed-resistant MPM patients’ cohort.

Background
EphB4, a receptor tyrosine kinase and its ligand EphrinB2 are both cell membrane bound proteins that regulate cell migration, boundary formation, venous or arterial specification, vessel formation and maturation. EphB4-EphrinB2 interaction leads to bidirectional forward and reverse signaling. EphB4 is induced in certain cancers where it regulates cell survival, growth and metastasis.

Methods
We have studied the expression of EphB4 in lung cancer. 89 cases of matched normal and lung tumor samples were analyzed by IHC using EphB4 specific monoclonal antibody. Biological function of EphB4 was studied specifically in Kras mutant lung adeno Ca due to induction of EphB4. In addition, an in vivo efficacy study was conducted with soluble EphB4 receptor fused in frame at the C-terminus with Albumin (sEphB4HSA).

Results
EphB4 is significantly over-expressed compared to paired normal tissues in adenocarcinoma (n=41; 4.3-fold mean difference), large cell carcinoma (n=15; 2.9-fold mean difference), small cell carcinoma (n=13; 2.4-fold mean difference), and squamous cell carcinoma (n=10; 2.7-fold mean difference) subtypes. Overall, lung tumors were found to express EphB4 3.2-fold more strongly than paired normal tissues. EphB4 gene amplification (>3 fold) was also seen in 23% of squamous cell carcinoma tissues. Knock down of EphB4 led to near 70% loss of cell viability indicating that EphB4 is downstream of Kras and plays essential role in Kras mutant lung adenoCa. sEphB4 blocks bidirectional signaling and albumin fusion provides long circulation time in vivo. Kras mutant human tumor xenografts showed tumor regression and combination with taxol resulted in complete regression in lung adenoca.

Conclusion
sEphB4HSA cGMP material toxico-kinetic studies in non-human primates were performed and found to be safe up to a dose of 30mg/kg IV weekly. A first in human, phase I clinical trial of sEphB4HSA is approaching completion.

Background
Lung fibrosis can be caused by irradiation in radiotherapy or bone marrow transplantation, and many reports have documented an association between diffuse pulmonary fibrosis and lung cancer. Although a large number of compounds showed good radioprotection in in vitro studies, most of them failed in vivo due to acute toxicity and side effects. We tried to induce lung fibrosis in mice by irradiation, and at the same time, examined some compounds which are clinically used and thought to work as radioprotectors.

Methods
C57BL/6J mice,4-6weeks old were exposed to X-ray radiation, dose rate 0.88Gy/min, in the following conditions; (1) local fractionate irradiation (limited to the thorax); 2Gy/day x 10 or 20 days. (2) local single doze irradiation; 10, 15, 20 Gy. (3) total body single doze irradiation; 4Gy. At the same time, we administrated to each mouse by subcutaneous or intraperitoneal injection prior to irradiation sterile saline or 11 compounds which are clinically used and consist of antioxidants, sulfhydryl compounds, immunomodulators and so on. We examined the lung tissue of each mouse 5-8 months after irradiation, by checking microscopic change with Masson trichrome staining, and measured the Sircol assay level of the lung.

Results
By Masson trichrome staining, lung fibrosis were seen in the tissues irradiated with 40Gy in 20 equal fraction and 15 and 20Gy single dose more than 7 months after irradiation. The Sircol assay level of the lung rose as the radiation dose increased except for 4Gy total body irradiation, suggesting lung fibrotic change. Among the 11 compounds, administration of ascorbic acid and AHCC (Active Hexose Correlated Compound) showed no fibrotic change by Masson trichrome staining and they suppressed all the Sircol level of the lung.

Conclusion
Lung fibrosis after irradiation was suppressed by ascorbic acid and AHCC. We might be able to prevent lung tissue impairment after irradiation by using ascorbic acid and/or AHCC, and find other compounds which can be safe radioprotectors by this method.

Background
We identified an intronic miRNA, miR-135b, up-regulated in aggressive non-small-cell lung cancer(NSCLC). Ectopically delivering mir-135b enhanced cell invasive and migratory ability in vitro and in vivo; whereas specific inhibition of miR-135b by miR-135b-specific molecular sponge and antagomirs suppressed cancer cell invasion, orthotopic lung tumor growth and metastasis in mouse model. We showed that miR-135b could directly repress the expression of Hippo pathway components. In this study, we design a tunable pH-responsive hydrogels to enhance the bioactivity of chemically modified antisense RNA oligonucleotide and SPION in tumor microenvironment for acidosis-related tumor therapy.

Methods
pH-responsive matrix of PEG-imidazole hydrogel releases chemically modified oligonucleotides (antagomir) and positively charged superparamagnetic iron oxide nanoparticles (SPION) were prepared. NOD-SCID mice were subcutaneously injected with CL1-5 cells and control antagomiR or antagomir-135b was intra-tumoural injected for 3 weeks. Body weight was determined. Blood was collected before euthanasia. Total tumor volume, metastatic nodules, and miR-135b expression are measured.

Results
By using pH-responsive release of SPION from hydrogels to release antagomiR, we found the hydrogel administered to natural physiology had a rate of slower release at pH6.7 than at pH7.4, which is sufficient to restrain cellular uptake of antagomir and the rate of release in acidic environments can be manipulated via the imidazole content. .In addition, systematic administrated antagomiR-135b through I.V. injection inhibited the orthotopic lung tumor growth and decreased the volume of lung metastases. Both results trigger us to examine the possibility of in vivo placing the antagomiR-containing hydrogels by the side of tumor, to evaluate the effect of localized releasing antagomiR on tumor growth.

Conclusion
Our results support that inhibition of miR-135b by pH control release nanoparticle may be promising to develop a new therapeutic strategy for NSCLC.

Background
The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Focal amplification of FGF receptor 1 (FGFR1) has been identified in a subset of squamous and small cell lung cancers and is associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in these cohorts of patients. A number of small-molecule FGFR targeted agents, with diverse kinase inhibitory and pharmacological profiles, are currently in clinical development.

Methods
Fragment-based drug discovery coupled to structure-based design was used to identify JNJ-42756493. Fragments were optimized into potent FGFR inhibitors with selectivity against VEGFR2, which shares 57% sequence identity with the kinase domains of FGFR1 and FGFR3, and 54% with that of FGFR4.

Results
JNJ-42756493 has a pharmacological profile that is differentiated from other agents in this class currently under investigation. JNJ-42756493 displays potent pan FGFR (1, 2, 3 and 4) tyrosine kinase inhibitory activity and is highly selective outside the FGFR family. JNJ-42756493 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines dependent upon deregulated FGFR expression. JNJ-42756493 demonstrated highly specific tumor inhibitory effects in FGFR1-4 dependent cell lines in vitro and xenografts in vivo, with no discernible activity in models that were not dependent on FGFR signaling. JNJ-42756493 showed favorable drug like properties and displayed a high distribution to lung tissue. JNJ-42756493 was well tolerated at efficacious doses and resulted in potent dose-dependent antitumor activity accompanied by pharmacodynamic modulation of tumor FGFR and downstream pathway components.

Conclusion
Data presented here highlights JNJ-42756493 as a novel, highly potent and selective small-molecule pan FGFR kinase inhibitor with potent antitumor activity against FGFR-deregulated tumors in preclinical models. These data, together with our ongoing Phase 1 clinical trial, position JNJ-42756493 as a differentiated selective pan-FGFR family inhibitor and support its continued clinical development in lung cancer and other malignancies associated with aberrant FGFR signaling.

Background
Mesothelioma is essentially incurable and new drugs to effectively treat it are urgently needed. Our strategy to achieve this aim was to identify candidate mouse and human genes that may have a role in mesothelioma growth and to inhibit their expression in fully transformed mesothelioma cell lines using siRNA.

Methods
The initial selection of candidate genes was made on the basis of their differential expression in transcriptome or CGH analyses when comparing malignant to normal mesothelial cells. This was combined with known functional information relevant to tumorigenesis. We also selected a small number of candidates from other published studies. A second set of candidates was chosen from expressed kinases with the idea that these genes are more likely to represent druggable targets given the broad range of kinase inhibitors that are widely available. Where possible, we identified mouse and human homologues of the 40 candidates and then generated both mouse and human siRNA libraries. We tested the effect of gene knockdown on the growth of mouse and human mesothelioma cell lines in vitro.

Results
We found knockdown was efficient and inhibition of a subset of the selected genes slowed cell growth significantly across a range of cell lines in both mouse and human systems. There was not complete concordance between the mouse and human: Incenp, Plk1 and Tpx2 were important pathways for murine cellular proliferation; whereas, AURKA, TPX2 and BIRC5 were relevant for human cellular proliferation only. KIF11 was identified in both studies.

Conclusion
These genes all have a function in chromosome positioning, centrosome separation and spindle assembly during cell mitosis. Our data show that targeting these gene products in mesothelioma cell line causes growth inhibition both in vitro and in vivo. These studies could provide new leads for drug development.

Background
Chemotherapy (cisplatin, pemetrexed) remains is the standard of care for mesothelioma (MM) in Australia, however novel immunotherapies are now emerging in clinical trial. Anti-PD-1 (MDX-1106) and anti-CTLA4 (tremelimumab) block different aspects of negative T cell regulation to prolong the activation and survival of anti-tumour cytotoxic T lymphocytes (CTL). While anti-CTLA4 is being tested in Phase II clinical trial, the efficacy of anti-PD-1 in MM patients is yet to be determined. The notion of combining chemo-immunotherapy has gained ground in recent years with the discovery that chemotherapy-induced tumour cell death can be immunogenic, and thus exploited with the right immunotherapy drug.

Methods
The murine mesothelioma line generated in our lab, AB1-HA, was inoculated subcutaneously into the flanks of Balb/c mice. Tumour growth and survival following treatment with anti-PD-1 and/or anti-CTLA4, plus chemotherapy (gemcitabine, cisplatin) was monitored. Tissues (spleen, lymph nodes) were harvested at various time-points for flow cytometric analaysis to investigate immune correlates of response.

Results
Dual checkpoint blockade (anti-CTLA4 + anti-PD-1) was effective at delaying tumour outgrowth and improving survival, over either treatment alone (anti-PD-1 had negligible effect on AB1-HA growth). Combining this with cisplatin chemotherapy achieved an even greater effect, however this was not the case with gemcitabine.

Conclusion
The effect of dual checkpoint blockade mirrors that which has recently been discovered in mouse models of melanoma [1]and colon cancer [2]. The ability to combine this with chemotherapy to our knowledge has not been previously identified. Furthermore, it is interesting that the triple combination was only successful with cisplatin rather than gemcitabine, which in our hands has been shown to be immunogenic and works synergistically with other immunotherapies, such as anti-CD40. Not only can this finding be directly translated to the clinic, it also prompts future investigation into how best to combine different therapies to tackle malignancies that may be refractory to standard monotherapy treatments. 1. Curran, M.A., et al., PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A, 2010. 107(9): p. 4275-80. 2. Duraiswamy, J., et al., Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors. Cancer Res, 2013. 73(12): p. 3591-603.

Background
Malignant pleural mesothelioma (MPM) is an aggressive cancer affecting the pleura. Treatment options are limited and most patients die within 24 months of diagnosis. The recommended first line chemotherapy for MPM is a combination of cisplatin/pemetrexed (or alternatively Raltitrexed), and there is no recommeneded second-line therapy. As such new therapeutic approaches are required for the management of MPM. Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones to facilitate the transcription of target genes. Various inhibitors targeting the activity of BET proteins have been developed and have shown potent antiproliferative effects in hematological cancers, and more recently been studied for in vitro efficacy in lung adenocarcinoma cell lines (1). We examined the expression of various members of the BET in MPM and assessed the effects of one of these inhibitors (JQ-1) to determine if this family could represent a novel candidate target(s) for therapeutic intervention in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of BRD2 and BRD4 by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The expression of a known target of the BET inhibitor JQ1, oncogenic transcription factor FOSL1 was also examined. The effects of a small molecule inhibitor of BET proteins (JQ-1) on cellular proliferation was examined (BrdU ELISA).

Results
We show that the expression of the BRD2 and BRD4 variants are detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of BRD2 was very significantly downregulated (p=0.0006). BRD4 comprises 2 transcript variants, a long variant (BRD4L – Refseq NM_058243.2), and a short variant (BRD4S – Refseq NM_014299.2). BRD4L was not significantly affected in malignant MPM compared to benign pleura, whereas BRD4S was significantly elevated in the tumours compared to the benign pleura (p<0.05). When separated across histological subtype BRD2 was significantly decreased across all histological subtypes (p=0.0009). FOSL1 a candidate target of JQ1 (1) was found to be significantly elevated in malignant MPM compared to benign pleura (p<0.05). Treatment of REN/ NCI-H226 cells with JQ1 caused significant inhibition of cellular proliferation, with NCI-H226 being more sensitive than REN to this compound.

Conclusion
The BET domain proteins are altered in MPM, and a small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM. Reference: 1. Lockwood WW et al., (2012). Proc Natl Acad Sci U S A. 109(47):19408-13.

Background
Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura associated with exposure to asbestos. Treatment options are limited, and the current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Despite this treatment option, almost all patients die within 24 months of diagnosis. Therefore, new therapeutic options are urgently required for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of KDMs are common in many cancers, and play important roles in tumorigenesis. The jumonji (JMJ) family of lysine demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. One such family, the KDM4/JMJD2 family, may therefore be altered in MPM and could represent a novel candidate target for intervention

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM4 family members by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The effects of a small molecule inhibitor of KDM4A/JMJD2A, 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA) on cellular proliferation and gene expression were examined.

Results
We show that the expression of the KDM4 family is ubiquitously expressed across our panel of cell lines. In primary tumours however, the expression of KDM4 members KDM4A, KDM4B and KDM4C were significantly elevated in malignant MPM compared to benign pleura. Treatment of REN/ NCI-H226 cells with the small molecule PA caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

Conclusion
The KDM4/JMJD2 family of lysine demethylases are significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. Targeting this protein may have important future implications for the management of MPM.

Background
Primary and acquired resistance to platinum agents is a serious clinical problem in lung cancer. Its mechanisms are probably multifactorial and remain poorly understood. Enhanced DNA repair can lead to increased cell viability in the face of DNA damage and has been proposed to be important in mediating platinum resistance. PARPs (poly(ADP-ribose) polymerases) are a family of nuclear enzymes that regulate the repair of DNA single-strand breaks (SSBs). Cisplatin sensitivity and DNA repair mechanisms following treatment with the PARP inhibitor, PJ34, was investigated in this study.

Methods
A panel of isogenic cisplatin resistant (CisR) NSCLC cells lines (MOR, SKMES-1, H1299) previously generated in our laboratory were used. The cisplatin resistant phenotype was initially assessed by treating CisR and parental (PT) cells with increasing doses of cisplatin (0-80uM) for 72h, after which time, cell proliferation was measured (BrdU). The effects of PJ34 on cell survival were also examined in a similar dose-response study. IC~25~ concentrations were calculated for each cell line using GraphPad statistical software. Cells were treated with PJ34 (IC~25~) alone, or in combination with cisplatin and cell survival/proliferation measured after 72h. Under similar experimental conditions, RNA was isolated from cells from which cDNA was reverse transcribed. All cell lines were screened for PARP1, PARP2, BRCA1, BRCA2 and ERCC1 mRNA at basal levels, and in response to treatment (RT-PCR). To investigate DNA double strand break (DSB) repair capacity in our panel of cell lines in response to PARP inhibition and cisplatin, phosphorylated γH2AX foci was examined by High Content Analysis (HCA) following treatment of cell lines for 24h. Cisplatin-DNA adduct formation (Pt-GpG) was studied following treatment of cells for 24h. Cells (1x10[6]/ml) were spotted on Superfrost® Gold glass slides. Immunofluorescence staining of specific DNA platination products, and quantification of adducts, was performed using an antibody that specifically recognises cisplatin-GpG DNA adducts.

Results
MOR and H1299 CisR cells were significantly more resistant to cisplatin (10µM and 20µM) compared to PT cells. SKMES-1 CisR cells were also significantly more resistant at 10µM, 20µM and 40µM cisplatin. While PJ34 had no effect on NSCLC cells when treated as a single agent, cell proliferation was significantly inhibited in MOR and H1299 cells when used in combination with cisplatin. No effect however was observed in our panel of CisR cell lines. While baseline expression levels of PARP1/2, BRCA1/2 and ERCC1 mRNA levels were similar in PT and CisR cell lines, BRAC1/2 mRNA expression was increased in cells treated with cisplatin alone, and in combination with PJ34 in PT cells but not in CisR cells. The formation of γH2AX foci and measurement of cisplatin-GpG DNA adducts in response to PARP inhibition and cisplatin are currently being investigated.

Conclusion
Data from this study show that inhibition of NSCLC cells with the PARP inhibitor, PJ34, sensitises lung cancer cells to the cytotoxic effects of the platinum drug, cisplatin. Further studies are warranted to investigate the role of PARP inhibitors in cisplatin resistant NSCLC cells.

Background
Blockade of genetic driver alterations in cell signaling pathways such as the epidermal growth factor receptor (EGFR) have led to dramatic tumor responses in the metastatic setting. However, these agents have unexpectedly failed to improve outcomes in clinical trails of early stage (BR.19) and locally advanced (S0023) NSCLC. In fact, survival was significantly worse among patients receiving gefitinib in the S0023 trial, and trended to be worse in BR.19. While it is clear that EGFR TKIs can reduce the tumor bulk and improve symptoms in the metastatic setting, these results raise the possibility that EGFR inhibition might somehow stimulate tumor growth either directly or indirectly.

Methods
We studied the fractions and numbers of ALDH+ cells and activation of stemcell signaling pathways in two EGFR mutated cell lines treated with erlotinib.

Results
Here, we report that treatment of EGFR-mutated lung cancer cell lines with erlotinib, while showing robust cell death, essentially increases the fraction and absolute number of ALDH+ clonogenic stem cell-like cells. This phenomenon can be abolished by inhibition of Notch3, while Notch1 inhibition has little effect or slightly increases ALDH+ cells. We demonstrate EGFR kinase activity-dependent coprecipitation of Notch and EGFR receptors and EGFR kinase dependent tyrosine phosphorylation of the Notch3 receptor. We further found that inhibition of EGFR activity leads to increased nuclear accumulation of gamma-secretase dependent Notch3 that correlates with the increase in ALDH+ cells.

Conclusion
These data suggest that while EGFR TKIs are very effective at debulking tumors in the metastatic setting, inhibition of EGFR paradoxically causes Notch activation and an increase in clonogenic stem cell-like cells. Therefore, curative-intent therapy may be best accomplished by dual targeting of EGFR and Notch3.

Background
Lipids play roles in several biological functions such as cell growth, division, and membrane stabilization in normal and cancer cells. There has been also interest in the relation of serum lipid levels and cancer in various studies. Epidemiological studies have demonstrated that high total cholesterol level is associated decreased cancer incidence. On time of diagnosis, HDL-cholesterol levels are reduced in lung cancer patients. We investigated the relation between lipid profile and weight loss in advanced stage lung cancer patients.

Methods
Forty-eight advanced stage lung cancer patients and 20 healthy subjects were included in the study. SCLC patients had extensive stage disease and NSCLC patients were stage IIIB and IV. All of study patients and control subjects were smoker and non-obese. Serum lipid profile, total protein, albumin, erythrocyte sedimentation rate (ESR) and clinical data were recorded.

Results
Lower HDL-cholesterol levels detected in advanced stage lung cancer patients. Serum total cholesterol, total protein, and albumin levels were also lower in cancer patients than controls. Serum LDL-cholesterol measurements were not different between patients and healthy subjects. However, ESR is higher in patients than controls. Twenty-four patients had weight loss. Total cholesterol, HDL-cholesterol, and LDL-cholesterol levels were lower in the patients with than without weight loss. However, total cholesterol, HDL-cholesterol, and LDL-cholesterol levels were not different between lung cancer patients without weight loss and control subjects. In lung cancer patients, serum HDL-cholesterol level was correlated with inversely ESR; directly with serum albumin level.

Conclusion
Although the weak association between HDL-cholesterol and cancer has been reported and the effect of HDL-cholesterol in carcinogenesis has been discussed, we not found difference in lipid profiles of lung cancer patients without weight loss. We consider that the reduction of lipid levels may be related to cancer cachexia. Moreover, serum albumin level and ESR, indirectly markers of inflammation, were correlated with HDL-cholesterol. It is known that inflammation reduce HDL-cholesterol. The cause of coincidence between reducing HDL-cholesterol and cancer may be inflammatory process. Further studies that investigate the clinical signification of reduced HDL-cholesterol and other lipids are necessary.

Background
Prognostic and predictive biomarkers are increasingly used to customize treatment of patients with solid tumors. Intra- and inter-tumor heterogeneous distribution of biomarker expression are potential confounders for use of biomarkers, as small biopsies may not necessarily truly reflect the pattern of biomarker expression. It may also be an important factor in chemoresistance, as tumors with heterogeneous biomarker expression may potentially harbor chemoresistant tumor clones.

Methods
Immunohistochemical evaluation of expression of excision repair cross complementation group 1 (ERCC1), epidermal growth factor receptor (EGFR), class III-β-tubulin (TUBB-3), Thymidylate synthase (TS), Ki-67 and ribonucleotide reductase M1 (RRM1) was performed in 15 separate areas in each of 6 small microscopically completely resected adenocarcinomas of the lung in order to elucidate any heterogeneous distribution.

Results
Clinically relevant biomarker heterogeneity with respect to expression of EGFR, ERCC1, RRM1, TUBB-3, and Ki-67 was observed in 4 (66%), 4 (66%), 2 (33%), 3 (50%) and 5 (83%) out of 6 tumors, respectively. Thus, heterogeneity could potentially allocate these tumors erroneously into high or low expressers by chance alone, according to previously reported cut-off values. In contrast, TS was almost completely homogenously distributed.

Conclusion
Most biomarkers examined, except for TS, showed clinically significant intratumor heterogeneity in 33% to 87% of tumors examined. This heterogeneity may influence results in studies investigating the therapeutic impact of predictive biomarkers in NSCLC.

Background
Selection of EGFR TKIs for lung cancer requires accurate detection of activating mutations. Traditional techniques are limited by small biopsy sizes. We compared droplet digital PCR (ddPCR, Bio-Rad) to Sanger sequencing, mutant-enriched PCR (ME-PCR) and high resolution melt (HRM) PCR.

Methods
A comparative effectiveness study was performed on 317 resected NSCLCs with salt extracted gDNA. EGFR exons 19 & 21 Sanger sequencing and (Shigematsu et al., 2005) and HRM/ME-PCR mutation detection (Sriram et al., 2011) was previously reported. ddPCR (Bio-Rad) was performed with competitive allele specific Taqman PCR (CastPCR; Life Technologies); EGFR L858R assay for exon 21 c.2573T>G and c.2572_2572CT>AG; exon 19 deletion assay for 20 common deletions (EGFR_ex19dels_mu, EGFR_6224_mu). 8ng gDNA was tested in 20uL reactions partitioned into 20000 droplets; valid reads contained ≥ 10000 droplets. Controls were 8ng gDNA from mutation positive cell lines (AJCC: H1975, H1650), human female DNA (Promega) and no template controls. QuantaLife (Bio-Rad) calculated Poisson statistics determined allele copy/uL; samples with minimum estimated mutant copy number ≥ 0.15/uL (3 copies/8ng) were “called” positive.

Results
Serial dilution of control assay demonstrated detection of template to 40pg input gDNA. 209 (66%) men and 108 (34%) women of mean age 63 years (range 36 to 83) were included. 295 (93%) had smoked and 18 (6%) were never smokers; 4 (1%) were unknown. 1 subject (0.3%) was Asian. pTNM stages were I (47%), II (31%) and III (22%) respectively (6[th] Ed). 171 (54%) were adenocarcinomas, 109 (34%) squamous cell carcinomas, and 37 (12%) other histologies. Figure 1 Exon 19 and 21 ddPCR assays yielded valid results for 300 and 301 samples respectively. ddPCR detected all mutations previously demonstrated by Sanger sequencing (13) and HRM (13) but not ME-PCR (13/15). Mean droplet counts were lower in ddPCR only called (30 droplets/ng) than those samples also called by other methods (3600 droplets/ng; p=0.039). Median percentage tumour and necrosis content of mutation positive samples only by ddPCR were 30% and 0% respectively, identical to those called by other methods.

Conclusion
ddPCR identifies mutations detected by Sanger sequencing and HRM. Limitations include nanodrop quantitation of input DNA and data replication is required. This technique demonstrates high sensitivity but limited specificity and requires further validation to examine the significance of low droplet number positive calls. The authors acknowledge the assistance of R Harrison and A Beckhouse, Bio-Rad. Financial support gratefully received from: NHMRC (MD PhD Scholarship), CCQ (MD PhD Scholarship), Cancer Australia, TPCH Foundation, Queensland Health.

Background
ROS1 (c-ros oncogene 1) is a receptor tyrosine kinase that can become constitutively active and drive cellular proliferation in a variety of cancers. Approximately 1-2% of patients with non-small cell lung cancer (NSCLC) harbor activating ROS1 gene fusions and these patients may benefit from ROS1-targeted inhibitor therapy.

Methods
Immunohistochemistry for ROS1 expression was performed on 33 NSCLC specimens previously characterized for the presence of genetic abnormalities. These specimens were selected for ROS1 gene rearrangements (6 specimens) detected by RT-PCR and FISH, ALK gene rearrangements (5 specimens), EGFR mutations (5 specimens), KRAS mutations (5 specimens), HER2 mutations (3 specimens), RET gene rearrangements (3 specimens), and pan-negative (6 specimens). Immunohistochemistry was performed in a CLIA-certified laboratory with manual application of the ROS1 DFD6 antibody (Cell Signaling Technology, Inc) for 1 hour. ROS1 protein expression was evaluated by a pathologist with a hybrid (H)-score scale of 0 (no expression in any tumor cells) to 300 (intense expression in all tumor cells). ROS1 over-expression was defined as an H-score greater than 100.

Results
ROS1 protein over-expression was detected by immunohistochemistry in all 6 of the NSCLC specimens with ROS1 gene fusions detected by RT-PCR (example in figure below). None of the remaining 27 lung cancer specimens with ALK gene rearrangements, EGFR mutations, KRAS mutations, HER2 mutations, RET gene rearrangements, or pan-negative exhibited ROS1 protein over-expression. Figure 1

Conclusion
Detection of ROS1 over-expression by immunohistochemistry exhibited 100% concordance with results of ROS1 gene rearrangement for 33 NSCLC specimens and did not overlap with any of the other genetic alterations. Six specimens were positive for ROS1 gene rearrangement by both RT-PCR and immunohistochemistry. Tumors positive for genetic alterations associated with the ALK, EGFR, KRAS, HER2, and RET genes were all negative for ROS1 gene rearrangement and ROS1 immunohistochemistry. ROS1 immunohistochemistry is a sensitive, specific and cost-effective method for identification of a subset of patients with lung cancer that may benefit from ROS-1 targeted-therapy.

Background
The early diagnosis of bone metastasis (BM) may bring improvements of life quality and treatment to cancer patients. Although single-photon emission computed tomography (SPECT) is the most frequently used method for BM screening, it still has some shortages. This study was initiated to investigate the clinical significance of serum BAP, TRACP 5b and ICTP as bone metabolic markers for BM screening in lung cancer patients.

Methods
Newly diagnosed advance lung cancer patients with (N=130) and without (N=135) BM were enrolled in present study. In addition, newly diagnosed primary lung cancer patients (N=38) were enrolled as control. Serum BAP, TRACP 5b and ICTP were measured using enzyme-linked immunosorbent assay (ELISA) before the initiation of treatment. The differences in concentration of BAP, TRACP 5b and ICTP were analyzed by one-way analysis of variance (ANOVA) (or Kruskal-Wallis tests when appropriate). The screening effectiveness of BAP, TRACP 5b, ICTP and the combination of TRACP 5b and ICTP was assessed by receiver operating characteristic (ROC) curves analysis in patients with and without BM.

Results
For concentrations of BAP, TRACP 5b and ICTP, significant differences was found between patients with and without BM (all P<0.0001), as well as patients with solitary and multiple BM (BAP: P<0.0001, TRACP 5b: P=0.0008, ICTP: P=0.0474). ROC curves analysis reveals the area under curve (AUC) of BAP, TRACP 5b and ICTP was 0.760, 0.753 and 0.835 (all P=0.0001), respectively. The optimal cut-off value for BAP, TRACP 5b and ICTP was 21.8 μg/L (sensitivity=63.1%, specificity=77.0%), 7.8 U/L (sensitivity=58.5%, specificity=80.7%) and 8.8μg/L (sensitivity=63.1%, specificity=90.4%), respectively. When TRACP 5b and ICTP was combined for BM screening , AUC was elevated to 0.895 (P=0.0001), and the optimal cut-off value was TRACP 5b > 7.6 U/L and ICTP >8.4μg/L (sensitivity=71.5%, specificity=93.3%).

Conclusion
Our research has demonstrated that serum BAP, TRACP 5b and ICTP may serve as a useful supplement for SPECT in lung cancer BM screening. If the 3 markers can be properly used together with SPECT, BM screening would turn to be more timely and accurate.

Background
Treatment strategy in lung cancer is lack of surrogate markers that may improve the clinical management in such an aggressive and deadliest tumour. Recently, CTC detection and characterization has been suggested as a promising and valuable outcome biomarker that is beginning to be elucidated in this context. The study investigates whether CTC reduction along treatment has a prognostic significance in previously untreated patients with advanced NSCLC receiving chemotherapy.

Methods
Patients with histologically confirmed stage III or IV NSCLC and suitable for chemotherapy treatment were selected for the study irrespective of other baseline characteristics. From each patient, two peripheral blood samples for CTC analysis were collected at baseline and concomitantly with first radiological evaluation, after three cycles of chemotherapy. CTC expressing EpCAM were detected in the semiautomated platform; the CellSearch® system.

Results
In this single institution prospective study, 25 consecutive patients were included between April 2011 and January 2013. The patients had a median age of 67 years (range 41-80), most were former or current smokers (60% and 32%, respectively), had ECOG 1 (80%), adenocarcinoma subtype (80%) and stage IV tumour at diagnosis (84%). First line platinum-containing chemotherapy was combined with antiangiogenics in 64% and with antifolates in 36% of patients. After 34 months of follow up, the median overall survival for the whole population was 10.9 months (95% IC 6.9-15 months). A non-significant survival benefit was identified in the group of patients for whom a reduction in CTC enumeration was achieved (N=12), in comparison to those with equal or greater number of CTC detected (N=13) between the first and second blood samples collected [11.2 months (95%IC 9.07 – 13.4) vs 7.2 months (95% IC 4.9 – 9.5); p=0.44] (figure 1). However, progression free survival was similar in both groups of patients (5.9 months vs 5.6 months, respectively). Figure 1 Figure 1. Kaplan-Meier curves for overall survival (OS) of patients with a reduction in the number of CTC (R-CTC) versus patients with equal or greater number of CTC (NR-CTC) detected in peripheral blood samples during chemotherapy treatment.

Conclusion
CTC serial isolation along treatment is a non-invasive tool that shows an encouraging prognostic value in advanced non-small cell lung cancer. Those findings strengthen the introduction of outcome markers in treatment decisions in this setting, but warrants further investigation for its validation in larger studies.

Background
Anti-cancer effect of 5-fluorouracil (5FU) is affected by the expressions of 5FU related enzymes, such as dihydropyrimidine dehydrogenase (DPD) and thymidine synthase (TS) and orotate phosphoribosyltransferase (OPRT), in each tumor. On the other hand, anti-cancer effect of epidermal growth factor receptor tyrosine kinase (EGFR-TKI) is affected by EGFR mutation status in each tumor. In 2007, Suehisa and colleagues reported that adjuvant chemotherapy with uracil-tegafur, a fluorouracil prodrug, significantly prolonged survival rates among patients with EGFR wild-type adenocarcinoma but not among patients with EGFR mutant tumors. In this study, the correlation between 5FU related enzymes and EGFR mutation status was analyzed.

Methods
We analyzed 49 patients with primary NSCLC who were postoperatively treated with S-1, an oral fluorouracil anticancer prodrug composed of tegafur, CDHP, and potassium oxonate in the molar ratio 1:0.4:1. We then evaluated the relation between the EGFR mutation status, each of the 5FU related enzymes and various clinicopathological factors. In vitro, DPD mRNA and protein expression was investigated in various cell lines.

Results
Among the 49 cases (thirty adenocarcinoma (ADC), sixteen squamous cell carcinoma (SQCC), two adenosquamous carcinoma, and one carcinoid), EGFR mutation was observed only in ADC (12 patients; 24.5%). In immunohistochemical examination, 10 patients were DPD immune-positive (20.4%), 31 patients were OPRT immune-positive (63.3%), and 16 patients were TS immune-positive (32.7%). Three year disease free survival rate of single S-1 adjuvant therapy was 77.6%, and three year overall survival rate was 89.7%. DPD immune-positive cases were significantly correlated with EGFR mutation status (p = 0.003). In vitro, EGFR mutated cell lines showed high DPD mRNA and protein expression.Figure 1

Conclusion
High DPD expression was shown to be correlated with EGFR mutation in adenocarcinoma cells and tissues. This result indicates that 5FU might be effective for EGFR wild type tumors than mutant type tumor, and EGFR mutation status might be a potential poor predictive marker for treatment with 5FU drugs.

Background
Polymerase I and transcript release factor (PTRF)/Cavin-1 was initially　identified as a regulator of rRNA transcription in the nucleus. It then was demonstrated to be essential to the formation of mature caveolae at the plasma membrane. Recently, downregulation of PTRF/Cavin-1 was reported in several types of cancers including non-small cell lung cancer compared to normal tissue. However, its precise expression pattern and clinical significance in lung adenocarcinoma remains unclear.

Methods
Proteomic analysis of 12 lung adenocarcinomas and the paired non-cancer lung tissue were preformed using iTRAQ coupled LC-MS/MS. To determine the expression pattern of PTRF/Cavin-1, we then performed immunohistochemical staining of PTRF/Cavin-1 on 186 adenocarcinoma tissues completely resected at Osaka City University Hospital from January 2005 to December 2008. To evaluate the clinical significance of PTRF/Cavin-1, the relationship between PTRF/Cavin-1 expression and clinicopathological parameters was analyzed.

Results
Proteomic analysis shows that expression level of PTRF/Cavin-1 is significantly lower in the cancer compared to the paired non-cancer lung tissue. This result suggests that PTRF/Cavin-1 may be involved in the development of lung adenocarcinoma. Immunohistochemistry analysis reveals that 30 cases (16%) were strongly positive for PTRF/Cavin-1 as observed in the non-cancer lung tissues, while 158 cases (84%) were negative. Furthermore, we found that overall survival rate of PTRF/Cavin-1-positve cases was significantly lower than that of negative cases (Log-rank test, p=0.0010). These findings imply that PTRF/Cavin-1 in cancer cells may facilitate the progression of lung adenocarcinoma progression.

Conclusion
These findings indicate that expression of PTRF/Cavin-1 in adenocarcinoma is associated with poor prognosis and might be a useful prognostic marker for lung adenocarcinomas.

Background
Bone morphogenetic protein-7 (BMP-7) is signaling molecule belonging to the transforming growth factor (TGF) - beta superfamily. Expression of BMP-7 is highest in the kidney and it is thought to be related to kidney and eye development and skeletal patterning. Recent studies demonstrated that BMP-7 was expressed in various human cancers. However, there have been few reports detailing this in non-small cell lung cancer (NSCLC). Then, the purpose of the present study was to investigate expression of BMP-7 in clinical samples of NSCLC to determine its clinicopathological and prognostic impact.

Methods
160 NSCLC patients who received complete resection at Kagoshima University Hospital from 2001 to 2007 were enrolled in the study. Two patients underwent pneumonectomy, 4 bilobectomy, 154 lobectomy. A total of 160 patients were classified, including 102 male and 58 female patients (range, 26- 84 years; average, 69 years). The final pathological examination disclosed that cases of stage IA, IB, IIA, IIB, IIIA NSCLC numbered 50, 52, 16, 15 and 27, respectively. The patients were histopathologically classified as 112 adenocarcinoma, 40 squamous cell carcinoma or 8 others (adenosquamous carcinoma, large cell carcinoma, mucoepidermoid carcinoma, pleomorphic carcinoma) according to the 7[th] Edition of General Rule for Clinical and Pathological Record of Lung Cancer (The Japan Lung Cancer Society, 2010). Expression of BMP-7 in cancer　tissue was evaluated by immunohistochemistry. Correlations between expression of BMP-7 and clinicopathological factors and prognosis were analyzed retrospectively. The study was approved by the Institutional Review Board of Kagoshima University and performed according to the Helsinki Declaration. A statistical analysis of group differences was performed using χ[2] test. The Kaplan-Meier method was used for survival analysis and evaluated by the log-rank test. The Cox proportional hazard model was used in multivariate analysis. p<0.05 was considered statistically significant.

Results
Immunohistochemically, in NCSLC, BMP-7 expression was identified in cell membranes but also in the cytoplasm of cancer cells. The patients were classified into two groups (BMP-7-positive group, 68 cases; BMP-7-negative group, 92 cases). Expression of BMP-7 correlated with T factor (p=0.047), N factor (p=0.013) and pathological stage (p=0.046). BMP-7 expression was significantly correlated with overall survival after the operation (p=0.003). Moreover, multivariate analysis revealed BMP-7-positivity as an independent prognostic factor (p=0.022).

Conclusion
We can use BMP-7 expression as a predictor of lymph node metastasis and postoperative outcome in NSCLC. The signals activated by BMP-7 are complicated and involve intracellular and extracellular factors, so further analysis seems to be necessary to determine the mechanism involved.

Background
Lung cancer is one of the leading causes of cancer death throughout the world. A more sophisticated understanding of the pathogenesis and biology of NSCLCs could provide useful information for predicting clinical outcome and personalized treatment. α1,6-FT is the only one enzyme responsible for the core α1,6-fucosylation of N-glycans of glycoproteins, including EGF receptor, TGF-β1 receptor, and integrin α3β1.

Methods
α1,6-FT expression was studied by immunohistochemistry in a cohort of 129 surgically resected NSCLCs, classified categorically based on the proportion of positively stained cancer cells (high, > 20%; or low, < 20%), and analyzed statistically in relation to various characteristics, including histology, survival and prognosis.

Results
High and low expression of α1,6-FT was found in 67 and 62 of 129 NSCLCs, respectively. Multivariate logistic regression analysis revealed a significant association between high α1,6-FT expression and non-squamous cell carcinoma (mostly adenocarcinoma), as compared with squamous cell carcinomas (odds ratio, 3.51; p = 0.008). Patients with tumors having high α1,6-FT expression had significantly shorter survival time than patients with tumors having low expression in potentially curatively resected NSCLCs (p = 0.03) and adenocarcinomas (p = 0.009), as well as in pStage I NSCLCs (p = 0.03) by the log-rank test. Surprisingly, in pStage I adenocarcinomas, none of 15 patients with tumors having low expression died of lung cancer, although 12 of 23 patients with tumors having high α1,6-FT expression died of lung cancer. High α1,6-FT expression was a significant and independent unfavorable prognostic factor in potentially curatively resected NSCLCs (hazard ratio, 1.81; p = 0.047) and in pStage I NSCLCs (hazard ratio 2.55; p = 0.03) by Cox’s proportional hazards model analysis.

Conclusion
These results suggest that α1,6-FT may play a pivotal role for the biological characteristics of NSCLCs. α1,6-FT expression is associated with histology of NSCLCs, and may be a new prognostic marker for overall NSCLCs and adenocarcinomas.

Background
The majority of newly diagnosed patients with lung cancer are at an advanced stage, implying small chances of cure. However, lung cancer treatment has in the last years taken advantage of newly developed targeted therapies. EGFR-mutations and ALK-translocations are druggable alterations used in treatment decisions. There are several additional druggable mutations in cancers, specially among kinases, but their frequency in lung cancer is not fully elucidated.

Methods
Blood samples and tumour tissue were obtained from 96 operated early stage lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2011. 48 were women, 21 squamous cell carcinomas, 73 adenocarcinomas and two large cell carcinomas. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80[o]C until DNA isolation. The tumour cell content in the specimens was found to be more than 70% in most samples. DNA was isolated from both tumour and corresponding blood sample according to standard procedures. Targeted resequencing was performed using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons for all known kinases, and selected cancer related genes and their associated UTRs, in total 612 genes. Targeted regions were sequenced at 50-60x coverage, allowing the detection of subpopulations down to 20%. The derived sequence was analysed based on a pipeline including calling variations, somatic mutations, DNA copy number changes, indels and genomic rearrangements, as well as functional annotations.

Results
There were significant differences in the number of somatic mutations detected within each tumour, ranging from 1 to 81, with a median of 14 mutations. Each mutation was supported by at least 20% mutant reads in the tumour, and the great majority corresponded to missense mutations. Over 1000 mutations were identified among all the samples analysed, but recurrent mutations were identified in specific pathways like the PI3K- and CHEK2-pathways. The TP53-gene was the most frequent mutated gene, in almost 50% of the samples, and these mutations have been validated by Sanger sequencing. Of the samples with more than 30 mutations, 55% revealed a mutation in the ATM-gene, whereas the frequency among the other samples was 14%, indicating a deregulation in DNA repair. Using the exon data from tumour and normal samples, we estimated DNA copy number changes, detecting gains and amplifications in cancer relevant genes i.e. KIT, ERK, EGFR.

Conclusion
In this pilot study, we have analysed 96 lung carcinomas by next generation sequencing, focusing on the kinome. We have identified several interesting mutational events, and analyses on different clinical subgroups are ongoing.

Background
Recently we found a disintegrin and metalloproteinase-9 (ADAM9) was highly expressed in resected stage Ⅰ non-small cell lung cancer (NSCLC), correlated with shorterned survival time. Here, we investigate the abnormal expression of ADAM9 in surgically resected advanced NSCLC, to elucidate the relationship between ADAM9 expression and lymph node metastasis, to further evaluate the significance of ADAM9 as a novel biomarker in predicting the prognosis, and predicting the necessity of personalized postoperative chemo-radiation for the resected NSCLC.

Methods
One hundred and twenty eight cases of completely resected stage Ⅰ, Ⅱ and Ⅲ NSCLC with mediastinal N2 lymph nodes dissected were immunohistochemically analyzed for ADAM9 protein expression. Survival analysis were conducted to asses the significance of ADAM9 expression and the relationship with other clinicopathological characteristics.

Results
Of the 128 NSCLC, 64 were stage Ⅰ, 19 stage Ⅱ and 45 stage Ⅲ; 66.4% (85/128) was found with ADAM9 protein highly expressed (ADAM9+), significantly higher when compared with normal control lung tissues (P=0.000). The ADAM9+ rate in adenocarcinoma was higher than in squamous cell carcinoma (75.5% vs 41.2%) (P=0.000). ADAM9+ rates in stage Ⅱ and Ⅲ NSCLC were 84.2% and 77.8%, respectively, significantly higher than 53.1% in stage Ⅰ (P=0.006). Stratified, ADAM9+ rates in N1 and N2 cases were 76.2% and 80.6%, respectively, significantly higher than 56.3%, the ADAM9+ rate in N0 NSCLC (P=0.025). There was no difference found between ADAM9+ rates in T factor groups (P>0.05). The overall 5-year survival rate was 54.6% for this group of 128 completely resected NSCLC. The 5-year survival rate in ADAM9 low expression (ADAM9-) group (43 cases) was 68.8%, however, the 5-year survival rate was sharply decreased to 47.7% in ADAM9+ group (85 cases), the difference was statistically significant (P=0.039). Linear correlation analysis discovered that the ADAM9 expression showed a significantly negative correlation with the survival time of the 128 cases of resected NSCLC (R=-0.217, P=0.014). Patients who received postoperative chemo-radiation therapy (41 cases) had a higher 5-year survival rate of 69.5% when compared with those who received surgery only but without adjuvant chemo-radiation (87 cases) whose 5-year survival was 47.9% (P=0.017). When stratified, in the 85 ADAM9+ cases, the 5-year survival rate for those who received postoperative chemo-radiation therapy was 63.7%, higher than 40.4% who did not receive adjuvant chemo-radiation (P=0.037); however, in the ADAM9- cases, postoperative chemo-radiation did not improve the 5-year survival rate with a statistic significance (P=0.198).

Conclusion
ADAM9 is highly expressed in human resected non-small cell lung cancer tissues, correlated with lymph nodes metastasis and pTNM stage; highly expressed ADAM9 predicts worse prognosis, suggesting that ADAM9 is a useful novel prognostic biomarker. Importantly, ADAM9 could become a novel useful predictive biomarker helping decide if postoperative chemo-radiation therapy should be selected or not; adjuvant chemo-radiation therapy might benefit ADAM9+ NSCLC much more, instead of ADAM9- NSCLC. (This study was partly supported by grant from the Nature Science Foundation of Liaoning Province, China, No.20102285; and the Fund for Scientific Research of The First Hospital of China Medical University, No.FSFH1210).

Background
The aim of the study was to determine whether circulating tumour cells (CTCs) can be detected and whether they provide predictive or prognostic information in a cohort of patients with locally advanced and metastatic non-small cell lung cancer (NSCLC).

Methods
Participants with locally advanced or metastatic NSCLC had blood samples collected and analysed for circulating tumour cells with the CellSearch® platform at baseline, prior to their third cycle of chemotherapy and two weeks following treatment.

Results
Of thirty-four participants, circulating tumour cells were detected in 15 (44%). Ten out of 19 adenocarcinomas had detectable CTCs. Three of nine squamous cell carcinomas had detectable CTCs. Two of six NSCLC “not otherwise specified” had detectable CTCs. Of the 15 detected CTC cases, 10 were stage IV NSCLC. No significant associations have been seen to date with histology type, stage, performance status, age at diagnosis, gender, history of weight loss at presentation, time to progression or overall survival.

Conclusion
Circulating tumour cells can be detected in advanced non-small cell lung cancer. These results are intriguing and require further investigation - plans are underway to extend the study to a larger sample size to determine if there is any prognostic or predictive value to circulating tumour cell detection.

Background
Evaluation of prognostic factors in carcinoid tumors of the lung is limited due to the rarity of disease. This study assessed Ki-67 expression and other clinical variables as prognostic factors in cohort of 262 patients seen at Mayo Clinic, and subsequently developed a nomogram for predicting recurrence-free survival (RFS).

Methods
A systematic search of Mayo Clinic lung cancer epidemiology and tumor registry databases from 1997 to 2009 identified 448 consecutive patients, with 262 having available tissue blocks [40 atypical carcinoids (AC) and 222 typical carcinoids (TC)]. Clinical data were collected by chart review. Tissue blocks were reviewed by 1 of 3 pathologists using WHO criteria. Tumors were tested for the Ki-67 index using digital image analysis (tumor tracing) by two operators. The associations of the factors with RFS were explored using multivariable Cox proportional Hazards models, including concordance (c) index. A nomogram was developed using the variables from the final multivariate model.

Results
Age, smoking history, lymph node (LN) involvement, tumor size, and Ki-67 index were significant prognostic factors for RFS from a multivariate model (Table 1). Median follow-up on alive-patients was 5.6 years (0.008-16.2). Median percentage of Ki-67 index of AC and TC were 1.61% and 0.56% (P<0.0001), respectively. The multivariable model with Ki-67 index showed a c-index of 0.79 which was identical to a multivariable model with pathological diagnosis (c-index 0.79). The nomogram showing the probability of 5-year RFS estimates is shown in Figure 1. Figure 1

Variables	Adjusted by Ki-67 and Clinical Variables HR; 95% CI (P)	Adjusted by Pathological Diagnosis and Clinical Variables HR; 95% CI (P)
Ki-67	1.25; 1.11-1.41 (0.0016)	--
AC vs. TC	--	2.01; 1.05-3.88 (0.0436)
Age	1.05; 1.03-1.08 (<0.0001)	1.06; 1.03-1.09 (<0.0001)
Smoking Never Former Current	(<0.0001) -- 3.11; 1.68-5.74 4.34; 1.94-9.74	(0.0003) -- 2.86; 1.56-5.24 3.74; 1.67-8.40
Size of Tumor	1.42; 1.20-1.67 (0.0002)	1.33; 1.13-1.56 (0.0012)
Metastatic LN Negative Positive	(0.0007) -- 3.05; 1.66-5.59	(0.0068) -- 2.51; 1.33-4.76

Conclusion
Ki-67 index is a valuable prognostic biomarker for pulmonary carcinoids based on this large cohort. The nomogram based on Ki-67 index, age, smoking history, LN involvement, and tumor size is a useful clinical tool for predicting the 5-year RFS rate. Updating this nomogram with additional clinical follow-up, as well as external validation of this nomogram is critical before routine clinical use.

Background
Bone and brain are frequent and problematic sites of metastasis in metastatic non-small cell lung cancer (mNSCLC). Conflicting studies exist whether patients with EGFR mutations develop brain metastases (BM) more often or have a longer survival after diagnosis of mNSCLC than EGFR/KRAS wild type (WT) or KRAS+ patients. For metastatic bone disease (MBD) this is not known. In this retrospective matched control study we compared in EGFR+, KRAS+ and WT patients time from mNSCLC to development of MBD/BM, skeletal related events (SREs) and subsequent survival.

Methods
In this retrospective case-control study all EGFR+ patients diagnosed at two molecular pathology departments were selected (VUMC 01-11-2004 to 01-01-2012, MUMC 01-10-2008 to 01-08-2012). For every EGFR+ patient a consecutive KRAS+ and WT mNSCLC patient was selected. Patients with another malignancy within 2 years of mNSCLC diagnosis or no follow up were excluded. Data regarding age, gender, histology, performance score, treatment, MBD and BM diagnosis, SRE and subsequent survival were collected.

Results
222 patients were included: 73 EGFR+, 76 KRAS+ and 73 WT (table 1). Respectively 56.2%, 51.3% and 50.7% had MBD (p=0.768) of which respectively 41.5%, 25.6% and 40.5% were diagnosed during follow up (p=0.262). Time to MBD was (mean, [SD]) respectively 13.4 [±10.6], 20.7 [±17.8], 16.8 [±9.6] months (p=0.360). Post MBD survival was (median, [95% confidence interval (CI)]) 15.0 [11.0-19.0], 7.1 [1.3-12.8], 3.2 [0.0-8.3] months respectively (p=0.008). Time to 1[st] SRE was not significantly different (p=0.164). Respectively 28.8%, 39.5% and 34.2% had BM (p=0.444) of which 76.2%, 60.0% and 48.0% were diagnosed during follow up (p=0.148). Mean time to BM was 20.3 [±11.7], 10.8 [±9.3], 14.3 [±10.8] months respectively (EGFR+-KRAS+ p=0.013, EGFR+-WT p=0.176). Post BM survival was 11.0 [2.2-19.8], 6.9 [0-14.1], 12.5 [5.6-19.5] months respectively (p=0.969). Results did not change significantly when patients with only best supportive care were excluded nor when in the EGFR+ group only exon 19/21 patients were included.

table: patient characteristics and results bone and brain metastasis

Characteristics	EGFR+ N = 73	KRAS+ N = 76	Wildtype N = 73	p-value
Female N (%)	51 (72.6)	44 (57.9)	29 (39.7)	0.001
Mean age, years (range)	59.6 (29.3-90.7)	60.6 (35.1-83.3)	62.5 (39.6– 81.8)	0.228
Never smoker N (%)	29 (45.3)	2 (2.7)	10 (15.2)	<0.001
WHO PS 0-2 N (%)	63 (98.4)	72 (97.3)	60 (92.3)	0.270
Adenoca N (%)	67 (91.8)	63 (84.0)	55 (76.4)	0.209
1[st] line no treatment 1[st] line chemo 1[st] line EGFR-TKI	3 ( 4.1) 23 (31.5) 47 (64.4)	10 (13.2) 64 (84.2) 2 ( 2.6)	14 (19.2) 54 (74.0) 5 ( 6.8)	0.069 <0.001 <0.001
MBD N (%) Yes - at diagnosis - during follow up No	41 (56.2) -24 (58.5) -17 (41.5) 32 (43.8)	39 (51.3) -29 (74.4) -10 (25.6) 37 (48.7)	37 (50.7) - 22 (59.5) - 15 (40.5) 36 (49.3)	0.768 0.262
SRE+ N (%)	22 (53.7)	23 (59.0)	21 (55.3)	0.887
BM N (%) Yes -at diagnosis -during follow up No	21 (28.8) - 5 (23.8) -16 (76.2) 52 (72.2)	30 (39.5) -12 (40.0) -18 (60.0) 46 (60.5)	25 (34.2) - 13 (52.0) - 12 (48.0) 48 (65.8)	0.444 0.148

Conclusion
Incidence of MBD or BM was not different between EGFR+, KRAS+ and WT patients. Time from diagnosis of mNSCLC to MBD, 1[st] SRE or post-BM survival did not differ. However, survival after MBD was significantly longer in EGFR+ patients. This stresses the impact of bone management in these patients and probably warrant more intense screening for MBD. In EGFR+ patients BM remain a serious event with short survival. This should stimulate investigators to search for BM specific treatments in order to prolong survival post BM in EGFR+ patients.

Background
Patients with NSCLC harbouring an ALK translocation exquisitely respond to ALK inhibitors. It is therefore important to know ALK status for newly diagnosed NSCLC patients. Our institution has become centre of reference in Spain for ALK determination by FISH for other hospitals. The aim of this work was to report the clinical and pathological characteristics of the samples with ALK results evaluated in our institution.

Methods
We entered clinical-pathological characteristics of external and in-house samples into a database. ALK was evaluated by FISH with the FDA approved test (Abbot Molecular Inc, Des Plaines, IL). Whole sections were analysed evaluating a minimum of 50 nuclei per case. The case was considered typically rearranged when separated green and orange/red signals (at least by three times the signal diameter) were identified and atypically rearranged when a single orange signal was observed. Gain (including both low or high genomic gain) was defined as a mean copy number of 3 to 5 fusion signals in >=10% of cells and amplification as the presence of >=6 copies of ALK per cell in >=10% of analysed cells (Salido et al, JTO 2010). To analyse correlations between ALK status and clinical-pathologic variables, we used the Chi-square test or Fisher’s exact test with a significance at p<0.05.

Results
A total of 471 cases were included in the database. Patients’ clinical characteristics are summarized in Table 1. ALK translocation was found in 15 of 471 patients (3.2%). Within the ALK translocated cases 8 were female, 11 were adenocarcinomas, 2 squamous cell histology, 1 large cell neuroendocrine carcinoma, and 1 not otherwise specified. There was a significant association between smoking status and ALK translocation (6.6% of translocations among non-smokers and 2% among smokers, p=0.042). Fourteen patients (3%) showed ALK amplification, 366 (77.7%) gain in ALK copy number, 50 (10.6%) were disomic and 5 (1%) monosomic for ALK and 20 cases were not evaluable (4.2%). EGFR mutation was found in 23 of 252 patients (9.1%) and non of these was observed in cases with ALK translocation. We observed an association between the type of sample and the ability to obtain an evaluable result for ALK with 97.5% assessable biopsies vs 84.4% citologies, (p<0.0001).

			N (%)
Median age (range)	62.46 (32-91)
Gender	Male			330 (70.1)
	Female			141 (29.9)
Smoking status	Never			121 (25.7)
	Current/Former			350 (74.3)
Sample origen	Lung			425 (90.2)
	Pleura			15 (3.2)
	Lymph node			15 (3.2)
	Other			16 (3.3)
Type of sample	Citology			66 (14)
	Biopsy			405 (86)
Stage	I			104 (22)
	II			40 (8.5)
	III			87 (18.5)
	IV			240 (51)
Histology	Adenocarcinoma			363 (77.1)
	Squamous cell carcinoma			44 (9.3)
	Large cell carcinoma			11 (2.3)
	Other			43 (11.3)

Conclusion
ALK translocation is present in about 3% in Spanish NSCLC patients and is associated with adenocarcinoma histology and non-smoking status. The performance of ALK FISH in biopsy specimens is significantly better than in citologies.

Background
Lung cancer is the leading cause of cancer-related death in Luxembourg with high metastatic potential and mortality rate. Despite tremendous efforts made in biomarker studies for this disease, none of the blood-based tests available in the clinic are able to detect the presence of lung cancer early enough or to predict outcome in patients subjected to targeted treatments. Analytical difficulties of plasma proteome due to the high complexity and large dynamic range, and the lack of accessibility to the high-quality clinical samples are major obstacles, requiring a strategic and efficient experiment design to develop potent diagnostic and predictive/prognostic biomarkers.

Methods
In this study, liquid chromatography-mass spectrometry (LC-MS) based targeted proteomics and high-throughput immunohistochemistry (HT-IHC) screening were applied to plasma and tissue samples derived from the matching patients in order to identify lung cancer biomarkers detectable in plasma. A total of 95 biomarker candidates potentially secreted or shed to blood were selected from the previously performed discovery studies. Two proteotypic peptides per target were selected as surrogate peptides and selected reaction monitoring (SRM) based LC-MS assays for 190 peptides were developed. The evaluated assays were multiplexed in two LC-MS methods and analyzed in depleted plasma samples of lung cancer patients. For HT-IHC, tissue micro arrays (TMAs) of matching patients were prepared from formalin fixed and paraffin embedded (FFPE) blocks acquired during the surgery of the patients whose plasma samples were used for proteomic analyses. Both tumor and adjacent non tumor area of the FFPE blocks were included in the TMAs and screened against 50 potential biomarkers.

Results
The complementary results of LC-MS based assays and HT-IHC were analyzed to find the correlation of the expression profiles of targets found in tumor tissues and plasma samples. Several targets in the IHC experiment exhibited significant scores in tumor compared adjacent normal. 17 plasma proteins were analyzed in the corresponding patients’ plasma samples to identify a panel of biomarkers. This panel of biomarkers were further used to monitor the responsiveness of tumors upon therapeutic and surgical interventions.

Conclusion
Targeted proteomics was successfully applied to lung cancer biomarker study in plasma samples. Expression profiles of cellular protein markers measured by HT-IHC were critical to group the patient samples to be analyzed. A panel of biomarkers is currently being tested as diagnostic, predictive, or prognostic markers in lung cancer, and preliminary results will be shown.

Background
Stage I adenocarcinoma is the most curable form of non-small cell lung cancer (NSCLC), yet recurrence following complete resection is >30%. To improve this, it is important to identify indicators of tumor biology, which can predict a more aggressive disease course so adjuvant therapies can be considered. Osteopontin (OPN) is a regulator of malignant function in NSCLC. In more advanced NSCLC patients, plasma OPN levels correlate with prognosis. We hypothesize that pre-operative plasma OPN in combination with clinical factors can predict recurrence following resection in stage I adenocarcinoma.

Methods
A cohort of completely resected stage I adenocarcinoma patients without adjuvant or neoadjuvant therapy was prospectively collected and followed through 3 years. Pretreatment demographics, operative variables, pathologic characteristics, and time to progression were recorded. Histology was classified as solid or mixed with noninvasive features. Pre-operative plasma OPN was measured blinded and in duplicate by ELISA (R&D, Minneapolis, MN) and is reported in ng/ml. Cut points to predict recurrence were determined by X-tile (Yale University, CT) plots.

Results
There were 141 patients (50M/91F), 103 were stage IA. Median follow-up was 43.9 months and was complete in all to 3 years. Thirty-nine patients (27.8%) recurred by 3 years. The median pre-operative OPN was 54.9 (range, 2.3 – 150.6). OPN levels correlated with tumor size (r=0.25, p=0.003), but not with age, pack years, t-stage, extent of resection, or invasive histologic component. Median OPN was higher in males than females (62.9 vs. 50.5, p=0.009), and in current smokers compared to former/never smokers (68.5 vs. 53.3, p=0.04). In Cox regression analysis, an increased risk for recurrence was associated with preoperative plasma OPN >49.6 (HR=3.8, CI:1.7-7.8, p=0.001), solid histology (HR=2.5, CI:1.3-4.9, p=0.008) and male gender (HR=2.5, CI:1.3-4.6, p=0.005), but not with size, stage, age, pack years, and extent of resection. A model incorporating preoperative OPN and invasive histologic component stratified recurrence risk for both genders, but was highly significant in females (p=0.006) (Fig). Receiver operator curve (ROC) incorporating sex, OPN and invasive histologic component had AUC=0.76 (CI: 0.6-0.8, p=0.03). Figure 1

Conclusion
Circulating OPN provides a view of the tumor micro-environment and can serve as an important indicator of the course of the disease in resected NSCLC. When combined with sex and measures of histologic invasive component, plasma OPN >49.6 form a highly predictive cumulative model to predict early recurrence in resected stage I adenocarcinomas and should be validated to assess its value in selecting patients for adjuvant and tumor prevention protocols.

Background
An association has been well-established between common EGFR mutations and response to reversible and irreversible direct EGFR tyrosine-kinase inhibitors (EGFR-TKIs); however, there is a significant lack of information about the impact of uncommon mutations on outcomes such as overall response (OR), progression-free survival (PFS) and overall survival (OS) rates after being exposed to EGFR-TKIs or platinum-based chemotherapy (CT).

Methods
Information regarding 186 NSCLC patients from three Latin-American countries was analysed. Tests were made for EGFR and KRAS mutations; the clinical and pathological characteristics and the presence of common and uncommon EGFR mutations were considered according to OR, PFS and OS rates concerning EGFR-TKIs and CT.

Results
79.5% of the patients had common EGFR mutations and 20.5% uncommon mutations, including complex alterations. Lepidic and acinar histological subtypes were associated with higher common EGFR mutation frequency (p= 0.010). Patients having an OR to EGFR-TKIs treatment also had an OR to CT (p< 0.001). Patients harbouring common EGFR mutations had greater sensitivity to EGFR-TKIs than those having uncommon mutations (63.8% [IC 95% 51.1-76.5] vs 32.4% [20.0-44.7] p< 0.0001). Median PFS regarding EGFR-TKIs (16.4 [12-21.1] vs 4.1 months [1.9-5.9]) and CT (16 [10.9-21] vs 4.3 months [0.9-12.9]) was better in patients having common EGFR mutations compared to patients carrying uncommon mutations. The median OS of patients treated with EGFR-TKIs that harbored common EGFR mutations (37.3 months [33.2-41]) was longer compared to those patients who harbored uncommon mutations (17.4 months [12.9-21.8]).

Conclusion
Our findings suggest that patients with EGFR uncommon mutations, could receive platinum-based chemotherapy as first line of treatment and EGFR-TKIs can be reserved as second or third line treatment options.

Background
Purpose: The immunophenotypes of cancer-stromal cells have been recognized as prognostic factors of cancer. Previous reports have indicated the prognostic value of stromal cells in adenocarcinoma or non-small cell lung cancer. However, the prognostic value of stromal cells in completely resected high-grade neuroendocrine carcinomas of the lung (HGNEC; both small cell carcinoma and large cell neuroendocrine carcinoma) has not been reported. The purpose of this study was to analyze the prognostic markers of HGNEC by examining the immunophenotypes of cancer-stromal cells, including tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and cancer-associated fibroblasts (CAFs).

Methods
Materials and Methods: One hundred and fifteen patients who underwent a complete resection of HGNEC were included in this study. There were 98 men (85%), and their median age at the time of surgery was 68 years (range, 22-86 years); 71 patients had p-stage I diseases. The histologic type was SCLC in 52 patients and LCNEC in 63. We examined the presence of CD204-positive TAMs, Foxp3-positive Tregs, and podoplanin-positive CAFs to evaluate the prognostic values of these markers.

Results
Results: The number of CD204-positive TAMs and Foxp3-positive Tregs did not influence the overall survival (OS) or the relapse-free survival (RFS) of the patients. However, patients with podoplanin-positive CAFs had a significantly better prognosis than those with podoplanin-negative CAFs (OS: p=0.002, RFS: p=0.002, 5-year overall survival (5 YR): 74% vs. 45%). According to subgroup analyses, patients with podoplanin-positive CAFs displayed a better prognosis for both small cell carcinoma (OS: p=0.046, 5 YR: 74% vs. 46%) and large cell neuroendocrine carcinoma (OS: p=0.020, 5 YR: 74% vs. 45%). A univariate analysis identified 4 significant risk factors for OS: sex (female), pN(+), lymphatic permeation (+), and podoplanin-negative CAFs. In a multivariate analysis using the Cox regression model, sex, the presence of lymphatic permeation (ly), and podoplanin-negative CAFs were shown to be statistically significant independent predictors for recurrence.

Conclusion
Conclusion: The current study reported that podoplanin-positive CAFs had prognostic value in both SCLC and LCNEC. Our results imply that podoplanin expression reflects a tumor-inhibitory phenotype of CAFs in HGNEC. Although the exact mechanisms responsible for this phenomenon are not fully understood, our results provide novel insights into the pathogenesis of a unique microenvironment of HGNEC as well as basic data for new treatment strategies for HGNEC.

Background
Lung cancer has the highest mortality of all cancers worldwide with a 5 year survival rate of <15%. The prognosis improves dramatically when the disease is detected at an early stage, and when curative treatment is possible. Current (low dose CT) screening and diagnostic procedures are suboptimal with low specificity. Thus, novel detection methods for lung cancer as stand alone or in combination with other methods are needed. DNA hypermethylation of biomarkers in sputum have shown to distinguish lung cancer cases from cancer-free controls. The aim of the present study was to validate the usage of DNA hypermethylation of biomarkers in sputum samples of lung cancer patients and controls for lung cancer diagnosis, in comparison with sputum cytology.

Methods
We prospectively collected sputum of lung cancer patients and controls during 3-9 days in the Amsterdam and Nieuwegein area, The Netherlands. From this sputum bank, a learning set (n=80 lung cancer patients, n=91 controls) and validation set (n=173 lung cancer patients, n=164 controls) were randomly composed. DNA promoter hypermethylation of the following biomarkers was assessed by means of quantitative methylation specific PCR: RASSF1A, APC, cytoglobin, 3OST2, PRDM14, FAM19A4 and PHACTR3. Cut-off values for positive hypermethylation were calculated using Youden’s index. Sputum cytology analysis was performed for all sputum samples. McNemar’s test was used to compare the difference between sensitivity of hypermethylation and sputum cytology for lung cancer diagnosis. A two-sided p-value <0.05 was considered significant.

Results
RASSF1A was best able to distinguish cases from controls, with sensitivity of 37-41% and specificity of 91-97% in both learning and validation sets. In multivariate analysis, a panel of RASSF1A, 3OST2 and PRDM14 showed highest sensitivity of 82% [95% confidence interval (CI): 76 – 88%] with a specificity of 68% [95% CI: 61 – 74%] in the learning set, with consistent results in the validation set. Molecular analysis was superior (P<0.001) over sputum cytology (sensitivity of 15%). The sensitivity of the biomarker panel did not improve when it was combined with sputum cytology. There was no association observed between DNA hypermethylation and clinical parameters such as age, smoking status, tumor stage, and histology.

Conclusion
This study validates hypermethylation analysis in sputum for the diagnosis of lung cancer. RASSF1A hypermethylation showed high specificity and thereby can have an important role in lung cancer diagnosis in symptomatic patients. A panel of biomarkers RASSF1A, 3OST2 and PRDM14 showed high sensitivity, but relatively low specificity.

Background
In patients with NSCLC, accurate and accessible EGFR mutation testing is important for guiding treatment decisions. Current procedures involve the testing of biopsy or cytology samples, which are not always available from all patients. However, plasma of patients with advanced NSCLC contains circulating-free tumor-derived DNA (cfDNA) that is suitable for mutational analysis. The large, multi-center, non-interventional, non-comparative ASSESS diagnostic study (NCT01785888) will evaluate the utility of plasma-based testing compared with tissue or cytology-based testing as a less invasive methodology by which to assess EGFR mutation status in patients with NSCLC.

Methods
A total of 1300 patients (age ≥18 years in Europe; ≥20 years in Japan) with newly diagnosed locally advanced/metastatic (Stage IIIA/B/IV) chemotherapy-naïve NSCLC who are not eligible for curative treatment, or patients with recurrent disease after surgical resection with/without adjuvant chemotherapy, will be screened for EGFR mutation status in tumor and plasma across Japan and 7 European countries over 18 months. To allow determination of sensitivity between tumor and plasma-based EGFR screening (95% confidence interval [CI] 40-60%, assuming 50% sensitivity), 100 patients each with mutation-positive NSCLC in Europe (EGFR mutation frequency: ~10%) and Japan (EGFR mutation frequency: ~30%) will be required; 1000 and 300 patients will therefore be enrolled from Europe and Japan, respectively. Provision of tumor (biopsy/cytology/other tumor cell sample) and plasma samples for EGFR mutation testing will be mandatory. Precise clinical phenotyping will be performed, and clinical information about first line (all patients) and second line (patients with mutation-positive NSCLC) therapy decisions will be recorded. EGFR testing will be performed according to local practices, with Exon 19 deletions and L858R point mutations assessed as a minimum. The primary objective is determination of concordance between EGFR mutation status obtained via tissue/cytology and plasma-based testing (concordance rate, sensitivity, specificity, positive and negative predictive values, and exact 2-sided 95% CIs). Secondary objectives: determination of EGFR mutation frequency (including mutation subtypes) in patients with adenocarcinoma/non-adenocarcinoma NSCLC; description of first-line (all patients) and second-line (all available patients) therapy following mutation testing; characterization of current EGFR testing practices; correlation between EGFR mutation status identified in tumor/plasma samples and demographic/disease status data. Pre-planned exploratory objective: investigation of exploratory biomarkers which may help to define molecular features of NSCLC (prevalence, co-occurrence, correlation with demographic data) using optional, additional tumor (biopsy/cytology/other) samples. The secondary analyses from the study will help define the current status of EGFR mutation testing procedures across Japan and Europe, and provide further information regarding mutation frequency across patient subgroups, and the relationship between EGFR mutation status and therapy decisions.

Results
Not applicable.

Conclusion
Not applicable.

Background
The study was supported by Ventana Medical Systems, Inc., a Member of the Roche Group Background: The reliable identification of NSCLC patients with anaplastic lymphoma kinase (ALK) gene rearrangement is crucial for the prescription of ALK tyrosine kinase inhibitors (e.g. crizotinib). Whereas the US FDA-approval (2011) is based upon FISH-testing, the European EMA-approval (2012) refers to the definition of “ALK-positive” NSCLCs without mandating a particular test. Therefore a reliable ALK-immunohistochemistry (IHC) could be a promising option in daily routine practice.

Methods
Material and methods: To test the reliability of ALK-IHC-diagnosis in a multi-centre environment (17 European institutes from Belgium, Denmark, France, Germany, Scotland, Spain, Sweden and Switzerland) two tissue microarrays (TMA) consisting of 15 NSCLC cases (all adenocarcinomas; 3 cores for each case) were independently tested for ALK-expression by each laboratory using Ventana Medical System’s ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView Amplification kits. Cases included in the study were unequivocal ALK-break positive or negative (by FISH), as well as so called “ALK-borderline” cases (low percentage of ALK-break positive cells by FISH, around the cut-off of 15%, therefore challenging in diagnosis, but PCR-confirmed as harbouring EML-4-ALK-fusion variants and thus eligible for therapy). Prior to the TMA-based case testing, each participating instrument was qualified using the VENTANA ALK 2 in 1 Control Slides. To provide a uniform baseline interpretation, a webinar-based training was given to all observers. This training included an overview of the ALK Interpretation Guide, a guided review of 50 patient cases using digital whole slide images, and a proficiency exam certifying each observer.

Results
Results: Detailed data analysis was only partly accomplished at the time of abstract submission and will be presented in detail at the “World Conference on LUNG Cancer” in Sydney. Besides the binary evaluation of the cases (ALK-negative vs. ALK-positive) observers were asked to estimate the staining intensity (0-3) within positive cases in correlation to the number of tumor cells and to generate the H-score.

Conclusion
Conclusion: Referring to the EMA-approval text our multi-centre study may contribute to validation and accuracy of IHC-based ALK-testing. Such a validated and reliable IHC-assay could be used: (a) as a good pre-screening method reducing time consuming and costly FISH analysis (shorten turn-around time for test results) and (b) as a final predictive approach in cases with reduced interpretability of FISH results (e.g. minimal tumor cell content in small biopsies, decalcified or artificial altered tissue, FISH in doubt/”borderline”).

Background
Recently, personalize therapy for non-small cell lung cancer (NSCLC) has been improving and significantly to extract various molecular target. However, development of molecular targeted drugs is proceeding only in lung adenocarcinoma to date, while there are few drugs for lung squamous cell carcinoma (SCC). Therefore, we tried to extract molecular targets for SCC by comprehensive gene expression analysis of clinical specimen.

Methods
The subjects of this study consisted of 215 patients with NSCLC who underwent complete resection since 2005 to 2011 in our hospital. They included 102 adenocarcinomas and 113 SCC. First, we tried to extract molecules specific to SCC by tissue array analysis of clinical specimen. We selected FAM83B as a candidate marker for SCC by using comprehensive gene expression analysis. Then, we examined the protein expression of FAM83B in NSCLC tissues by immunoblot and immunohistochemical analysis (IHC). The relationship between the FAM83B expression and clinic-pathological factors was statistically analyzed.

Results
FAM83B expression at mRNA level was significantly higher in SCC than in normal lung or adenocarcinoma (P<0.0001). Immunoblot analysis also confirmed this tendency. In IHC, FAM83B was diffusely localized in the cytoplasm and/or plasma membrane. When more than 10% positive area for FAM83B were judged as “positive”, 94.3% (107/113) of SCC and 14.7% (15/102), of adenocarcinoma were positive. If the patients were divided into two subgroups by IHC (54 high-expression patients and 53 low-expression patients), high-expression group was associated with a better disease free survival rate (P=0.042, log-rank test). Figure 1

Conclusion
Our results indicated that FAM83B could be a reliable diagnostic and prognostic biomarker for SCC. Biological function of FAM83B in lung cancer is not well known. Further analyses should be required to identify its clinical significance and biological function.

Background
Lysosomal proteolytic enzymes play an important role in carcinogenesis and metastasizing processes. Activation of lysosomes may result in an increased exfoliation of cancer cells, which in vivo may promote metastatic progression. It was also observed that increased activity of lysosomal enzymes is connected with an increased permeability of cellular membranes and vascular endothelium, in turn associated with promoting metastasizing, also in lung cancer.

Methods
We evaluated the activity of selected lysosomal enzymes and one of protease inhibitors in serum, lung parenchyma and lung tumour, in 41 patients operated on with radical intent due to non-small cell lung cancer (NSCLC). Control group consisted of 44 healthy individuals. Cathepsin D, acid phosphatase, arylsulfatase and alpha-1-antitrypsin serum concentration was measured in patients before surgery, and on day 7, and 30 after operation. The concentration of these enzymes was also measured in the tumor and in healthy lung parenchyma. Obtained results were compared with control group, where concentration of enzymes was measured only in serum.

Results
In NSCLC patients an elevated serum concentration of cathepsin D (p<0.001), acid phosphatase (p<0.001) and arylsulfatase (p<0.001) was observed, compared with the control group. Serum concentration of acid phosphatase (p=0.033) and arylsulfatase (p=0.004) was elevated in patients with metastases to regional lymph nodes. Concentration of acid phosphatase (p<0.001), arylsulfatase (p<0.001) and alpha-1-antitrypsin (p<0.001) was higher in pulmonary tumor than in the healthy lung parenchyma. Concentration of acid phosphatase (p=0.002) and arylsulfatase (p<0.001) in pulmonary tumor was also elevated in patients with metastases to regional lymph nodes. In lung cancer patients, postoperative concentration of acid phosphatase and arylsulfatase decreased significantly, as comperative values. Figure 1 Figure 1. Chosen biomarkers activity comparison. (A) Comparison of cathepsin D (Cat D) activity in NSCLC patients with (N1+N2) and without (N0) lymph node metastases at baseline, POD 7 and POD 30. (B) Comparison of arylsulfatase (AS) activity in NSCLC patients with (N1+N2) and without (N0) lymph node metastases at baseline, POD 7 and POD 30. P values for each comparisons were obtained with Mann-Whitney U tests; POD, post-operative day.

Conclusion
Serum concentration of cathepsin D, acid phosphatase, arylsulfatase and alpha-1-antitrypsin is useful in the diagnostics of NSCLC. Moreover, serum acid phosphatase and arylsulphatase concentrations are useful in postoperative monitoring of these patients.

Background
Use of adjuvant chemotherapy in non-small cell lung carcinoma (NSCLC) is based upon pathological stage and is not generally recommended for patients with Stage I disease despite a five-year overall mortality of 30% in Stage IA and 50% in Stage IB. Molecular biomarkers have the potential to guide treatment by identifying patients at highest risk for recurrent cancer. An evaluation of prognostic breast RNA profiles revealed a common component of cell cycle regulated mRNAs which contains the major prognostic power of each expression profile. The expression levels of cell cycle progression (CCP) genes measure tumor growth irrespective of the underlying genetic aberrations. CCP has been shown to be a highly significant predictor of cancer specific mortality at five years in three individual datasets. From these data a prognostic model was generated incorporating the CCP expression signature with pathological stage. The study herein will assess the validity of this combined clinical and gene expression score to predict five-year risk of lung cancer death in patients with early stage lung adenocarcinoma. A high combined prognostic score will identify patients with an increased risk for relapse whom may benefit significantly from adjuvant chemotherapy.

Methods
A cohort of patients with NSCLC adenocarcinoma was assembled with the following clinical covariates: age at diagnosis, gender, smoking status, tumor size and grade, pleural invasion, TNM Stage, adjuvant treatment status, and EGFR mutation status (if known). Outcome variables include cause of death and time to recurrence and death. An event is defined as death due to lung cancer within five years of surgery. If cause of death is unknown, death following recurrence will be used as a surrogate. A cohort with 150 events will have 99% statistical power at the 5% significance level to demonstrate an association between CCP and death from lung cancer outcome. A CCP score will be calculated from the mRNA expression levels of 31 proliferation genes in this cohort and combined with stage in a final prognostic score.

Results
To date, 631 Stage I and Stage II adenocarcinomas have been assembled. Two hundred and fifty-five deaths have occurred in the cohort with more than 100 deaths caused by lung cancer. Also, there have been over 150 instances of lung cancer recurrence documented. Two hundred and thirty-four samples have been processed with CCP scores ranging from -3.20 to 2.20. The distribution of CCP scores is consistent with those observed in previous cohorts of early stage lung adenocarcinoma. Complete analysis will be presented.

Conclusion
This validation cohort will provide adequate events to significantly demonstrate whether the prospectively defined prognostic score can define a high–risk group of early stage NSCLC patients with a high risk of death from lung cancer. This information may help guide adjuvant treatment decisions.

Background
This study was conducted to analyze a comprehensive panel of single nucleotide polymorphisms (SNPs) in genes in DNA repair and apoptosis pathways and determine the relationship between polymorphisms and treatment outcomes of patients with non-small cell lung cancer (NSCLC) treated with first-line paclitaxel-cisplatin chemotherapy.

Methods
Three hundred eighty two patients with NSCLC were enrolled. Seventy-four SNPs in 48 genes (42 SNPs in 27 DNA repair pathway genes and 32 SNPs in 21 apoptotic pathway genes) were genotyped and their associations with chemotherapy response and overall survival (OS) were analyzed.

Results
Among SNPs in DNA repair genes, BRCA1 rs799917 was significantly associated with both chemotherapy response and OS. XRCC1 rs25487 exhibited a significant association with chemotherapy response and ERCC2 rs1052555 with OS. Four SNPs in apoptotic genes (TNFRSF1B rs1061624, BCL2 rs2279115, BIRC5 rs9904341, and CASP8 rs3769818) were significantly associated with OS, but not with response to chemotherapy. When the six SNPs which were associated with OS in individual analysis were combined, OS decreased as the number of bad genotypes increased (P~trend~ = 2ⅹ10[-6]). Patients with 3, and 4-6 bad genotypes had significantly worse OS compared with those carrying 0-2 bad genotypes (adjusted hazard ratio [aHR] = 1.54, 95% CI = 1.14-2.08, P = 0.005; aHR = 2.10, 95% CI = 1.55-2.85, P = 2ⅹ10[-6], respectively).

Conclusion
In conclusion, these findings suggest that the SNPs identified could be used as biomarkers predicting chemotherapy response and survival of NSCLC patients treated with first-line paclitaxel-cisplatin chemotherapy.

Background
In-frame deletion at exon 19 and point mutation at exon 21 are the two most common activating mutations in EGFR tyrosine kinase accounting for >85% of all clinical relevant EGFR mutations. In this study, we aim to develop a highly sensitive method to detect and quantify these two mutations in plasma of patients with advanced non-small cell lung cancer (NSCLC) using droplet digital PCR (ddPCR).

Methods
We analyzed 208 plasma samples from patients with advanced NSCLC from the ASPIRATION study using the QX100 ddPCR system (BioRad). ASPIRATION study is a single arm study on the use of first line erlotinib in patients with EGFR mutation (confirmed from tissue samples) and test the concept of treatment beyond RECIST progression. 36 archived plasma samples with known EGFR wild type were used as control. ddPCR simultaneously performs PCR reactions in 20,000 DNA containing droplets, and for each plasma sample we measured the absolute quantities of circulating EGFR mutant and wild-type sequences partitioning in discrete droplets.

Results
Specific ddPCR assays were developed for detecting EGFR exon 19 deletion and L858R mutation independently. 126 (61%) of 208 plasma samples were positive for EGFR mutation (63 cases for Exon 19 deletion, 63 cases for L858R). Table 1 summarizes the concordance of the tissue and plasma analysis. Sensitivity is 61%, specificity is 94% and positive predictive value is 98%. The mean absolute concentration for detectable exon 19 deletion and L858R in plasma is 1,060 and 1,510 copies/ml plasma respectively. Mean fractional concentration is 11%. Further correlation between plasma EGFR mutation results and clinical data will be performed.

Table 1

EGFR mutation	Tumor tissue +ive	Tumor tissue -ive
Plasma ddPCR +ive	126	2	128
Plasma ddPCR -ive	82	34	116
	208	36	244

Conclusion
Droplet digital PCR analysis is a novel sensitive detection method for EGFR mutation in plasma of patients with advanced NSCLC. Quantification of low level of circulating EGFR mutant DNA is feasible. Future investigation aims to correlate the quantified plasma EGFR mutation DNA with clinical outcomes.

Background
Bevacizumab (BEV), an inhibitory monoclonal antibody to VEGF, is widely used to treat patients with non-small cell lung cancer (NSCLC), but biomarkers that predict BEV response are controversial. Reportedly, hypertension is linked to response to BEV therapy, possibly because BEV might suppress vascular nitric oxide (NO) production. However, the usefulness of serum NO (NO~s~) as a predictive biomarker for BEV therapy has not previously been shown. Here, we studied the predictive value of NO~s~ in BEV-treated patients with NSCLC.

Methods
Fifteen patients with advanced or recurrent NSCLC treated with BEV-based regimens were evaluated retrospectively. Blood samples were taken before treatment (Pre), and after the 1st and 2nd chemotherapy courses (Post~1~ and Post~2~, respectively). NO~s~ (NO~2~[–]/NO~3~[–]) was assayed by the Griess method. Relationships between clinical parameters (e.g., clinical responses, adverse events) were analyzed against NO~s~. This study was approved by the ethics committee of Fukushima Medical University.

Results
Median Pre NO~s~ was 62.7 ±42.9 μmol/L (range: 1.9–138.8 μmol/L). NO~s~ tended to decrease at Post~1~ (46.6 ± 30.8 μmol/L; P = 0.246) and Post~2~ (37.6 ± 29.4 μmol/L; P = 0.072) compared to Pre values. Post/Pre NO ratios correlated with hypertension onset (Post~1~/Pre: P = 0.316; Post~2~/Pre: P = 0.148) and clinical response (Post~1~/Pre: P = 0.389; Post~2~/Pre: P = 0.163). Decrease at Post~2~ might correlate with progression-free survival (P = 0.127). NOs level of patients with treatment responder increased at Post PD (P = 0.101).

Conclusion
NO~s~, could be a predictive biomarker for response to BEV in patients with NSCLC. Prospective confirmation is needed; we are conducting a prospective translational study of NOs in BEV therapy. Figure 1Figure 2

Background
Development of Non-Small Cell Lung Cancer (NSCLC) requires multiple genetic and epigenetic alterations, with some differences according to etiology and histology. The most frequently mutated genes in these tumors are EGFR and KRAS (present mostly in adenocarcinomas), however, the prognostic value of KRAS mutations in NSCLC is still controversial.

Methods
Fresh tumor tissue samples (n=150) were obtained from resectable NSCLC patients. DNA was extracted by standard methods based in TriZol® and analyzed for KRAS mutational status by RTqPCR with ARMS technology and Scorpions probes. Non-parametric methods were used fos statistical analysis. Progression free survival (PFS) and overall survival (OS) were evaluated by Kaplan-Meier method (log-rank test). A p value ≤ 0.05 was considered statistically significant.

Results
Baseline characteristics of the patients were: median age, 64 years [26-82]; 86.0% male; 71.3% ECOG-PS 0; 40% adenocarcinomas (ADC). KRAS mutations were detected in 10.7% of the tumors (n= 150). Table 1 summarizes the mutations found in our cohort. In the subgroup of ADC + ADC-SCC samples, mutant KRAS represents 20% of the tumors. Considering only the never-smoker group of patients, 31.6% of the samples were mutated for KRAS. Our results showed that patients with KRAS mutated tumors had significantly shorter PFS than patients with wild type KRAS (11.633 vs 45.833 months, respectively, p= 0.043) and a trend to a shorter OS (23.067 vs 66.967 months, respectively, p= 0.074). Table 1: Distribution of KRAS mutations in our cohort

	n	%
Wild Type	134	89.3
12SER	1	0.7
12CYS	5	3.3
12ASP	7	4.7
12VAL	3	2.0
TOTAL	150	100.0

Conclusion
KRAS gene mutation is a poor prognostic factor for PFS in our cohort of resectable NSCLC; therefore, the determination of the mutational status of KRAS gene might be implemented routinely in clinical practice. This work was supported in part, by a grant [RD06/0020/1024 and RD12/0036/0025] from Red Temática de Investigación Cooperativa en Cáncer, RTICC, and Instituto de Salud Carlos III (ISCIII).

Background
The value of CTC has not been fully examined in patients (p) with NSCLC, in particular in those with locally advanced disease

Methods
A prospective study to evaluate CTC in NSCLC p is been conducting. Peripheral blood samples have been collected for CTC analysis basally in all stages and after finishing chemotherapy and radiotherapy in stage III. CTC analysis is performed using CellSearch (Veridex).

Results
One hundred and twenty nine patients were enrolled between January 2009 and May 2013. CTC was positive (CTC ≥1) in 21% (27/129 p). The number of CTC varied between 1 and 136 (7 p had only 1 basal CTC, 4 p had 2, 4 p 3 , 2 p 4, 2 p 5 , 2 p 6 and 1 p 7, 8 11,14, 37 and 136 CTC respectively). Basal positive CTC according to the stage were: 4% stage I and II p (1/26), 13% stage III p (6/45) and 35% stage IV p (20/58). In p with positive basal CTC, no differences were found in terms of histology (adenocarcinoma 22% p (18/81), squamous 20% p (7/34), others 14% p (2/14)), smoking status (current smoker 16% (10/61 p), non-smoker 21% (3/14 p), former smoker 26% (14/54 p)), EGFR status (EGFR + 17% (2/12 p), EGFR wt 25% (25/117 p), but a statistically significant difference was found in terms of ECOG (17% ECOG 0-1 (16/97 p) versus 34% ECOG 2 (11/32 p); p: 0.044). In 58 p with stage IV no differences were found related to location of metastasis (mts): M1a 32% (5/16 p), M1b 33% (8/24 p) although none of p with brain mts showed basal CTC. With a median follow-up using inverse Kaplan-Meier of 315 days, no differences were found in terms of survival based on basaline CTC status. Data of dynamic changes are still pending

Conclusion
Although this study is still ongoing, the role of basal CTC in stage III NSCLC is still unclear with only 13% of p positive at diagnosis. The value as predictive factor will depend on the data of dynamic changes that will be presented at the meeting

Background
Over the last decade encouraging new targeting agents have afforded benefits to patients with adenocarcinoma (ie. bevacizumab, erlotinib, gefitinib, crizotinib) but, very few advances were made in the treatment of squamous-cell lung cancer (SqCLC). However, many genomic abnormalities (PTEN, PI3KCA and FGFR1 etc.) are present in SqCLC and there is growing evidence of their cell survival, proliferation, and growth. These expressions have also been related to the resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in preclinical models. The objective of this study is to investigate the molecular and clinical factors that predict EGFR-TKI efficacy as a second-line or higher therapy in previously treated patients with SqCLC. We especially focused on the protein expression of EGFR, PTEN and PI3KCA gene amplification.

Methods
This retrospective study included 67 SqCLC Korean patients with available tumor tissue and data on EGFR-TKI treatment response and survival. EGFR protein expression in tumor tissue was evaluated by immunohistochemistry (IHC) with a specific antibody that detects the intracellular domain (ID) of EGFR. In addition PTEN expression in tumor tissue was assessed by IHC. PI3KCA gene amplification by quantitative real-time polymerase chain reaction (PCR) and mutational analyses of EGFR exon 19 and 21 by a PCR-based assay were performed.

Results
The median age was 70 years. The proportions of males and ever smokers were 85% and 82%. Patients had received a median of 2 prior chemotherapy regimens for advanced disease before treatment with EGFR-TKI. Eighty-four percent (n=56) of the patients received erlotinib treatment and the other (n=11) received gefitinib. Of the 54 patients available for response evaluation at 12 weeks, disease control rate was 35% (2 patients in partial response; 17 patients in stable response). The median progression free survival (PFS) and overall survival (OS) were 1.8 and 4.63 months, respectively. Positive EGFR protein expression in tumor tissue was present in 56 patients (85%), loss of PTEN expression (PTEN-negative) in 26 (39%) and PI3KCA gene amplification in 12 (21%). No cases exhibited EGFR activating mutation. But positive EGFR expression correlated with improved PFS (1.87 vs. 0.9 months, p=0.049). Negative PTEN expression was associated with a significantly higher risk of death (3.7 vs. 5.7 months median OS, p=0.028). Multivariable model confirmed that positive EGFR expression correlated with improved PFS (HR = 0.435, 95% CI = 0.21-0.89, p = 0.024) and positive PTEN expression was associated with an increased OS (HR = 0.437, 95% CI = 1.17-4.45, p = 0.015) after adjusting sex, age, performance status and number of previous chemotherapy regimens. The patients with EFGR-negative / PTEN-negative had poorer clinical outcomes than those with positive EGFR or positive PTEN expression: with shorter median PFS (2.1 vs. 4 months, HR = 1.713, p = 0.036). PI3KCA gene amplification was not related to clinical outcomes.

Conclusion
EGFR and PTEN protein expression could be used to identify which patients with SqCLC are likely to gain a benefit from EGFR-TKIs. The potential clinical application of specific EGFR-ID antibody for prediction of clinical outcomes in SqCLC needs validations.

Background
Background HGF, a ligand of the c-met proto-oncogene, exhibits activating effects on human lung cancer both in vitro and in vivo (Hosoda H, 2012). The major mediator of angiogenic and permeability-enhancing effects of VEGF-A is the tyrosine kinase receptor VEGFR2.(Ferrara N. 2001, Takahashi H 2005). Some data indicate negative impact on survival of HGF and VEGF/VEGFR2 in lung cancer patients (Yang F, 2011;Kumara 2009, Han Y,2011 Ikeda N, 2010). EGFR-mutated lung cancer patients have longer survival under treatment with anti-EGFR TKI than non-mutated tumors. The biological reasons for long-surviving advanced lung cancer patients (ALCP) with not EGFR mutated tumors are not described.

Methods
Methods The plasma samples of ALCP without EGFR-mutation who survived more than 20 months (mo) are taken for analyses from serum/plasma bank storage at -80C. The measurement of HGF and VEGFR2 are done according to the manufacturing instructions of eBioscience Instant ELISA test and Platinum ELISA eBioscience test. Statistical analysis is made by SPSS.9.0.

Results
Results 30 plasma samples from 13 ALCP taken before treatment and at response evaluation thereafter are analyzed in duplicate. ALCP, mean age 60.4(range 44-75) years, 10/3 man/woman, 7/6 smokers/nonsmokers, 3/7/3 squamous/adeno/small cell, 10/3- ECOG PS 0/1, 6/7 died/censored, have mean PFS (measured from diagnosis till first progression) of 24.07 months (SD 5.2) and mean OS of 37.5 mo (SD 4.7). ALCP have disease confined to the thorax (pulmonary mets, pl. effusion) with 5 pts with bone and 2 with suprarenal mets. IHC confirmation of diagnosis is done in 60% of pts. The first line chemotherapy is platinum based with addition of VP-16 for small cell, Gemcitabine for squamous and Pemetrexed for adeno-histotype. Ten pts receive maintenance treatment and 9 had more than three lines of treatment. None of pts receive anti-angiogenesis therapy. The mean baseline values (13 samples) of VEGFR2 and HGF are 520,4 pg/ml(SD262,2) and 104,4 pg/ml(SD91,5), while at second (10 samples) and third (7samples) measurements mean values are 544,9 pg/ml (SD 95,5) / 49,2 pg/ml (SD 20,4) and 563,65 pg/ml (SD 152,1)/ 147,13 pg/ml(SD53,6). Strong negative Pearson correlation between plVEGFR2 and Hb levels is found (p=0.007). No correlation between albumin, LDH, WBC , PLT and cytokine baseline levels are found. According to the median baseline value of VEGFR2, ALCP with values bellow 422.7 pg/ml have significantly longer PFS (34,6mo) and OS (47,1mo) than those with VEGFR2 values above 422,7 – PFS -11,8 mo and OS-26,2 mo. (p=0.023 and p=0.020, ANOVA test). Median baseline HGF values of 88.3 bellow/above separate ALCP with longer/shorter OS 46/32,1 mo but without reaching statistical significance (p=0.17)

Conclusion
Conclusions Classical clinical prognostic factors cannot identify ALCP with long survival –PS and numbers of therapeutic lines have positive effect on prognosis. Circulating baseline angiogenesis-related cytokines particularly VEGFR2 and HGF might be used for biological determinates of long survivors identification among patients with advanced lung cancer. However further studies with enlarged patients number with long survival are needed.

Background
The MET receptor tyrosine kinase and its ligand are associated with the malignant phenotype. In non-small cell lung cancer (NSCLC) MET expression increases with disease stage and is involved in de novo and acquired resistance to tyrosine kinase inhibitors. Despite this, in early stage NSCLC, conflicting data series have reported MET expression and copy number to be prognostic in some studies but not others[1,2]. We investigated a large cohort of patients who underwent curative surgical resection at our institution to determine whether MET receptor or gene amplification was prognostic.

Methods
Tissue Microarrays (TMAs) were constructed using 1mm cores of FFPE primary NSCLC tissues in triplicate. TMAs were stained with the MET SP44 clone and a H-score calculated based on % cells stained and intensity; (%cellsx1)+(%cellsx2)+(%cellsx3) with a minimum of 0 and maximum of 300. The mean of triplicate values was calculated. MET gene amplification was detected using Ventana’s MET DNP probe with ultraView SISH DNP silver detection, performed on Ventana’s XT autostainer. DNA was isolated and subjected to mutational profiling using Sequenom’s LungCarta panel.

Results
Data for 508 patients, 352 (69%) male, were available for analysis including 329 pathological node negative (pN0), 67 pN1, 104 pN2 and 8 patients with resected primaries and solitary brain metastases (M1). Most patients were smokers with only 33 (6%) non-smokers. The median MET H-score was 100 and consistent across N0, N1 and N2 patients, although was higher in M1 patients. Median H-scores were significantly higher in adenocarcinoma compared to squamous cell carcinoma (140 vs 91.5, p<0.0001). Increased MET expression (H-score>100) was seen in 227 (45%) patients. High quality DNA was isolated in 443/508 (87%) of samples. The commonest mutations were in KRAS (21%), TP53 (10%), EGFR (5%), PIK3CA (4%) MET (3%) and NRF2 (3%). No mutation was found in 44% of samples. EGFR and KRAS mutations were associated with significantly higher MET expression, whereas TP53 was associated with significantly lower expression (Chi square p=0.0005). These differences may reflect the higher rates of adenocarcinoma in both EGFR and KRAS mutated tumours. Increased MET copy number by SISH was only observed in 6 samples. MET expression was not associated with cancer specific survival across all stages. In tumours harbouring mutations and in wild type tumours, there were no significant differences in survival according to MET expression.

Conclusion
Although increased MET expression was associated with both KRAS and EGFR mutations, it was not prognostic in this large cohort of resected NSCLC. MET expression may be both predictive and prognostic in advanced NSCLC, but its role in early stage NSCLC is unclear. References: 1. Dziadziuszko R, et al. Correlation between MET Gene Copy Number by Silver In Situ Hybridization and Protein Expression by Immunohistochemistry in Non-small Cell Lung Cancer. Journal of Thoracic Oncology. 2012 Feb;7(2):340–7. 2. Cappuzzo F, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. Journal of Clinical Oncology. 2009 Apr 1;27(10):1667–74.

Background
Bevacizumab is a recombinant monoclonal humanized antibody targeted against vascular endothelial growth factor (VEGF) that improves Time to Progression (TTP) in patients with advanced non-squamous NSCLC in combination with a doublet of platins, but currently no proven predictive markers exist. The VEGF-A 165 splice variant has been described as the most abundant and active isoform in cancer. Exon 8 distal splice site modifications of VEGF 165 generates the VEGF-A 165a family of isoforms, which has a pro-angiogenic effect, and the VEGF-A 165b family, with an anti-angiogenic activity in in vivo models. This study is aimed to explore the role of VEGF165a and VEGF165b isoform expression in tumors as predictive biomarkers of efficacy in patients with non-squamous NSCLC treated with a doublet of platin plus bevacizumab.

Methods
22 patients were included (20 adenocarcinomas and 2 large cell carcinomas): 5 received carboplatin-taxol-bevacizumab, 14 carboplatin-taxotere-bevacizumab and 3 cisplatin-gemcitabine-bevacizumab. Total RNA was isolated from clinical samples by RNeasy FFPE procedure (Qiagen). VEGF~165~a and VEGF~165~b expression was analyzed by RT-qPCR using appropriate specific primers and probes in LightCycler 480II platform at 45 cycles. Individual VEGF~165~a and VEGF~165~b family of isoforms expression was calibrated to normal tissue and the ratio between both isoforms was calculated.

Results
From studied cases, VEGF~165~a overexpression was detected in 14 (63.6%) cases and VEGF~165~b overexpression in 15 (68.2%) tumors. Individual overexpression for each family of isoforms was not predictive of benefit to bevacizumab therapy (p=0.933 and 0.166). However, the ratio between VEGF~165~a and VEGF~165~b was associated with TTP, correlating a predominant expression of the pro-angiogenic VEGF~165~a in tumor with a significant benefit compared with cases with predominant VEGF~165~b expression (median TTP, 15 vs. 8 months respectively, p=0.005). The expression of both family isoforms did not impact on overall survival (p=0.477).

Conclusion
The overexpression of VEGF~165~a family of isoforms associated with a low expression of VEGF~165~b correlated with benefit to anti-angiogenic therapy in this small cohort of advanced NSCLC patients, supporting a potential use as predictive biomarkers for bevacizumab treatment in stage IV non-squamous NSCLC.

Background
Lung cancer (LC) is the second most common cancer diagnosed in men and women. The age-adjusted incidence rates are 62.6 per 100,000 populations per year. LC is also the most common cause of cancer mortality in the United States with 56% of the cases being metastatic at the time of diagnosis. Hyponatremia has classically been associated with Small Cell LC. Studies have shown Non Small Cell LC (NSCLC) to be associated with Hyponatremia. Hyponatremia is a predictor of mortality and worse prognosis in NSCLC. We studied the association of sodium (Na) levels with histological type, level of differentiation and staging in NSCLC.

Methods
We retrospectively enrolled 490 patients with NSCLC from the tumor registry data of our hospital from 2001 to 2011. One hundred one patients were excluded based on the following criteria: 1) patients without biopsy proven NSCLC, 2) medical records unavailable, and/or 3) age < 18 years at time of diagnosis. The following variables were collected: TNM staging (Stage 1, 2,3 as low stage and 4 as high stage),histological type, level of differentiation (well differentiated, moderately differentiated, or poorly differentiated) and Na levels at the time of diagnosis. Na level of 136meq/l or lower was used as a cut-off between high and low sodium groups. Data was analyzed using Chi Square statistical analysis and the Fishers Exact Test. Inferences were tested to be significant at P value of 0.05 or less.

Results
There were 234 (59.7%) males out of 389 patients. The mean age was 67.3 ± 11.4 years. The distribution of race was 227 (58%) white patients, 91 (23%) black patients, and 71 (20%) other races. Patients in low stage constituted 218 (55%) of the patients. Low Na levels were significantly more common in patients with high stage NSCLC [Odds ratio 1.85, CI 95% (1.19, 2.87), P<0.006]. On further sub-group analysis, low sodium levels were found to be significantly associated with higher stage in patients with Adenocarcinoma (n=183, 47%), [Odds ratio 2.07, CI 95% (1.12, 3.84), P<0.021]. No statistical association was seen between Na levels and other tumor variables.

Conclusion
We demonstrated higher stages of NSCLC and Adenocarcinoma Lung were associated with lower sodium levels. Possible mechanisms explaining this phenomenon includes Syndrome of Inappropriate Anti Diuretic Hormone secretion, increased levels of Atrial Natriuretic Peptides, release of Neuropeptide Y stimulating the posterior pituitary & metastases to adrenal or brain. Previous studies have demonstrated that lower sodium levels are associated with poor prognosis in LC. Serum sodium is an inexpensive and a routinely ordered test. Further prospective and larger studies are needed to substantiate the role of serum sodium levels as an easily assessable biomarker for advanced disease. It might also be useful to explore its potential as a marker of the disease progression.

Background
Tumor vasculature is a very important target for the management of NSCLC, with antiangiogenic therapy becoming part of standard antitumor treatment. Angiopoietin-2 (ANG-2) is a functional ligand of the Tie2 tyrosine kinase receptor expressed on endothelial cells. The dominant biologic role of ANG-2 as a destabilizing agent of established blood vessels as a prerequisite to sprouting angiogenesis includes it among the most intensely explored target molecules for the development of second-generation antiangiogenic drugs. The aim of this study was to evaluate peripheral blood leukocytes’ ANG-2 mRNA expression levels and serum circulating levels of ANG-2 as prognostic factors for NSCLC patients.

Methods
The study included 150 Caucasian NSCLC patients from the North region of Portugal, with a mean age of 64.0 years. The samples were collected at the time of diagnosis, before treatment, and included 52 epidermoid, 76 adenocarcinomas, 20 undifferentiated NSCLC, 2 large cells and 2 mixed carcinomas, of which 76% were male and 73,5% smokers or former smokers, divided in 77 non-metastatic and 73 metastatic cases. ANG-2 circulating levels were evaluated in subject samples with R&D Quantikine ELISA Kit, and mRNA expression levels (92 patients) with High Capacity RNA-to_cDNA[™] kit and Taqman[®]Gene Expression Assay, by real-timePCR, both from Applied Biosystems, according to manufacturer’s instructions.

Results
Our results demonstrate that patients with high ANG-2 circulating levels present a worst overall survival (OS) than patients with lower circulating levels (21.0 months vs 42.6 months, respectively; Log Rank Test, p=0.001). Equally, patients with high mRNA expression levels have diminished overall survival when compared to those with low mRNA expression (20.3 months vs 34.3 months, respectively; Log Rank Test, p=0.016). Moreover, Cox Regression analysis adjusted to tumor stage, smoking status and histological type indicates that both high ANG-2 circulating levels and high mRNA expression levels are independent prognostic factors in NSCLC (HR=1.83, CI95%=1.19-2.80, p=0.006; HR=1.82, CI95%=1.02-3.23, p=0.043, respectively).

Conclusion
ANG-2 is considered as a major player of the angiogenic switch in the course of tumor progression, and it is found to be particularly increased in highly vascularized tumors. In fact, in some tumor models, the mRNA induction of ANG-2 in tumor endothelium has made it a very attractive circulating biomarker of angiogenesis activation. Several studies are currently investigating the promising role of ANG-2 as a target of antiangiogenic inhibitors in several cancers, as an alternative to acquired resistance to currently used anti-VEGF molecules. Our results indicate that higher levels of ANG-2 mRNA in peripheral blood leukocytes and circulating ANG-2 are associated with worst survival in NSCLC patients. Assuming that blockage of ANG-2 is achieved in tumor stroma in a near future, this might represent a new breakthrough in cancer treatment and its circulating levels and mRNA expression levels may be helpful as predictive factors of treatment response, surpassing the need to obtain tumor tissue samples to assess its levels of expression.

Background
Circulating tumor cells (CTCs),which can be detected from peripheral blood, offer the potential for the assessment of clinical outcome. Whole genome sequencing of CTCs may provide comprehensive information related to tumor invasion and metastases, but has been hampered by their low abundance

Methods
From 7.5 ml peripheral blood, we captured with the CellSearch platform a small numberof CTCs, which, after further isolation with 95% specificity, were subject to whole genome amplification with Multiple Annealing and Looping-Based Amplification Cycles (MALBAC). The individual CTCs’ copy number variations (CNVs) were determined by whole-genome sequencing, and their single nucleotide variations (SNVs) and insertions/deletions (INDELs) were detected by exome sequencing.

Results
We sequenced 24 CTCs from four patients with advanced lung adenocarcinoma and compared them with the matched primary/metastatic tumors. Patient 1 with EGFR mutation experienced a phenotypic transition from lung adenocarcinoma to small cell lung cancer in liver, and resistance to EGFR-TKIs. Individual CTCs from each patient exhibited reproducible copy number variation (CNV) patterns, which resembled those of the metastatic tumors. CTCs from different patients showed similar CNV patterns on certain chromosomes. Some rare single nucleotide variations (SNVs) and insertions/deletions (INDELs) in primary tumor, including those that may relate to drug resistance and phenotypic transition, were enriched in CTCs.

Conclusion
CTCs exhibit highly reproducible CNV patternswhich offer a potential biomarker for cancer diagnosis and classification. The SNVs/INDELS in individual CTCs can be detected and provide molecular targets for personalized treatment.

Background
Ki-67 is a nuclear proliferation marker that reflects growth of tumor. Recently, the predictive implication of ki-67 labeling index (LI) for response to chemotherapy has been evaluated. Since St. Gallen International Expert Consensus in 2009, the ki-67 LI have been used one of factors that should help decide whether chemotherapy is given in breast cancer. In the present study, we examined the predictive value of ki-67 LI for chemotherapy for non-small cell lung cancer (NSCLC) patients using the histoculture drug response assay (HDRA).

Methods
Surgically resected fresh tumor specimens were obtained from 92 NSCLC patients at our institution from January 2007 to June 2011. The patients comprised 56 male patients and 36 female patients who ranged in age from 39 to 84 years (median= 73 years). The specimens examined were 57 adenocarcinomas, 26 squamous cell carcinomas, 4 adenosquamous carcinomas, 3 pleomorphic carcinomas and 2 other histological types. HDRA were used as an in vitro drug sensitivity test. HDRA technique was the same as we previously reported (JTCVS 133: 303-8, 2007). The inhibition rate of cisplatin and docetaxel were measured. Immunohistochemical staining for ki-67 was done and measured ki-67 LI. Relationships between ki-67 LI and the inhibition rate were examined using Spearman’s correlation coefficient test by rank test and chi-square test. Values of p<0.05 were considered to be significant.

Results
Immunohistochemical staining of ki-67 and the HDRA for cisplatin and docetaxel were successful in all specimens. Ki-67 LI was significantly correlated with the inhibition rate of cisplatin (rs=0.24, p=0.025) and docetaxel (rs=0.29, p=0.005) evaluated by HDRA. Ratio that have positive sensitivity for cisplatin in higher ki-67LI (ki-67 LI≧70) patients (52.6%) was significantly higher than that in lower ki-67LI (ki-67 LI≦30) patients (21.2%) (p=0.04). Ratio that have positive sensitivity for docetaxel in higher ki-67LI (ki-67 LI≧70) patients (40.0%) was significantly higher than that in lower ki-67LI (ki-67LI≦30) patients (10.8%) (p=0.025).

Conclusion
Our result revealed the ki-67 LI was the predictive marker for chemosensitivity in NSCLC. The high expression of ki-67 indicates positive sensitivity to cisplatin and docetaxel in patients with NSCLC.

Background
Tyrosine kinase inhibitors (TKIs) are widely used in advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations. A research recently found that some patients, including NSCLC patients failed their TKI therapy due to a Bim deletion polymorphism. We here try to distinguish the prevalence and clinicopathologic characteristics of the Bim deletion polymorphism in Chinese NSCLC patients.

Methods
300 patients were included in the study for Bim polymorphism analysis. PCR and direct sequencing were applied to determine the polymorphism status of tissue or blood sample extracted from these patients. 187 patients who received TKI therapy were further analyzed for relationship between clinicopathologic characteristics, therapeutic effects of TKI and Bim polymorphism status.

Results
40 of 300 (13.3%) patients were detected of Bim deletion polymorphism. Further analysis among the 187 patients indicated that this polymorphism distributed randomly in clinical characteristics including age, gender, smoking history, histological type and disease stage. However, patients harboring the Bim polymorphism had significantly shortened progression free survival (PFS) than those without the polymorphism (3.0±1.4 m vs. 7.5±0.9 m, p=0.012). Objective response rate (ORR) in Bim polymorphism carrying patients and wild typed patients also showed significant difference (21.7% vs. 50.6%, p=0.009). In further stratified analysis by EGFR mutation status, the PFS and ORR differences in Bim polymorphism and wild type patients remained significant. Disease control rate (DCR) of the polymorphism carriers also showed a tendency of inferiority (39% vs. 75%, p=0.061), though without a significant difference.

Conclusion
Chinese NSCLC patients carrying Bim deletion polymorphism had inferior response to TKI therapy despite EGFR mutation status. And Bim polymorphism could serve as an inferior prognostic factor in NSCLC TKI therapy.

Background
Human Mena and the isoform hMena[+11a] are cytoskeleton regulatory proteins involved in adhesion, motility, regulated in the epithelio-mesenchimal transition. Here, we investigated their potential prognostic value in node-negative non-small-cell lung cancer (NSCLC) patients.

Methods
Pan-hMena, hMena[+11a], E-cadherin, vimentin, ER-beta, EGFR, HER-2, pAKT, detected immunohystochemically on duplicate TMA and clinical factors (sex, age, histology, grading, T-size, number of resected nodes, RN) were correlated to 3-yr disease-free (DFS), cancer-specific (CSS), and overall survival (OS) using a Cox model. ROC analysis provided optimal cut-off values and model validation. A logistic equation including regression analysis coefficients was constructed to estimate individual patients’ probability (IPP) of relapse. Internal cross-validation (100 simulations with 80% of the dataset) and external validation was accomplished.

Results
In a training set of 248 patients (median follow-up: 36 months, range 1-96), Pan-hMmena and hMena+11a were the only biological variables displaying significant correlation with outcome(s), confirmed by the cross-validation (replication rate: 78%, 83%), with a prognostic model accuracy of 61% (standard error 0.04, p=0.0001). Patients with high pan-hMENA expression had a non-significant trend towards a worse outcome, while patients with high hMena+11a expression had a significant and borderline significant advantage in DFS (p=0.03) and OS (p=0.056), respectively, and a non-significant trend towards a better CSS. Univariate and multivariate 3-yr median individual patient probabilities of recurrence were 70.9 (range 40.3-94.4) and 41.2 (range 13.6-86.5), respectively (data not shown). The subgroup of patients with High Pan-hMena/Low hMena11a relative expression fared significantly better than any of the other 3 groups (p≤0.002 for all outcomes). On the basis of the combination between this molecular hybrid variable and T-size and RN, a 3-class risk stratification model was generated; the derived 3-risk class survival model strikingly discriminated between patients at different risk of relapse, cancer-related death, and death for any cause, with a prognostic accuracy of 61% (standard error 0.03, p=0.01), according to ROC analysis. The 3-risk class survival model was externally validated in an independent dataset of 133 patients, and significantly discriminated between patients at Intermediate- and High-Risk of relapse and cancer-related death.

Conclusion
The expression of the hMena and its isoform may represent a powerful prognostic factor in early NSCLC and usefully complements clinical parameters to accurately predict individual patient risk..

Background
Klotho is a type I transmembrane protein which is encoded by the KL gene; it is associated with many metabolic processes in differing neoplasias, including lung adenocarcinoma. Its abnormal expression conditions irregular endothelial growth and hyperactivation of proliferation via the PI3K/Akt signalling pathway.

Methods
Information concerning 84 patients with epidermal growth factor receptor (EGFR) mutations was taken to explore Klotho’s cytoplasmic positivity using an anti-Klotho antibody (Calbiochem, BD Biosciences, San Jose, CA, USA; 1:100 dilution). The results were correlated with multiple outcomes, including differing clinical characteristics, response rate, progression-free survival (PFS) and overall survival (OS).

Results
Mean age was 60.7 years (SD±13.1) and Klotho expression in the population of patients having EGFR mutations was considered positive in 35.7% of them (n=30), negative in 31.0% (n=26) and unknown in the remaining 33.3% (n=28). Positive Klotho expression was not influenced by gender (p=0.51), histological pattern (p=0.063), base functional state (p=0.49) or a history of smoking (p=0.19); nevertheless, Klotho overexpression was greater amongst exon 19 deletion carriers (p=0.030) and in patients having the L858R mutation (p=0.009) compared to the group of subjects having infrequent mutations. EGFR mutation patients’ overall response was greater in those having increased Klotho expression compared to the population of subjects lacking reactivity or in those where evolution following the administration of any type of reversible tyrosine-kinase inhibitor remained unknown (p=0.011). Overall population PFS was 16.7 months (12-21 95%CI) after directed therapy was started; PFS lasted longer in the Klotho positive group (positive expression 21.1 months vs. negative 13.7 months; p=0.032). Median OS was 28.5 months (25.8-31.2 95%CI), longer for patients having increased Klotho expression (31.9 versus 22.0 months; p=0.039).

Conclusion
Klotho expression revealed by immunohistochemistry in lung adenocarcinoma patients having EGFR mutations facilitated stratifying prognosis.

Background
Platinum-based doublet chemotherapy (CT) is the standard treatment in non-small cell lung cancer (NSCLC) patients, but less than 30% respond to CT, and survival remains between 10-12% at five years. The most important prognostic factor in survival is the stage, although there is significant variability in survival among patients with similar disease. It is postulated that different single nucleotide polymorphisms (SNPs) in DNA repair genes may play a role in the effectiveness of the platinum-based chemotherapy. The purpose of this study was to evaluate the association of 17 SNPs in 8 genes involved in DNA repair mechanisms, with the response to treatment with platinum-based chemotherapy in NSCLC patients.

Methods
The genomic DNA was automatically extracted from blood samples using the salting out procedure (Autopure, Qiagen) and was quantified using the BioSpec-nano spectrophotometer. We analyzed 17 polymorphisms belonging to 8 genes, using 48.48 dynamic array on the Biomark™ system (Fluidigm): six genes belong to the Nucleotide Excision Repair pathway (ERCC1, ERCC2/XPD, ERCC3/XPB, ERCC4/XPF, ERCC5/XPG and XPA), and two genes belong to the Base Excision Repair pathway (XRCC1, XRCC2).

Results
We included 161 patients with stage IIIA-IV. The median age was 63.7 years; 77.6% were men, and 54% had stage IV disease. All patients received a platinum agent (cisplatin: 95, carboplatin: 66) in combination with a third-generation drug. Patients with stage IIIA and IIIB also received concomitant or sequential radiotherapy. In patients with stage IIIA and IIIB (n=74), the multivariate analyses showed a significant association between the following SNPs and response: rs11615 (ERCC1) (p=0.0448 in a recessive model), rs3738948 (ERCC3) (p=0,0049 in an additive model). In patients with stage IV (n=87), the multivariate analyses showed a significant association between the following SNPs and response: rs1799793 (ERCC2) (p=0.013 in a recessive model), rs179801 (ERCC4) (p=0.033 in a dominant model) and rs25487 (XRCC1) (p=0.002 in a recessive model).

Conclusion
These results confirm the association between polymorphisms in genes ERCC1, ERCC2 and XRCC1 and response to treatment with platinum compounds as previously described. In our cohort, response to treatment was also associated with genes ERCC3, ERCC4, also involved in DNA repair processes. Prospective studies are needed in order to validate the role of polymorphisms as predictors of response to chemotherapy in NSCLC patients.

Background
PNA-LNA PCR clamp is highly sensitive, real-time PCR-based laboratory technique developed to enable reliable detection of EGFR gene mutations in wide spectrum of tissue/biopsy samples from NSCLC patients. The aim of the study was to assess diagnostic reliability of PNA-LNA PCR clamp assay in EGFR mutations detection in different NSCLC samples.

Methods
Evaluation was performed: (i) in reference NSCLC tissue FFPE samples (n=10), (ii) in comparison to direct sequencing in resected NSCLC tissue (n=199) and biopsy material specimens (n=179) characterized by different tumor cells content (TCC) and fixation [Table 1].

[Table 1.] NSCLC samples

Resected tissue		Biopsy material
fresh -frozen	FFPE	FFPE	Cytology smear
84	115	115	64

Results
(i) PNA-LNA PCR clamp correctly detected all exon 19 deletions and L858R mutations in the reference FFPE materials, including those with meager TCC (5% and 10%). (ii) In total of 378 samples analyzed with PNA-LNA PCR clamp method EGFR mutations were detected in 36 (9.5%). No significant differences in detection efficiency were observed in reference to material (resected tissue vs biopsy, p=0,3972; OR=0,7405; CI=0,3694-1,4844) and fixation procedure (FFPE vs fresh-frozen tissue, p=0,5459; OR=0,7304; CI=0,2635-2,0248; biopsy material FFPE vs cytology smear, p=0,4366, OR=0,6908; CI=0,272-2,7544). (iii) PNA-LNA PCR clamp method and direct sequencing presented high conformity (overall percent agreement, OPA=99%; Cohen’s Kappa score of 0.94 (95% CI=0.9, 0.99) in n=100 samples with >50% TCC. (iv) PNA-LNA PCR clamp presented higher sensitivity in samples with TCC <50% (p=0.004). Reevaluation with direct sequencing proved positive only in 24 out of 36 (67%) mutation positive samples [Table 2].

[Table 2. ]Comparison of PNA-LNA PCR clamp vs direct sequencing EGFR mutation detection sensitivity in 36 EGFR mutation positive materials with different %TCC.

	PNA-LNA PCR clamp	direct sequencing
≥50%	25/25 (100%)	22/25 (88%)
20<50%	6/6 (100%)	2/6 (33%)
≤20%	5/5 (100%)	0/5 (0%)
total	36/36 (100%)	24/36 (67%)

Conclusion
PNA-LNA PCR clamp method is characterized by high sensitivity of EGFR exon 19 and 21 mutations detection in tissue and biopsy material, particularly in samples with TCC lower than 50%. Fixation procedures did not affect PNA-LNA PCR clamp method mutation detection effectiveness.

Background
Radiation pneumonitis (RP) is a major dose-limiting toxicity after thoracic radiotherapy (RT), with no good models available to accurately predict the individual risk.MicroRNAs (miRNAs) are found to be stable in serum and other body fluids,with exciting potential as novel non-invasive biomarkers. This study is to investigate serum microRNAs associated with RP grade ≥ 2 in inoperable/unresectable NSCLC patients treated with definitive RT.

Methods
134 patients with inoperable/unresectable NSCLC treated with definitive RT (18-month minimum follow-up) were eligible. Serum samples were collected prospectively before treatment. 100 patients who had enough serum and reliable miRNA profile quality were included in this study. MiRNA profiling was performed using real-time PCR-based array, containing a panel of 84 miRNAs detectable in human bodily fluids. Spiked-in cel-miR-39 was used for normalization. The primary endpoint was symptomatic RP (grade 2 and higher). 2-sample mean comparisons were used between the RP and non-RP subgroups.Stepwise Logistic regression model building was used to build a miRNA signature. Receiver operator characteristic (ROC) analysis was used to assess the predictive ability of single-marker and signature of RP.

Results
Of 100 patients enrolled, 17 (17.0%) patients developed symptomatic RP. Patients received a median of 70 Gy (34-85.6Gy) of RT with a mean lung dose (MLD) of 16.9 Gy (2.1-25.5 Gy). Serum miRNA profiling identified pre-treatment expressions of 9miRNAs were significantly associated with risk of RP (p<0.05). Significant correlations were not found for any clinical or dosimetric parameters including age, gender, stage, MLD (p>0.05). Stepwise regression modeling identified only has-miR-191 as significant predictors of symptomatic RP (HR=4.94, 95%CI:1.46-16.66, p=0.01). Using ROC curves, we found has-miR-191 was independent predictors of symptomatic RP (p=0.01). A model of combining has-miR-191 and MLD had AUC of 0.72 (p=0.004) comparing to 0.64 of MLD alone (p=0.08).

Conclusion
In our preliminary analysis, baseline serum has-miR-191 may help predictingsymptomaticRP. However, analysis on larger and independent datasets will be required to verify our findings.

Background
Measurement of CTCs is being increasingly recognized as a promising tool in oncology. Several studies have evaluated CTC in early and locally advance disease; however, few studies have evaluated the prognostic impact of the quantification of CTCs in advanced disease. The aim of this work was to quantify CTCs in peripheral blood through the simultaneous use of three epithelial markers in patients with stages IIIB (pleural effusion) and IV in NSCLC.

Methods
Seventy advanced NSCLC patients were included in the study. All patients received platinum-based chemotherapy in first line treatment. Peripheral blood was obtained of each participant, circulating tumor cells were quantified by RT-PCR using three markers: CK-18, CK-19 and CEA. The expression levels of CEA, CK-18 and CK-19 mRNA were quantified from a standard curve using the cDNA obtained from A549 cells. The protocol was registered in ClinicalTrials.gov (NCT01052818).

Results
We found a significant statistical correlation between levels of CK-18, CK-19 and CEA mRNA. CTC was lower in patients with oligometastatic disease; higher CTCs determinate by CEA mRNA levels was associated a worse progression-free survival to platinum-based chemotherapy and overall survival.

Conclusion
Detection of high CTC numbers by RT-PCR using CEA as a biomarker is useful as a prognostic marker in patients with advanced NSCLC.

Background
Expression of thyroid transcription factor 1 (TTF1) is commonly assessed to diagnose lung adenocarcinomas. TTF1 may also be an oncogenic driver. The prognostic impact of TTF1 expression in lung cancers has been evaluated. However, small sample sizes, population heterogeneity, and lack of control for genotype or targeted therapies have limited the interpretation and use of TTF1 as a prognostic variable.

Methods
We examined 638 consecutive patients with newly diagnosed (i.e. not recurrent disease) stage IV lung adenocarcinomas between 01/2009 and 09/2011. TTF1 was assessed by immunohistochemistry (8G7G3/1, DAKO, dilution 1:100); binary results were recorded (positive = any nuclear reactivity; negative = no reactivity). The association between TTF1 status and clinical variables (Chi-squared and t-tests), median survival (Kaplan-Meier methods, compared using logrank test), and outcomes with specific chemotherapies (Cox proportional hazard) and were assessed. Multivariate analysis of overall survival (Cox proportional hazard) was performed.

Results
TTF1 was assessed in 484 (76%) patients; 80% were TTF1+. TTF1 positivity associated with improved survival in all cohorts examined, although the EGFR cohort is limited by the small number of TTF1 negative tumors. TTF1+ was more common in EGFR (93%) than KRAS (76%) mutants (p<0.01). Figure 1 To reduce confounding from the effect of targeted therapy on survival, subsequent analyses excluded those with EGFR (n=129) mutations or ALK (n=12) rearrangements: In multivariate analysis, the HR for survival in TTF1+ patients was 0.42 (p<0.001), exceeding the prognostic impact of good performance status (KPS≥80, HR=0.54, p<0.001). There was no association between TTF1 and age (p=0.96), sex (p=0.41), smoking status (p=0.68), or performance status (p=0.07). TTF1 status did not predict improved outcomes with specific chemotherapies.

Conclusion
TTF1+ robustly and independently associates with improved survival in advanced lung adenocarcinomas. TTF1 exceeds the prognostic impact of clinical features (e.g. KPS) more commonly used to stratify patients. TTF1 should be assessed in all lung adenocarcinomas and should be used to stratify patients enrolled in clinical trials. Randomized trials are needed to conclusively assess if TTF1 predicts differential sensitivity to chemotherapies.

Background
Unfortunately, non-small cell lung cancers are often diagnosed at an advanced stage. Early stage, and particularly T1aN0 NSCLCs, still represent a small percentage of all lung cancers at the moment of diagnosis. Research on early stage lung cancer may lead to discover new molecular insights which, hopefully, will reflect on new treatment opportunities: in particular, MicroRNAs (miRNAs) play a key role in cancer pathogenesis. We retrospectively reviewed our recent experience on surgically resected T1aN0 non small cell lung cancers, focusing on their surgical, histological, and molecular characteristic.

Methods
From 2000 to 2010 we operated 114 T1aN0 non small cell lung cancers (81 male and 33 female). Most of them (90; 78,94%) underwent a lobectomy, 11 (9,65%) a segmental resection and in 13 cases (11,40%) a wedge resection; systematic lymphadenectomy was always performed. Operation was performed in 104 (91,23%) cases by thoracotomy (either posterolateral or lateral), 3 (2,63%) by VATS surgery and in 7 (6,14%) cases by robot assisted technique. All specimens were reviewed by two pathologists: 48 (42,10%) were invasive adenocarcinoma, 14 (12,28%) in situ/minimally invasive adenocarcinoma, 51 (44,74%) squamous cell carcinoma and 1 (0,88%) anaplastic carcinoma. Furthermore we evaluated Let-7g, miR-21 and miR-205 expression and their prognostic and predictive value.

Results
With a mean follow-up of 67 months, the 5-year overall survival is 75,00%. Recurrence occurred in 25 cases (21,93%), with a average disease-free interval of 26 months: 7 cases had a local recurrence, while 18 patients had distant metastasis. No correlation between survival, the kind of intervention performed, histology and cancer grading was found. Furthermore, maximum diameter of cancer do not affect survival. In average 8 ± 5,5 (range 3-28) lymphnodes were resected in 3 ± 1,3 stations (range 2-7): neither numbers of lymphnodes resected nor number of stations examined affect survival. All MicroRNAs considered were compared to the pathological and clinical variables.

Conclusion
T1N0 non small cell lung cancer have a good survival with a low recurrence rate. In our experience histology, grading and the kind of resection (wedge resection and segmentectomy vs lobectomy) do not seem to influence recurrence rate and the prognosis. MicroRNAs tools have a good potential role as prognostic and predictive factors in lung cancer.

Background
The Tn antigen (GalNAc alpha-O-Ser/Thr), a product of incomplete O-glycosylation, is expressed in about 90% of human carcinomas, but not in normal human tissues, being associated with poor prognosis in breast cancer. There is no information about the relationship between Tn antigen expression and clinical outcome of patients with lung cancer. Aim: To study the frequency of expression of the Tn antigen in a large set of surgically resected NSCLC tumor tissues, and its association with clinical, pathological and molecular characteristics including patient’s recurrence-free survival (RFS) and overall survival (OS).

Methods
We used tumor tissue microarrays containing 426 NSCLCs, including 281 adenocarcinomas (ADC) and 145 squamous cell carcinomas (SCC). We performed immunohistochemistry using the murine monoclonal antibody 83D4. The expression of the Tn antigen was quantified using a four-value intensity score (0, 1+, 2+, and 3+) and the percentage (0-100%) of tumor stained cells. The final score obtained was in the range 0-300. The patients were divided into 2 groups: those who received neoadjuvant chemotherapy (WNA) (n=67), and those without this treatment (WONA) (n=359).

Results
We found frequent Tn antigen expression in NSCLC. ADCs expressed high levels of the Tn antigen in 72.7% of cases while in SCCs Tn antigen was found in 27.3% of cases (p<0,004). In relation to smoking, patients with positive smoking history (smokers and former smokers) presented statistically higher expression of Tn than nonsmokers, (p = 0.001). We observed a trend of the Tn antigen expression in favor of male, Caucasian, under 70 years, adjuvant treatment and stage higher than I, not statistically significant. In patients with ADC but without neoadyuvant treatment, we found a statistically significant correlation of Tn antigen expression with positive smoking history too (p = 0.001) and its expression is different according the histology pattern, showing higher value in solid histology pattern and lower in lepidic, papilar and acinar histology pattern. Using Spearman Correlation test, Tn antigen correlated significantly with EpCAM-N (n = 393, r = 0.20, p = 0.001), EpCAM-C (n = 391, r = 0.12, p = 0.01), TTF-1 (n = 250, r = -0.29 p = 0.001), mutated EGFR status (p = 0.001) and KRAS (p = 0.01) and not with EML4-ALK fusion gene (p = NS). Interestingly, in the ADC-WONA subset, the high level of Tn antigen, are significantly associated with poor prognosis in RFS (p <0.04, HR = 1.45) and strong tendency in OS (p = 0.06, HR = 1.47). In the group ADC-WNA, high level of Tn antigen was significantly associated with poor prognosis in OS (p <0.02, HR = 2.84) and no difference in RFS. Patients with SCC in both groups, with or without neoadjuvant, showed no difference in prognosis regarding Tn antigen expression.

Conclusion
Tn antigen is frequently expressed in NSCLC and associates with worse prognosis in patients with ADC. Our data showed a significant correlation between the Tn antigen expression and other useful molecular markers in lung cancer (EPCAM, TTF-1, EGFR and KRAS), opening a new possible candidate for targeted therapy.

Background
Life Technologies Clinical Services Laboratory has developed a 14-gene molecular assay (Pervenio[TM] Lung RS) that provides mortality risk stratification in resected early-stage non-squamous non-small cell lung cancer (NSCLC). The test classifies patients as low, intermediate, or high-risk (for death), informing decisions about use of adjuvant chemotherapy. Accordingly, a high-risk sub-group can be identified to receive chemotherapy, and a low-risk sub-group can avoid chemotherapy-associated morbidity and costs. The objective of this study was to evaluate the cost-effectiveness of the Pervenio[TM] assay in Stage I/II NSCLC relative to standard care.

Methods
We developed a Markov model to estimate life expectancy, quality-adjusted life-years (QALYs), and costs for Pervenio[TM] testing versus standard care. Risk-group classification was based on Pervenio[TM] validation studies, and chemotherapy uptake was based on a study of pre/post testing recommendations from 58 surgeons and oncologists. We derived overall chemotherapy benefit from Lung Adjuvant Cisplatin Evaluation (LACE) database disease-free survival hazard ratios. In the Pervenio[TM] strategy, we differentially distributed chemotherapy benefit across risk groups, with high-risk patients deriving the greatest benefit, intermediate-risk patients deriving moderate benefit, and low-risk patients experiencing the least benefit (Table 1). Included costs were those related to the Pervenio[TM] test, chemotherapy with cisplatin+vinorelbine, monitoring, post-recurrence care, and chemotherapy adverse events. We calculated the incremental cost-effectiveness ratio (ICER), and evaluated uncertainty using one-way and probabilistic sensitivity analyses. We also evaluated non-predictive and strong predictive (based on Zhu et al.’s JBR.10 reanalysis) chemotherapy benefit scenarios. Our analyses used a lifetime horizon, a payer perspective, and a 3% discount rate.Figure 1

Results
The Pervenio[TM] and standard care strategies resulted in 55% and 33% of patients receiving chemotherapy, respectively. Life year, QALY, and cost outcomes are displayed in Table 1. The corresponding base case Pervenio[TM] strategy ICER was $22,270/QALY (Stage I: $29,210/QALY; Stage II: $12,190/QALY). One-way sensitivity analyses demonstrated that the proportion of high-risk patients receiving chemotherapy and the high-risk recurrence hazard ratio were the most influential inputs. Probabilistic sensitivity analyses demonstrated that the Pervenio[TM] strategy was cost-effective at a willingness to pay threshold of $50,000/QALY in 68% of simulations.

Conclusion
The results of our analysis suggest that in the U.S., the Pervenio[TM] Lung RS assay may be a cost-effective alternative to a standard care strategy in early-stage NSCLC. Future studies should evaluate the presence and magnitude of a differential chemotherapy benefit by risk group and post-testing chemotherapy preferences,as these were key determinants of model results.

Background
Pemetrexed disodium is a novel folate antimetabolite approved for first-line treatment in combination with a platinum doublet, for second-line treatment as a single agent and, more recently, as maintenance treatment after first-line chemotherapy in patients with non-squamous non-small cell lung cancer (NSCLC). Circulating factors associated with folate metabolism and/or phenotypic plasticity (e.g. the epithelial-to-mesenchymal transition (EMT)) may have predictive value in selecting advanced NSCLC for first-line pemetrexed. The objective of this study was to identify serum biomarkers capable of predicting improved outcomes for pemetrexed added to first-line platinum based chemotherapy relative to standard platinum doublet.

Methods
Pretreatment serum from a total of 72 patients with non-squamous stage IV NSCLC was evaluated with 76 biomarkers using Luminex immunobead assays. Patients were treated either with platinum combined with pemetrexed (P: n= 26) or with other agents (O; n=51) at the discretion of the treating physician. Patients were evaluated for disease progression using RECIST criteria. Biomarker data was processed using Ingenuity Pathway Analysis (IPA) Suite to identify interactions with folate metabolism. Cox Proportional Hazard (PH) regression model was used to assess the association between H-scores and progression-free/overall survival (PFS/OS) distribution estimated by the Kaplan-Meier method. PH interaction model was used to capture the differential effects of the biomarkers on the O vs. P treatment groups.

Results
Univariate PH regression analysis identified 10 biomarkers that were negatively associated (p<0.05) with progression-free survival (PFS) in either the O (sTNF-RI, sTNF-RII, Tenascin C, sIL-2Rα, spg130, sIL-6R, CA-125, and CA 19-9) or the P subgroups (total PSA, amphiregulin). Four other biomarkers (MMP-1, MMP-2, sVEGFR2, and PDGF-B) were all significantly (p<0.05) positively associated with PFS in the P group. Similarly, seven biomarkers were strongly negatively associated (p<0.01) with overall survival (OS) in the O group, including osteopontin, sTNF-RI, sTNF-RII, CA 15-3, sIL-2Rα, CYFRA 21.1, and IL-6; whereas the P group possessed both negative (osteopontin and amphiregulin) and positive (sVEGFR2, MMP-1, MMP-2, and sRAGE) associations (P<0.05) with OS. In our assessment of differential association with PFS, we found two serum biomarkers (PSA (total) and amphiregulin) with significant positive interaction terms, thus indicating differentially increased hazard of progression in the P group with higher level of the biomarker. MMP-1, HGF, and Tenascin C, sVEGFR2 were similarly noted to have significant negative interaction terms for PFS. Evaluations of the differential associations with respect to OS, demonstrated five biomarkers with significant (MMP-1, MMP-2, sVEGFR2, sTNF-RI, and Tenascin C; p≤0.05) and three strongly associated (osteopontin, HGF, s-IL-6R; p≤0.01) negative interaction terms, demonstrating a decreased hazard of progression in the P group.

Conclusion
Serum biomarkers with potential predictive (PFS, OS) value for selecting patients most likely to benefit from pemetrexed have been identified. Pathway analysis demonstrates interactions of biomarker candidates identified with folate metabolism. This study is currently being expanded with additional front-line patients (P=90; n=56) from our institutional archives to further evaluate their potential predictive value.

Background
Thoracic chemoradiotherapy (CRT) with or without surgery (S) is the standard-of-care in management of stage III NSCLC. However, it appears that plateau has been reached. New treatment strategies are needed. The objective of this retrospective study was to evaluate the relationships between patient outcomes and expression of biomarkers associated with either the epithelial-to-mesenchymal transition, or EMT ( E-cadherin and vimentin), or a lung cancer “stem-cell” phenotype (CD133), DNA repair enzyme (ERCC1), and cell survival/apoptosis (BCL-2, surviving and PTEN) in attempt to identify new therapeutic strategies.

Methods
Stage III NSCLC pts who were treated with chest radiation (40-65Gy) and concurrently with platinum doublet and who had sufficient pretreatment tissue were included in this study. Surgical pts received 40-45 Gy of radiation preoperatively and non-surgical patients received 60-65 Gy. Immunohistochemistry was used to detect nuclear and cytoplasmic expression of ERCC1, bcl-2, survivin, PTEN, vimentin, E-cadherin, and CD133. Scores were calculated using the Allred scoring system. The log-rank tests used to evaluate progression free survival (PFS) and overall survival (OS) with Kaplan-Meier plots used to plot group characteristics.

Results
A total of 119 patients receiving chemoradiotherapy with adequate tumor specimens for analysis were enrolled in this study; 61 had definitive chemoradiation whereas 58 had pulmonary resection after chemoradiation. Patients (n=79) with low nuclear survivin immunostaining (score ≤6) had significantly improved PFS as compared to those patients (n=34) with higher expression levels (14.1 vs. 10.5 months, p=0.042). Patients (n=72) with a cytoplasmic vimentin score ≤ 5 had superior PFS than the with higher expression levels (n=33) (13.17 vs. 9.99 months, p=0.045). High nuclear ERCC1 values (n=72) were associated with a worse OS than those patients (n=44) with low immunostaining (22.7 vs. 59.1 months; p=0.023). Patients with low cytoplasmic E-cadherin (n=25) had a significantly better OS than those patients (n=85) with immunostaining scores (62.6 vs. 24.6 months, p=0.036). The cytoplasmic vimentin/ E-cadherin ratio provided the most impressive separation of cohort performance with high V/E ratios being associated with a poor PFS (12.6 vs. 3.1 months; score ratio 10 cutoff; p=0.00073). No significant associations with cytoplasmic CD133 were observed in this cohort for either PFS or OS.

Conclusion
The association of inferior overall survival in locally-advanced NSCLC patients whose tumors express high ERCC1, high cytoplasmic E-cadherin (which is associated with mesenchymal phenotype and lower adherence of cells which are able to metastasize easier), and lower progression free survival with high survivin and high vimentin/ E-cadherin ratio suggests that combining inhibitors of survivin, DNA repair, EMT pathways might improve outcomes in molecularly defined subset of stage III NSCLC patients.

Background
Personalised strategies that tailor radiotherapy (RT) dose and schedule according to clinical and molecular factors, novel drug-RT combinations and/or advanced RT techniques are needed to optimise outcomes from RT for NSCLC. Development of such approaches would benefit from objective measures to inform on survival, chance of response and/or toxicity. We evaluated 26 circulating proteins and cytokines implicated in angiogenesis, metastasis, apoptosis, hypoxia and inflammation for prognostic (survival), predictive (RT response/toxicity) and/or pharmacodynamic significance in the context of RT for stage I-III NSCLC.

Methods
NSCLC patients donated blood prior to, serially and on completion of RT treatment. Samples were analysed for cell death (M30, M65, CYFRA), hypoxia (osteopontin, CA-IX), angiogenesis (Ang2, FGFb, HGF, VEGFA, VEGFC, PDGF, IL8, PlGF, KGF, VEGFR1, VEGFR2, Ang1, Tie2), metastatic (EGF, E-Selectin, VCAM1) and inflammatory (IL1b, IL10, IL12, TNFa, IL6) cytokines using single- or multi-plex ELISAs (SearchLight multiplex Aushon BioSystems, Peviva, R&D Systems). Clinical data were collected for age, gender, performance and smoking status, ace27 co-morbidity score, weight loss, TNM stage, haemoglobin, treatment received, various lung function and RT treatment parameters, histology, treatment received, toxicity, response and survival. Standard statistical methods were used to explore for associations and prognostic significance (Stata version 10).

Results
Seventy-eight patients were enrolled from March 2010 to August 2011: 61% male; majority (44%) squamous histology; 82% PS 0 or 1; 72% ex-smoker; 9% stage I/II, 44% stage IIIA, 47% IIIB; median age 66 years (range 31-86), 42% age > 70; 20% weight loss of 5-10%, 11% weight loss >10%; 62% prior chemotherapy/sequential RT, 20% concurrent chemoradiotherapy, 18% radiotherapy alone. RT treatment doses administered ranged from 50-55Gy in 20 fractions to 60-66 Gy in 33 fractions. Significant associations (p<0.05) were observed for EGF levels with gender and age, for FGFb with co-morbidity score and for IL8, IL1B and KGF with smoking status. Positive correlations of biomarkers at baseline (p<0.001) were observed for TNFa with FGFb, IL1b, IL8, IL12; FGFb with IL1b, IL8, IL12 KGF; IL1b with IL8, IL12; IL8 with KGF, IL12; KGF with IL12. In the overall population, at day 8 during RT significant decreases were observed for Ang2, EGF, E Selectin, FGFb, HGF, VCAM1, VEGFC & VEGFR2. Post completion of RT Ang2, EGF, E Selectin, FGFb, HGF & VEGFC levels remained significantly lower than at baseline prior to RT, and in addition significant global decreases in Ang1 and VEGFA were observed. The median survival overall was 16.8 months at a median follow up of 12 months. In univariate analysis there were non-significant trends to worse survival for older patients, weight loss >5%, higher TNM stage, higher co-morbidity scores and current smokers. Better survival was observed for patients with higher baseline levels of IL1b (p = 0.005) and TNFa (p = 0.022).

Conclusion
Preliminary analysis demonstrates appreciable changes of various circulating biomarkers during RT and identifies interleukin-1 beta and tumor necrosis factor alpha as potential prognostic factors. Multivariate analyses and correlation of biomarkers with response and toxicity are ongoing and will be presented.

Background
Targeting oncogene dependency for effective therapy has been one of the most successful strategies for managing metastatic non-small cell lung cancer (NSCLC). Although validating therapeutically tractable oncogenic driver mutations are a major focus, non-driver mutations may also confer dependencies that may also be exploitable. The prosurvival BCL2 protein, MCL1 prevents mitochondrial apoptosis by blocking interaction of proapoptotic BH3 only proteins with their multidomain proapototic counterparts, BAX and BAK. MCL1 is often mutated in cancers, and ranks as one of the most frequently amplified loci at 1q21.2. MCL1 amplified tumours exhibit addiction to this oncogene. Anthracyclines have been shown to transcriptionally suppress MCL1. Phase IIA studies in NSCLC have shown that epirubicin has useful single agent activity in unselected patients, with a significantly greater response rate than that achieved with standard chemotherapy. We therefore set out to evaluate MCL1 addiction in NSCLC, its correlation with anthracyline induced apoptosis and the prevalence of 1q21.2amplification to support a planned 1q21.2 stratified phase II trial in NSCLC, (EORTC-1303-LCG).

Methods
RNAi targeting MCL1was conducted in NCI-H460, NCI-H1299, NCI-H28 and NCI-H23 cell lines. Doxorubicin activity was measured by viability assay and apoptosis was assessed by western blot. gDNA from cell lines was obtained by Phenol-Chloroform extraction. The QIAamp DNA FFPE Tissue Kit was used to extract gDNA from FFPE tissues. MCL1 amplification was quantified by real-time PCR with a set of two primers and one probe (minor groove-binding (MGB) hydrolysis probe assay) for the gene of interest MCL1 and the two reference genes CCT3 and H6PD. Tonsil samples were used as a control diploid population.

Results
MCL1 silencing efficiently induced apoptosis in a subset of NSCLC cells, however we identified two cell lines that were resistant to MCL1 knockdown (NCI-H1299 and NCI-H28). Doxorubicin efficiently induced apoptosis in MCL1 addicted cells but exhibited significantly less activity in cells that were not addicted. We developed a genomic DNA based quantitative real time PCR assay to evaluate copy number variation (CNV) at the 1q21.2 locus. A clear correlation r[2] >0.91 was observed for 1q21.2 CNV compared with reference Conan Copy Number Analysis Tool (Cancer genome project, Sanger). Increased 1q21.2 copy number was consistently associated with MCL1addiction; however addiction also occurred in cells lacking 1q21.2 CNV, suggesting that MCL1 amplification represents a subset of MCL1 dependence. The concentration of doxorubicin was titrated against MCL1 protein downregulation into therapeutically sub-micromolar concentration range and we observed that MCL1 downregulation occurred coincidently with cleavage of poly-ADP ribose polymerase. We then screened DNA isolated from 19 adenocarcinomas, and identified 1q21.2 CNVs in 36.8%, with high level amplification (CNV >5) in 1q21.2 in 10.5%.

Conclusion
Targeting MCL1 addiction in 1q21.2 amplified NSCLC induces apoptosis and this dependence can be exploited by anthracyclines at therapeutically relevant concentrations. Given its significant prevalence in NSCLC, our data suggests that 1q21.2 amplification could be a novel non-driver mutation predictive for anthracycline response.

Background
Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer. Treatment options are limited and drug resistance is common. Thus, there is a need to identify novel therapeutic targets in this disease in order to improve treatment options and survival times. Macrophage stimulating protein (MSP) is the only ligand recognised to bind to the RON receptor (MST1R). RON is a member of the MET proto-oncogene family. The MSP-RON signalling pathway has been implicated in a variety of cellular functions such as macrophage morphogenesis and phagocytosis. De-regulation of this pathway has been linked to tumour progression and metastasis in a number of cancers. We have previously identified RON as frequently activated in MPM and high positivity for RON by IHC was an independent predictor of favourable prognosis.

Methods
A panel of mesothelioma cell lines were screened for the expression of MSP and RON at the mRNA (RT-PCR) and protein (Western blot) level. The effect of MSP, IMC-RON8 (a humanised IgG1 monoclonal antibody), LCRF004 (a small molecule inhibitor) and NRWHE (a small peptide) was examined in the H226 cell line using proliferation (BrdU ELISA), apoptosis (Multi-parameter apoptosis assay) and migration assays (xCELLigence). A phospho-kinase proteome profiler array was utilised to detect the downstream signalling pathways activated upon MSP stimulation. The expression of MSP and the macrophage marker, CD-68, was examined by IHC using MPM TMAs. Studies are ongoing to determine the effect of the LCRF004 compound in vivo using a xenograft murine model with the H226 cells.

Results
The mRNA and protein levels of RON and MSP were differentially expressed in a panel of MPM cell lines. Treatment with LCRF004 resulted in significantly decreased proliferation and increased apoptosis in the H226 cells. MSP was unable to rescue the cells from the effects of LCRF004. NRWHE and RON8 had little effect on either proliferation or apoptosis. All of the compounds examined inhibited the migration capacity of the H226 cells. The combination of LCRF004 and MSP produced a synergistic effect, showing greater inhibition of migration than either compound alone. However, MSP treatment resulted in the up-regulation of a number of phosphor-kinases including Akt, ERK and the Src family. Currently, a number of proteins identified in the array studies are undergoing validation. Results of an in vivo H226 murine model using the LCRF004 compound will be presented at the meeting.

Conclusion
From previous work performed in this laboratory, we have determined that high expression of RON in MPM is an independent predictor of favourable prognosis. IHC was performed on a TMA of MPM patient samples and high expression levels of MSP correlated with better survival. There was no association between CD68 staining and MSP, nor correlation of CD68 expression with survival. Targeting the RTK domain of the RON receptor with a small molecule inhibitor is an effective interventional strategy in MPM. The seemingly counter intuitive results obtained from the MPM TMA studies and the in vitro experimental data, may be RON isoform dependant. Additional studies are ongoing to further delineate the RON-MSP axis in MPM.

Background
Circulating Tumour Cells (CTCs) have been the subject of much interest as a potential biomarker however methods for isolating CTCs are still in their infancy. A promising method of CTC detection is ScreenCell. This technique uses polycarbonate filtration membranes containing multiple tiny pores. When blood is made to flow across the membrane, tumour cells are captured due to their greater size. Another such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype.

Methods
Peripheral blood samples were obtained from patients with advanced NSCLC using both Screencell & VitaAssay. In addition healthy blood samples spiked with NSCLC cells were also analysed. ScreenCell: Peripheral blood is diluted with specified buffer and drawn across the Screencell filter using a vacuum tube. The filters with captured fixed cells are then stained with H&E and/or immunocytochemistry. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12-18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs due to their invasive phenotype. Captured cells are fixed and stained using immunocytochemistry.

Results
Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. CTCs were detected in 70% of patient samples with. (n=10) Numbers of CTCs detected ranged from 6-82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli (CTM) were detected in 50% of patient samples. (An example of CTM is illustrated in Fig. 1) Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 & H2228 cells, approximately 20% (range 9%-26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified. (range 14-52, n = 6) (An example of CTCs detected by immunocytochemistry is illustrated in Figs. 2 & 3) Figure 1

Conclusion
ScreenCell & VitaAssay techniques both appear to be viable methods of isolating & enumerating CTCs, in both model cell-spiking experiments and in NSCLC patient samples, as determined by morphology and antigen expression detected with immunocytochemistry. Of particular interest many of the CTCs isolated using Screencell, were detected as clusters or microemboli. Additional samples are being taken to compare CTC & CTM numbers with clinical outcomes.

Background
KRAS mutations are reported in 20-25% of non-small cell lung cancer (NSCLC). Little is known about the prognostic/predictive role of KRAS in advanced NSCLC, with conflicting results among small studies, while recent evidence showed that they could predict for worse outcome in patients treated with platinum-based adjuvant chemotherapy. Evidence is also inconclusive on the prognostic role of EGFR mutations. Given that ethnicity may play a role on the mutational profiling of NSCLC, we report here on the first large scale mapping of NSCLC in Greek patients.

Methods
KRAS and EGFR genotypes were evaluated in 634 NSCLC patients with available clinical data, diagnosed from March 2000 to December 2012 (tissue blocks from the HeCOG tumor repositories). KRAS and EGFR mutations were associated with clinicopathological parameters (mutated vs. wild-type). Outcome comparisons were performed in 469 metastatic patients with available treatment data, following 1[st ]line chemotherapy without tyrosine kinase inhibitors.

Results
The majority of the patients were male (78%), current smokers (47%), with adenocarcinoma (AC) histology (68%). EGFR mutations were found in 14% and KRAS mutations in 15% of all histological types, while in AC they were 17% and 22%, respectively. Most EGFR mutations were classical (79%), while the most common KRAS mutations were p.G12C (35%), p.G12D (25%) and p.G12V (11%). Five tumors had concurrent EGFR and KRAS mutations. EGFR mutations were significantly associated with female gender, AC histology and non-smoking status, as previously described. KRAS mutations were associated with AC histology and younger age (<60). At a median follow-up of 39 months, EGFR status was prognostic for improved PFS (HR=0.52, 95% CI 0.35-0.78, p=0.001), in patients treated with 1[st] line chemotherapy and no TKIs, and OS (HR=0.64, 95% CI 0.43-0.95, p=0.028). KRAS mutations did not show any significant associations with OS or PFS, although a trend for worse outcome in KRAS mutated patients was observed. Furthermore, there was a significant difference in response to 1[st] line treatment according to KRAS status, with KRAS mutations associated with worse outcome (Clinical benefit, CR+PR+SD: 64.4% in wildtype vs 48.8 in mutant, and PD 23.4% in wildtypet vs 39.5% in mutant). No significant interaction between KRAS/EGFR status (EGFRmut vs. KRASmut vs. any wt) and platinum-based treatment was observed (p=0.975 for PFS and p=0.892 for OS).

Conclusion
EGFR and KRAS genotype incidences are presented for the first time in Greek metastatic NSCLC patients. In this setting, the presence of EGFR mutations shows prognostic significance in patients treated with 1[st] line chemotherapy, without TKIs, while the presence of KRAS mutations seems to adversely affect the response to 1[st] line chemotherapy.

Background
The inherent molecular heterogeneity prevents the efforts to improve outcomes for patients with non-small cell lung cancer (NSCLC). Platinum doublets are the standard option for the treatment of advanced NSCLC, but none of the platinum-based combinations used offer a significant advantage over the others. Pemetrexed is an antifolate antimetabolite that inhibits several key folate-dependent enzymes, mainly thymidylate synthase (TS). A phase III trial conducted in the first-line setting of advanced NSCLC demonstrated that survival was statistically superior for cisplatin plus pemetrexed in patients with adenocarcinoma (12.6 versus 10.9 months; HR 0.84, P = 0.03), and large-cell carcinoma (10.4 versus 6.7 months; HR 0.67; P = 0.0 3 compared with cisplatin plus gemcitabine (1). Preclinical data have indicated that overexpression of TS correlates with reduced sensitivity to pemetrexed (2). Baseline expression of the TS gene is superior in squamous cell carcinoma compared with adenocarcinoma (P < 0.0001) (3). BRCA1 is a component of multiple DNA repair pathways and functions as a molecular determinant of response to a range of cytotoxic chemotherapeutics agents. The analysis of BRCA expression levels in patients who had received neoadjuvant gemcitabine/cisplatin chemotherapy found that patients with low levels of BRCA1 had longer survival (P = 0.01) compared to those with high expression levels (4). RAP80 is an interacting protein that form complexes with BRCA1 and could modulate the effect of BRCA1. In patients with non-squamous lung carcinoma, survival was influenced by RAP80 expression (5). Taking into account this background, the Spanish Lung Cancer Group has started a phase IIA study of pemetrexed plus cisplatin as first line treatment for advanced/metastatic non-squamous lung carcinoma. The availability of tissue samples for analysis of expression of BRCA1, RAP80 and thymidylate synthase is mandatory. The primary objective is response rate adjusted for different expression levels of BRCA1, RAP80 and TS. Secondary objectives are OS, TTP and toxicity profile of the combination and its relationship with the biomarkers. The expected total number of patients accrued will be 90. Forty-nine patients have been included up to now. References Scagliotti GV, Parikh P, Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naïve patients with advanced-stage non–small-cell lung cancer. J Clin Oncol 2008. Sigmond J, Backus HH, Wouters D, et al. Induction of resistance to the multitarged antifolate pemetrexed in WiDr human colon cancer cells is associated with thymidylate synthase overexpression. Biochem Pharmacol 2003. Ceppi P, Volante M, Saviozzi S, et al. Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer 2006. Taron M, Rosell R, Felip E, et al. BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer. Hum Mol Genet 2004. Rosell R, Perez-Roca L, Sanchez JJ, et al. Customized treatment in non-small cell lung cancer based on EGFR mutations and BRCA1 expression. PLoS ONE 2009.

Methods
Not applicable

Results
Not applicable

Conclusion
Not applicable

Background
Patients (pts) with KRAS mutant lung cancers have a shorter survival compared to pts withKRAS/EGFR wild type tumors(Johnson et al, Cancer 2013). Whether outcomes for patients with KRASmutant metastatic lung cancers differ by smoking status or specific amino acid substitution is unknown. In order to understand the impact of KRAS mutation subtype in the metastatic setting, we analyzed a large cohort of patients with KRAS mutant metastatic lung cancer.

Methods
We identified all pts with KRAS mutant metastatic or recurrent lung cancers from Feb 2005 to Aug 2011. KRAS mutation type, clinical characteristics, and outcomes from diagnosis were obtained from the medical record. A multivariate cox proportion hazard model was used to identify factors associated with overall survival.

Results
KRAS mutations were identified in 677 pts (53 at codon 13, 624 at codon 12). Median age: 66 (range 31-89), women: 62%, never smokers: 7%. Pts with transition mutations (n=157) were more likely to be never-smokers (p<0.0001). There was no difference in outcome for pts with KRAS transition versus transversion mutations (p=1) or when comparing current/former smokers to never smokers (p=0.33). There was no difference in overall survival (OS) when comparing specific amino acid substitutions (G12C=366, G12V=141, G12D=114, G12A=68, G13C=27, G13D=23, G12S=19, G12F=11)(p=0.20). Pts with KRAS codon 13 mutant tumors had inferior OS compared to pts with codon 12 mutant tumors, median 13 months (mo) (95% CI 13-17 mo) and 16 mo (95% CI 9-16 mo), respectively (p=0.009). There was no difference in frequency of receiving platinum-based chemotherapy or chemotherapy of any kind between pts with codon 12 and 13 mutant tumors. In a multivariate Cox model which included age, gender and smoking status, KRAS codon 13 mutation was associated with worse overall survival than KRAS codon 12 mutation (HR 1.52 95% CI 1.11-2.08 p=0.008).

Conclusion
Among pts with KRAS mutant metastatic lung cancers, smoking history, and specific amino acid substitution do not affect outcome. Among patients with KRAS mutant metastatic lung cancers, those with codon 13 mutations have shorter survival compared to pts with KRAS codon 12 mutations.

Background
The purpose of this study was to determine the impact of pulmonary fibrosis on postoperative complications and on long-term survival after surgical resection in lung cancer patients with chronic obstructive pulmonary disease.

Methods
A retrospective chart review was conducted of 380 patients with chronic obstructive pulmonary disease who had undergone pulmonary resection for lung cancer at Chiba University Hospital between 1990 and 2005. The definition of chronic obstructive pulmonary disease was a preoperative forced expiratory volume in one second /forced vital capacity ratio of less than 70%; pulmonary fibrosis was defined as obvious bilateral fibrous change in the lower lung fields, confirmed by computed tomography. Statistical comparisons were carried out between the groups, and multiple logistic regression analysis was used to evaluate for independent risk factors for decreased survival.

Results
Pulmonary fibrosis was present in 41 patients (10.8%) with chronic obstructive pulmonary disease; the remaining 339 patients (89.2%) did not have pulmonary fibrosis. The preoperative forced vital capacity and forced expiratory volume in one second were significantly lower in patients in the group with pulmonary fibrosis than in the group without (p < 0.05). Acute lung injury and home oxygen therapy were significantly more common in the pulmonary fibrosis group; however, the 30-day mortality was similar between the groups. The cumulative survival at 3 and 5 years was 53.6% and 36.9% in the pulmonary fibrosis group and 71.4% and 66.1% in the non-pulmonary fibrosis group (p = 0.0009). The group without pulmonary fibrosis had significantly better survival, due to a lower rate of cancer recurrence. Increased age, decreased body mass index, advanced pathologic stage and the existence of pulmonary fibrosis were identified as independent risk factors for decreased survival.

Conclusion
Pulmonary fibrosis is a risk factor for decreased survival after surgical treatment in lung cancer patients with chronic obstructive pulmonary disease.

Background
Lung cancer is the most common cause of cancer related death among both men and women all over the world. Skeleton is one of the most common metastatic sites. Most of the patients with bone metastasis should be treated with systemic therapy or symptom-based palliative approach without surgery. We try to improve the therapeutic effect by synchronous surgeries in resectable non-small cell lung cancer(NSCLC) patients with solitary bone metastasis.

Methods
Five patients have undergone synchronous lung cancer resections and solitary bone metastasectomies between 2009 to 2011 in our hospital. All of them have received 18FDG-PET-CT or Bone Scintigraphy (BS) to demonstrate solitary bone metastasis and exclude other site metastases. They received standard lung cancer resections and mediastinal lymph node resections. Meanwhile, bone leasions were assessed by orthopedists and operated with standard procedures synchronously. After operations they all had standard chemotherapies. Perioperative indicators including time of thoracic drainage, hospital stays, incidence of postoperative complications and progress free survival (PFS) were observed.

Results
The average time of postoperative drainage is 4.6±1.1 days, postoperative hospitalization is 8.8±2.2 days. All of the procedures were carried out safely with no serious complications. The PFS of these patients is 13.2±7.7 months. Two patients with spine metastasis died about one year after operation, and the other three patients with limb bone metastases have survived more than 16 months in average after operation and still alive.Figure 1

Conclusion
Synchronous metastasectomy and lung tumor resection is safe to patients. The PFS time and survival results show that in the rare situation in which a patient has a solitary bone metastasis, aggressive surgical treatment may be an available choice.

Background
As society ages, the incidence of lung cancer is increasing. Elderly patients with lung cancer are also more susceptible to other diseases than are younger patients.

Methods
In this study, 357 patients with non-small cell carcinoma, who underwent pulmonary resection at our hospital, were retrospectively reviewed. These patients were classified into 3 groups: Group A, 121 patients aged <64 years; Group B, 149 patients aged 65–74 years; Group C, 87 patients aged >75 years. The causes of death were investigated with a special focus on other diseases.

Results
The follow-up rate of all cases was 95.8%. One patient died of pulmonary embolism, and the operative mortality rate was 0.26%. Out of the 357 cancers, 71.1% had stageⅠ, 8.1% stageⅡ, and 20.1% stageⅢ. In Group A, 9.1% of the cases underwent wedge resection, 18.9% in Group B, and 47.7% in Group C. The proportion of wedge resection cases increased with age. In Group A, cancer-related survivals were 59.5%, 61.1% in Group B, and 66.7% in Group B, and there was no statistical significance between the groups. The overall survivals were 57.9％ in Group A, 55% in Group B, and 51.7% in Group C. There were significant differences between cases in Group C and those in other groups (p = 0.019, log rank test). Within 5 years of the operation, the mortality rates due to other disease were 1.6% in Group A, 6% in Group B, and 14.9% in Group C. Chi-squared test showed significant differences between cases in Group C and those in other groups. Twenty-four patients died due to other diseases: 5 from cardiovascular disease, 5 from respiratory disease, 5 from other malignant diseases, 3 from gastrointestinal disease, 1 from cerebrovascular disease, and 5 from other causes.

Conclusion
Since after lung-cancer surgery, the mortality due to other diseases increases in elderly patients, a postoperative survey or therapy for other diseases are important especially in elderly patients. Therefore, in order to prevent death due to other diseases or to enhance the quality of life until their death, a less-invasive surgery or limited resection to preserve respiratory function are more important in elderly patients than in younger patients

Background
Primitive neuroectodermal tumor (PNET) is a rare undifferentiated and highly aggressive tumor , most commonly arising in chest wall in teen age patients. Treatment of these patients is challenging and multimodality treatment had a major impact on their outcome. We present our experience of post neo-adjuvant chemotherapy , radical chest wall resection and reconstruction and surgical outcomes.

Methods
A retrospective review of a prospectively maintained computerized database of patients was performed and patients with histologically proven chest wall PNET undergoing surgery were identified and analyzed for clinical profile , surgical details and peri-operative outcomes.

Results
A total of 71 patients had surgery for chest wall tumor between 2000 to 2009. Fifteen out of 71 were diagnosed as having PNET chest wall. The mean age of presentation was 21 years (15 - 30 years) and there was a slight male preponderance (1.16 : 1). Most common presenting symptom was chest wall swelling and pain. Mean pre chemotherapy tumor size was 20cm. As per our institutional protocol , all patients received neoadjuvant chemotherapy comprising VAC + IE regime followed by surgery. The number of resected ribs ranged from 2 to 5 and the mean chest wall defect was 15cm. Majority required resection of adjoin pleura and in 5 patients segment of adherent lung was resected. A composite chest wall reconstruction was performed using bi-layered synthetic mesh and latissimus dorsi (10) , pectoralis major (3) and serratus (2) muscle flaps. All patients had an uneventful post operative recovery and the peri-operative mortality was nil. Six patients had complete pathological response to chemotherapy and 9 patients had residual tumor and were given post operative radiotherapy. At a median follow up of 36 months , 8 patients are alive and disease free.

Conclusion
Multimodality management and advances in surgical techniques had revolutionized the approach to chest wall PNET in the recent past. Our experience has shown that radical chest wall resection and composite reconstruction can be accomplished with excellent outcomes even in patients with advanced PNET and post intensive chemotherapy sessions.

Background
Lung cancer is still one of the deadly types of cancer. Once the disease has relapsed, long survival cannot be anticipated. However, prognostic factors after postoperative recurrence in non-small cell lung cancer (NSCLC) have not been well elucidated. In the present study, to improve the prognosis for NSCLC, we focused disease free survival (DFS) interval length and newly examined predictive survival factors after recurrence for NSCLC for over 10 years.

Methods
Consecutive 419 patients with NSCLC were performed curative surgical operation between January 2001 and March 2012 at the Department of Thoracic Surgery, Hamanomachi Hospital, Fukuoka, Japan. Out of 419 patients, 116 cases had been recurrent. Predictive prognostic factors for 116 recurrent NSCLC cases were retrospectively examined.

Results
DFS time which is longer than 18 months as well as female gender and adenocarcinoma histology were independent better prognostic factors for 116 recurrent patients. For 74 patients with adenocarcinoma, DFS time which is longer than 20 months as well as female gender, younger age and EGFR mutation status were independent better prognostic factors in multivariate analysis.

Conclusion
In the present study,we for the first time demonstrated that DFS time is an independent prognostic factor for recurrent NSCLC.We have also shown that female gender, younger age and adenocarcinoma histology were independent predictive prognostic factors for all cases. Among several prognostic factors, in this study, however, we emphasis DFS interval time as an independent prognostic factor for postrecurrent survival. The recurrent patients whose DFS time is shorter than 20 months should be taken care intensively.

Background
The number of patients with malignant tumors receiving long-term hemodialysis (HD) has been increasing. Patients on HD who undergo surgery represent a high-risk group requiring careful perioperative management to avoid electrolyte imbalance and hemodynamic instability. This retrospective study analyzed the postoperative outcome in terms of complications and survival of a group of patients on HD who underwent pulmonary resection for non-small cell lung cancer.

Methods
Between January 1995 and March 2013, 10 patients (7 men, 3 women; median age, 71.5 years) with non-small cell lung cancer who were also receiving HD underwent radical pulmonary resection by open thoracotomy or video-assisted thoracic surgery at Tokyo Medical University Hospital. We retrospectively evaluated their postoperative clinical outcomes and survival results. Most patients had comorbidities, including cardiovascular disease (5), diabetes (3), and brain infarction (1). The distribution of clinical staging was IA in 2 cases, IB in 5, IIB in 1, and IIIA in 2. Procedures included 8 lobectomies and 2 segmentectomies. We performed 4 systematic lymph node dissections and 6 selective lymph node dissections.

Results
The median intraoperative time was 215.5 minutes (range, 101-308). The median blood loss was 55 mL (range, 0-478 mL). Blood transfusion was not necessary. There was no intraoperative mortality. There were major perioperative complications in 4 patients, including atrial fibrillation (3), cardiac failure (1), shunt failure (1), and pneumonia (1). The median length of hospital stay was 21 days (range, 11-47). Thoracic drainage removal was at 4.5 postoperative days (range, 3-9). Pathological staging was IA in 3 cases, IB in 2, IIA in 2, IIB in 1, and IIIA in 2. Two cases were upstaged from the preoperative period to the final period. Seven of the 10 patients are currently alive and recurrence-free. Two patients had mediastinal lymph node and lung recurrence. One patient died from mediastinal lymph node recurrence at 8 months after surgery, and the other patient died at 26 months after surgery from malignant lymphoma.

Conclusion
Patients with chronic renal failure on HD who undergo lung resection have a high rate of postoperative complications (40%). Surgical treatment remains one of the effective treatments for patients on HD with lung cancer.

Background
Several reports have described intraoperative intrapleural hypotonic cisplatin treatment as effective for suppressing the appearance of pleuritis carcinomatosa in resected patients who demonstrated positive findings from pleural lavage cytology. Furthermore, fibrin glue may allow the efficacy of cisplatin to be prolonged. We investigated the effectiveness and safety of intrapleurally administering a combination of cisplatin and fibrin glue.

Methods
This study retrospectively analyzed 6923 patients who underwent resection of primary lung cancer in Niigata Prefecture between January 2001 and December 2010. Sixty-four patients with positive pleural lavage cytology underwent complete resection and showed p-stage I. Of these, 17 consecutive patients (8 men, 9 women) received intraoperative intrapleural administration of a combination of cisplatin and fibrin glue (treatment group; mean age, 68.6±7.9 years; range, 55-85 years). The control group received no intraoperative treatment of the pleural space. Intrapleural administration treatment involved spraying the entire thorax with cisplatin (25 mg) and fibrin glue before closure of the open thorax. Histopathological tumor types included adenocarcinoma in 16 cases and squamous cell carcinoma in 1 case. According to the TNM classification, 2 cases were stage IA and 15 cases were stage IB.

Results
No complications were seen with intrapleural administration. In the treatment group, median time to follow-up was 42 months and the 5-year survival rate was 75.0% Figure 1, respectively. Two of these 17 patients showed distant recurrence (brain metastasis, n=1; axillary lymph node metastasis, n=1), and none had locoregional recurrence. In the control group, median time to follow-up was 33.8 months and the 5-year survival rate was 45.1%. Recurrence developed in 16 patients (locoregional recurrence, n=7; distant recurrence, n=4; unknown lesion, n=5). No significant difference was observed between groups (p=0.0565), but 5-year survival rates for patients with treatment tended to be better than in the control group.

Conclusion
Intraoperative intrapleural administration with a combination of cisplatin and fibrin glue for patients with positive results from pleural lavage cytology was found to effectively suppress the appearance of locoregional recurrence without severe complications.

Background
This article describes the application of SOFT COAG electrosurgical output mode of VIO (ERBE Elektromedizin GmbH, Tubingen, Germany) in combination with triangle-tensioning (HIMEJI method, Yamamoto et al, 2010, Annals of Thoracic Surgery). The method is to expand the operative field in 3 directions, centering the targeted area. With the combination 2 advantages are considered; operative ease by the method and additional safety by SOFT COAG that generates no electric sparks for unintended tissue damage.

Methods
By the method each apex is tensioned in radial fashion towards 3 directions, making effective operative field. For resection of pulmonary artery A2 in upper lobectomy, the artery is pulled to its original direction with the lung fully stretched. The principal surgeon and his assistant oppositely stretch the tissue around the artery in parallel directions to the longitudinal axis of the artery. Connected with SOFT COAG, the endoscopic scissors are applied slightly open, to capture, coagulate, and dissect the capsule. Including the bronchial artery, the method can be used to treat other arteries in VATS lobectomy. One of the fine applications is to treat thin pulmonary arteries with ERBE’s BiClamp® forceps which enables ligation and clip-less procedure.

Results
In consecutive anatomical resections of 58 cases: 49 lobectomies (right upper: 18, right middle: 4, right lower: 12, left upper: 6, left lower: 9) including 20 mediastinum lymph nodes dissection: 9 segmentectomies are included. Blood-loss and mean operation time are: 257 minutes and 130ml with mediastinum lymph nodes dissection and 223 minutes and 86ml without. All cases went safe with no severe complications.

Conclusion
The triangle-tensioning method in VATS resection is rational and practical. In addition, the combination with SOFT COAG enables safe and simple dissection.

Background
Previously, we reported the utility of the da Vinci[®] Surgical System (dVS: Intuitive Surgical, Inc., Sunnyvale, CA) for various types of anterior and middle mediastinal tumors in clinical practice. We evaluated the feasibility, safety and appropriate settings of this system for the surgical treatment of these tumors. One review reports about the importance of the appropriate settings according to tumor location in robot-assisted thoracic surgery (RATS), because no target always exists in the same location within the thoracic cavity. In this report, we evaluated the efficacy of a high-speed three-dimensional (3D) image analysis system (SYNAPSE VINCENT; Fuji Photo Film Co., Ltd.) for preoperative simulation and navigation during a RATS procedure.

Methods
In this study, a high-speed 3D-image analysis system was used to decide the best positioning of robotic-arms and instruments preoperatively. Moreover, this system has capable of detecting the tumor location and extracting surrounding tissues quickly, accurately and safely. Accurate and speedy set-up of the da Vinci S[® ]Surgical System was possible for this operation. Synapse Vincent facilitated determining the best positioning of robot arms and instruments, and was an excellent device for navigation in real time. All patients who underwent RATS in our institution provided written informed consent to receive robotic surgery using the dVS, and the institutional review committees of each institution gave their permission. In this report, a representative mediastinal tumor which was located in the upper thoracic cavity was selected to establish the merits of this procedure.

Results
The patient, a 38-year-old woman, had a posterior mediastinal tumor located at the upper level of Th 1 to 3. Accurate and speedy set-up of the dVS was capable on this operation. It was feasible to decide the best positioning of robot-arms and instruments, and excellent device for the navigation on real time. The total operation time was 270 minutes, the time of the dVS setting was 21 minutes, and the console time (the dVS working time) was 132 minutes. The amount of bleeding was 167 mL and the drainage time was 2 days after the operation and this patient had no complications. The pathological report revealed a schwannoma (85 × 42 × 20 mm) with no malignancy.

Conclusion
For the optimal performance of RATS, the positioning of all units and the locations of instrument ports need suitable directional setting. Preoperative simulation and navigation during of operation using SYNAPSE VINCENT for the RATS has efficacy for planning the setting, especially in deciding the points of instrument ports and the angle of robot arms, and very useful as a device of the navigation software and education use operating on it.

Background
The rate of fluorodeoxyglucose uptake measured as standardized uptake value (SUV) on positron emission tomography (PET) of the primary tumor has been correlated with tumor aggressiveness and poor survival in patients with lung cancer. A retrospective review of patients with lung cancer who were treated with surgical resection at MD Anderson Cancer Center (MDACC) was performed to determine if the pre-operative SUV uptake of N1 disease has any prognostic significance in patients with pathologic stage II lung cancer.

Methods
We reviewed all patients who underwent surgical resection for lung cancer at MDACC from 1998 to 2011. We evaluated non-small cell lung cancer patients who had at least a lobectomy at MDACC as first mode of surgical therapy who had pathologic stage T1-2 and N1 disease and pre-operative PET-CT scan. We determined the clinicopathologic characteristics of patients who had PET-positive N1 disease and compared them to patients who had PET-negative N1 disease. We also performed Kaplan Meier analysis to determine the survival between the two groups.

Results
Among patients who underwent surgical resection for lung cancer at MDACC during this time period, 120 patients met the inclusion criteria for the study. There were 100 stage IIA or T1aN1, T1bN1 or T2aN1 and 20 stage IIB or T2bN1 patients in the study. There were 62 patients (50% of the patients) who had a primary tumor in the periphery of the lung and 58 patients (50% of the patients) who had a primary tumor in the central portion of the lung. Within this group of 120 patients, only 29 patients (24% of the patients) had PET-positive N1 disease. Only 16 out of 58 patients (28%) in the central group and only 13 out of 62 patients (21%) in the peripheral group had PET-positive N1 disease. There was no clinical or pathological difference between the patients who had PET-positive N1 disease and PET-negative N1 disease. The average maxSUV of the primary tumor was 13 ± 10.7 and average maxSUV of the PET-positive N1 disease was 6.3 ± 4.1. Kaplan Meier analysis showed that there was no significant difference in survival between the patients who had PET-positive N1 disease and PET-negative N1 disease.

Conclusion
Among patients with pathologic stage II non-small cell lung cancer, preoperative PET scan was very poor at predicting positive pathologic N1 disease. Since it is difficult to predict pN1 disease, operative patients with clinical stage I non-small cell lung cancer should have surgical resection oppose to ablative therapy. Moreover, SUV uptake of N1 disease in patients with pathologic stage II lung cancer did not predict worse survival in pathologic stage II patients. Thus, patients with cN1 disease should undergo surgical resection after appropriate mediastinal staging.

Background
Prolonged pulmonary fistula is a common complication in pulmonary resection, which happens in 8% to 26% in patients undergoing routine pulmonary resection. And its management is difficult in many cases. Air leakages are associated with prolonged hospital stays, infectious, cardiopulmonary complications, and reoperation occasionally despite of the progress of the recent conservative cure. Blood coagulation factor XIII (BCF XIII) is known to play a role in wound healing. However little is known about the role of BCF XIII in the field of thoracic surgery. BCF XIII is known to fasciculate closure of fistula in gastro-intestine surgery. This time, we examine the relationship for prolonged air leakage and BCF XIII diabetes, chronic obstructive pulmonary disease (COPD), and total protein amount of postoperative.

Methods
In 32 patients who underwent pulmonary resection for lung cancer at Bell-land general hospital or Osaka city university hospital and experienced air leakage for at least 2 days after operation. Pre-operative HbA1c and BCF XIII and pulmonary function measured within 2 weeks pre-operatively .Post-operative total protein (TP) and BCF XIII measured at 5 days post-operatively. We evaluated the relationship between BCF XIII or HbA1c or TP or COPD and duration of chest drain placement respectively.For statistical analysis, t-test was used

Results
Six patients experienced a decrease in factor XIII to 70% or under normal range that was indication for administration of BCF XIII. The mean duration of chest drain placement was 5.2± 2.8 days in patients with post-operative BCF XIII level of ≥71% compared to 8.3 ± 2.8 days in those with post-operative BCF XIII level of ≤70%. Patients with post-operative BCF XIII level of ≤70% required drain placement for a significantly longer period (p<0.05). In this analysis, we did not recognize significant difference in other factors (HbA1c≦6.5% group and HbA1c≧6.6% group, Post operative TP ≧6.6 g/dl group and TP ≦6.5 g/dl , forced expiratory volume 1.0%(FEV1.0%)≧70% group and FEV1.0%＜70% group).

Conclusion
Factor XIII promotes crosslink of fibrin in the early stages of wound healing. Thus, factor XIII is considered to be consumed for lesion repair. In this study, we were considered the possibility BCF XIII is related to lung healing fistula. In diabetic patients, the occurrence of delayed wound healing has been reported frequently. No significant difference was noted between diabetic and non-diabetic patients in this study. We continue to increase the study case in the future, we want to evaluate the relationship between BCF XIII, diabetes or nutrition and the drainage period.

Background
Relieving the intense pain associated with a thoracotomy incision improves patient well-being and pulmonary function, resulting in a more comfortable and ambulatory patient. Epidural analgesia and opioids have been classically employed to treat post-thoracotomy pain (“Gold Standard”), however these techniques have important secondary effects and drawbacks. Pain management strategies have evolved within the last years and significant advances have been achieved with the appearance of multimodal pain treatment. This new concept might be safe and efficacious in controlling post-thoracotomy pain and reducing the amount of systemic opioids consumed.

Methods
From 2007 to present date we have incorporated into our general thoracic surgery protocol a collaborative multimodal post-thoracotomy pain management strategy involving Thoracic Surgery, Anaesthesiology and Physiotherapy Departments. Patients eligible for this protocol were those scheduled for a thoracotomy for non-small cell lung cancer resection. Two sorts of thoracotomies were employed depending on the tumour location: Anterior Thoracotomy (AT) for tumours in the upper or middle lobe, and Postero-lateral thoracotomy (PT) for tumours in the lower lobe. At the end of surgery a paravertebral catheter (PC) was inserted under direct vision in the thoracic paravertebral space at the level of incision. Postoperatively patients received 300 mg/day of local anaesthetic (ropivacaine) through the PC combined with an intravenous non-steroidal anti-inflammatory drug (NSAID) (metamizole 2gr) every 6 hours and daily Transcutaneous Electrical Nerve Stimulation (TENS) sessions. A subcutaneous opioid (meperidine) was employed as rescue drug. Drugs dosages were controlled by the Anaesthesiology Department. The level of pain was measured with the visual analogic scale (VAS) at 1, 6, 24, 48 and 72 h after surgery. The need of meperidine as rescue drug and secondary effects were also recorded.

Results
A total of 270 patients entered the protocol. We did not register secondary effects in relation to the PC, NSAID or TENS. Thirty-five patients (13%) needed meperidine as rescue drug. Mean VAS values were the following: all the cases (n=270): 4.9+/-2.0, AT (n=150): 4.3+/-2.1, PT (n=120): 5.8+/-1.8. VAS 1 hour: AT 2.9, PT 4.3; VAS 6 hours: AT 6.7, PT 7.5; VAS 24 hours: AT 6.0, PT: 6.8; VAS 48 hours: AT 4.0, PT 6.0, VAS 72 hours: AT 3.0, PT: 4.7. Patients submitted to AT experienced less pain than those with PT in mean and at any time (p< 0.01).

Conclusion
Paravertebral block is an effective alternative to epidural analgesia in the management of post-thoracotomy pain. The analgesic scheme combining paravertebral block (PVB) through a PC placed by the thoracic surgeon, NSAID and TENS performed by the physiotherapist has proven to be effective for postoperative pain control after thoracotomy. Inasmuch as surgical extirpation of lung cancers remains the best hope of survival for many patients, a multimodal postoperative pain management plan avoiding the use of epidural analgesia and opioids is feasible and provides and optimal pain management.

Background
Developing "minimally invasive small incision, muscle- and rib-sparing thoracotomy (miMRST), minimally invasive lung cancer radical surgery", to cure aging, cardiopulmonary dysfunction patients with lung cancer, who could not tolerate traditional large-incision posterolateral thoracotomy. Typical cases will be discussed here.

Methods
Man, aged 64, left lower lobe lesions 1.0cm, localized in central, deep part, not suitable for needle-biopsy, nor for wedge resection; smoking for 44 years, with serious chronic bronchitis 15 years, asthma episodes per year; coronary heart disease 13 years, coronary stenting 10 years; anticoagulation 10 years; serious gastric ulcers, colorectal polyps 2 years. Consulted in hospitals in Shenyang and Beijing for months, advised for follow-up considering his current cardiopulmonary condition and no malignant evidence. Then referral to China Medical University Lung Cancer Center in Dec 26, 2012. Surgical resection was advised at once. Preoperative examination: pulmonary function test revealed airway dysfunction, low blood oxygen. Anti-inflammatory, antispasmodic strategy and preoperative pulmonary function exercise did not improve lung function as expected. The patient was discussed not suitable for regular thoracotomy, unable to tolerate the damage from traditional large-incision posterolateral thoracotomy. “miMRST, minimally invasive lung cancer radical surgery” was scheduled.

Results
About 10cm lateral chest incision was enough for most lung cancer resection and mediastinal lymph nodes dissection. Latissimus dorsi and serratus anterior muscles were protected, chest cavity entered through intercostals space, no rib cut. Widespread intrathoracic adhesions, localized severe adhesions, and undifferentiated lung fissures were confirmed. The lesion was found in left lower lobe, adjacent to pulmonary vessels not suitable for wedge resection; swollen lymph nodes adhered around pulmonary vessels were confirmed. Left lower lobe resection, and No.3A,4,5,6,7,8,9,10,11,12,12u,13,14 group regional and mediastinal lymph nodes and surrounding adipose tissue were dissected. When awake after surgery, operative lateral upper limb recovered freedom of movement; the patient got out of bed in the 2nd postoperative day with catheter unplugged in the same day; the chest tube pull out in the 3rd postoperative day; no complications happened. Pathological examination reported lung squamous cell carcinoma, no lymph nodes metastasis. The patient recovered much better and quickly than other patients who received lung cancer resection via traditional standard posterolateral thoracotomy.

Conclusion
"miMRST", "minimally invasive small incision, muscle- and rib-sparing thoracotomy, minimally invasive lung cancer radical surgery", shows advantage of small incision, less pain; less damage; quick recovery, better recovery; operative side upper extremity activities early, pulling out catheter early, get out of bed early, being out of ICU early, chest tube pulled out early; stopping antibiotics early, discharge early; no need using expensive rib nails because of no-rib-cut; no need using expensive thoracoscopic vessel staples; almost no complications; significantly less cost. "miMRST ", is minimally invasive thoracic surgery, very suitable for aging, cardiopulmonary dysfunction patients with lung cancer, who could not tolerate traditional large-incision posterolateral thoracotomy. "miMRST ", is also economical, no need using expensive thoracoscopic devices, to some degree, very suitable for lung cancer surgery in developing countries. (This study was partly supported by the Fund for Scientific Research of The First Hospital of China Medical University, No.FSFH1210).

Background
Technological advances and increased life expectancies have resulted in increasingly complex procedures being performed more frequently on patients with advanced age. As surgeons gain competency in robotic-assisted surgery, surgeons are extending the benefits of these minimally-invasive procedures to geriatric patients. Thus, we investigated the complication rates after robotic-assisted pulmonary lobectomy in patients with advanced age.

Methods
We retrospectively analyzed 180 consecutive patients who underwent robotic-assisted lobectomy by one surgeon between September 2010 and February 2013. Patients were grouped by age >77 at the time of operation (Group A) versus age <77 (Group B). Clinically significant perioperative complications were noted, including minor complications, such as wound infection and anemia requiring transfusion, and more serious major complications, such as empyema and deep venous thrombosis/pulmonary embolus (DVT/PE). Rates of perioperative complications, conversion to open lobectomy, chest tube days, hospital length of stays (LOS), and in-hospital mortality were compared between the two groups, with p-value <0.05.

Results
A total of 180 patients were included (mean age 67yr). Group A had 31 patients with advanced age >77yrs (range 77-86yr; 16 men, 15 women); Group B had 149 patients (range of 29-76yr; 74 men, 75 women). Overall intraoperative complication rate was 17/180 (9%), overall postoperative complication rate was 87/180 (48%), and overall in-hospital mortality was 5/180 (3%). Group A had 7/31 (6%) intra-operative complications, compared to 10/149 (3%) for Group B (p=0.006). The most common intraoperative complication in both groups was bleeding from the pulmonary artery, with 3/31 (10%) in Group A and 3/149 (2%) in Group B. The overall rate of conversion to open lobectomy was 7/31(23%) in Group A versus 13/149 (2%) in Group B (p=0.026); although the rate of emergent conversion to open lobectomy was 3/31 (10%) in Group A compared to 3/149 (2%) in Group B. There were 19/31 (61%) patients in Group A with minor and/or major post-operative complications, compared to 68/149(46%) in Group B (p=0.11). The most common post-operative complications experienced by Group A were prolonged air leak 8/31 (26%), atrial fibrillation 6/31 (19%), pneumonia 4/31 (13%) and mucus plugs requiring intervention 4/31 (9%; p=0.24), while those for Group B were prolonged air leak 26/149 (17%; p=0.28), pneumonia 19/149 (13%; p=0.98), atrial fibrillation 16/149 (11%; p=0.23) and anemia 9/149 (6%). Group A had medians of 5+2.8 (S.E.M.) chest tube days and 7+1.3 (S.E.M.) hospital days, compared to 4+0.3 chest tube days and 5+0.4 hospital days for Group B (p=0.09 and p=0.004, respectively). Interestingly, Group A had 0/31 (0%) in-hospital mortality, compared to an in-hospital mortality rate of 5/149 (3%) for Group B (p=0.30).

Conclusion
Patients with advanced age >77 yr and who undergo robotic-assisted lobectomy have a higher risk of perioperative complications and conversion to open lobectomy. In addition, advanced age also resulted in longer hospital LOS. However, advanced age was not associated with increased in-hospital mortality and was actually associated with decreased mortality. Thus, our study suggests that robotic pulmonary lobectomy is feasible and safe in patients with advanced age.

Background
About 60-70 % patients present with advanced disease in carcinoma oesophagus with limited curative option. There are high local recurrence rate of 32%-45% when treated with surgery alone and 77% when radiation therapy only. So, the results of the treatment of ca oesophagus have been poor inspite of advances in various treatment modalities. A high tumour dose is needed to achieve adequate local control, which is possible by an intraluminal boost following teletherapy. The advantage of intraluminal brachytherapy as a means of dose escalation following EBRT based on inverse square law and quick dose fall off which results in relative sparing of surrounding normal tissues, and potentially improving the therapeutic ratio. Dysphagia is a potential problem. Brachytherapy is effective in palliation of dysphagia by delivering high tumoricidal dose may achieve excellent local control rate and disease free survival with acceptable toxicities.

Methods
Total of 40 atients with histologically proven carcinoma, tumour ≤ 5 cm in length, KPS > 50 with no prior malignancy or N0 status and unfit for surgery were taken in to the study. Intraluminal high dose rate (HDR) brachytherapy treatment was performed with the remote after loading HDR microselecton unit which contained a single cylindrical high-activity 192Ir source. Dose fractionation used 15 Gy in 3 # at 1 week interval. The prescription point was 0.5 cm from mucosal surface from the mid source. The improvement in dysphagia free scores, patterns of failure and treatment related toxicities were assessed. After treatment all patients were followed up with UGIE, barium swallow and chest X-ray at 2 months.

Results
Out of 40 cases analyzed, the lesion was present in mid 1/3[rd] in 18 patients, upper 1/3[rd] in 12 and involving GE junction in 10 cases. The median length of treatment was 5 cm. Gr II dyspahagia was in 34% and Gr III in 66% patients were seen. After treatment 40% of patients had improvement in dyspahgia. Stricture was found in 4 patients, ulceration in 7 and bleeding in 3 and 2 patient had trachea-esophageal fistula. Eight patients lost to follow up. Patients who had Gr II dysphagia initially had no progression in the complain. The median overall survival is 12.1 months and the median PFS was 18 months. who had initially Gr II dysphagia had no progression of dysphagia.

Conclusion
With dose fractionation of 5 Gy / # for 3 # and CT based planning enabled good optimisation along with decreased risk of high dose to mucosa by using 6 Fr tube, this schedule has shown effective palliation in dysphagia and few complication rates and comparable survival benefits. However a larger number of cases and a longer follow up is required.

Background
We need to separate the interlobar fissures during pulmonary lobectomy / segmentectomy. Accordingly, we can select from among several different devices to separate the interlobar fissures. But it is difficult to determine which devices are most beneficial. On one hand, there is an opinion that it is not recommended to use staplers for the interlobar fissures dissection because of a reduction of pulmonary compliance. On the other hand, some surgeons feel that staples do not affect postoperative pulmonary function. The purpose here is to elucidate on whether the use of staplers for the interlobar fissures dissection affects postoperative respiratory function.

Methods
The study consisted of 41 patients who were examined for pulmonary function test before and after surgery. They had undergone a lobectomy / segmentectomy for lung cancer between April 2009 and April 2013 at Ayabe City Hospital. Video assisted thoracic surgery (VATS) was performed in all 41 patients. They were classified into 2 groups: the stapler group underwent the routine surgical procedure (ST group), and the other group did not have staplers applied; other devices were used (OD group). Postoperative respiratory function after pulmonary resection was analyzed mainly. We also analyzed other things between the ST group and the OD group (for example; gender, laterality, smoking, synechia, excision site, and the number of staplers). Postoperative respiratory function was analyzed by means of the ratio between predicted postoperative FEV1.0 (Forced Expiratory Volume in first second) and postoperative FEV1.0. The predicted postoperative FEV1.0 was calculated utilizing the methodology of Juhl B. et al（Acta Anaesthesiol Scand, 1975）.

Results
There were 25 men and 16 women with a mean age of 69.6 years old (51-83 years old). Forty-one patients underwent 39 lobectomies and 2 segmentectomy. All patients recovered and were discharged home. There was no operative mortality, and no hospital deaths. No significant difference of Postoperative respiratory function was observed between the ST group and the OD group (106.6±15.4 vs 105.1±20.7 %; p=0.833). However, Postoperative respiratory function of laterality was significantly lower for the right side than the left side (101.8±16.3 vs 114.9±12.1 %; p=0.012). Moreover, the operative time was significantly longer in the ST group compared with the OD group (275±74.8 vs 206±31.6 min; p=0.02). There was no statistically significant difference between the two groups regarding the postoperative hospitalization length (5.6±2.8 for ST vs 5.1±1.4 days for OD; P=0.639) and the duration of the chest tube placement (3.5±2.9 for ST vs 3±1.8 days for OD; P=0.67).

Conclusion
Persistent air leaks require prolonged chest tube drainage time, which increases the risk of pleuropulmonary infections, associated pain, and consequently longer hospital stays. Several tools and techniques have been used to prevent postoperative air leaks, but in this study, the utilization of staplers for the interlobar fissures dissection did not affect postoperative respiratory function when patients underwent lung resections.

Background
Malignant cells in the pleural effusion are classified as stage Ⅳ in the 7th edition of the TNM-staging of lung cancer. The prognosis of non-small cell lung cancer patients with malignant pleural effusion is reported to be poor as the patients with malignant pleural effusion are generally not subjected to surgery. However, clinically relevant question whether or not the primary tumor should be resected when malignant pleural effusion is first detected at thoracotomy, is controversial. Our purpose is to address the role of surgical resection for main tumor in such patients.

Methods
A retrospective review was conducted with clinical charts of 155 patients with non-small cell lung cancer who had pleural effusion detected at radical surgery between January 2006 and December 2012 at Kansai Medical University Hirakata Hospital. We compared prognosis of the patients with or without surgical tumor resection.

Results
Of the 155 patients with pleural effusion, 30 patients had malignant cells and 125 did not. Of the 30 patients, 18 were men and 12 were women. Twenty-five tumors were adenocarcinoma, 3 were large-cell neuroendocrine carcinoma, 1 was small cell carcinoma and 1 was squamous cell carcinoma. Seven patients were treated with lobectomy, 12 were treated with wedge resection and 11 were with exploratory thoracotomy. Five-year survival rate was 35.0% in patients with primary tumor resection, whereas none of the patients without surgical resection of tumors survived 5 years. Two-year survival rate was 22.7% in patients with exploratory thoracotomy.

Conclusion
The prognosis of patients with malignant pleural effusion detected at surgery was not such poor compared to that of generally reported stage IV patients. Patients with surgical resection of main tumor showed better survival compared to those without surgical resection, suggesting that cytoreductive surgery contributed to multimodality treatment in patients with malignant pleural effusion. Based on our series of patients, status of N0 may be candidates for primary tumor resection even in patients with malignant pleural effusion.

Background
It is by now widespread that surgical resection is standard curative treatment for lung cancer, however in super-elderly cancer patients (over 85 years old) there is no clear evidence of safety and efficacy of surgical approach. This study attempts to clarify the benefit of surgical treatment for them.

Methods
Between January 2002 and December 2012, among 1229 consecutively treated patients with primary lung cancer who underwent surgical resection, 29 patients (2.4%) were over 85 years old. Clinicopathological information on these patients was retrospectively reviewed and the surgical outcome was investigated.

Results
There were 21 men and 8 women, with a mean age of 86.5 years old (range, 85-91 years old). All patients were selected as operable candidates based on the results of routine staging procedures consisting of physical examination, blood chemistry, chest roentgenograms, bronchoscopy, computed tomography of the thorax, abdomen, MRI of brain, and radionuclide bone scanning. Indications for surgery included clinical stage I,II, or IIIA (except bulky N2 ) disease. The surgical procedure was lobectomy in 19 patients, sleeve lobectomy in 2 patients, segmentectomy in 1 patient, and wedge resection in 7 patients. Curative operation ratio was 75.9%. The median intraoperative blood loss and operative time were 96 ml and 165 min. There was no blood transfusion case in this series. Postoperative pathological stage was stage IA in 11 patients, IB in 8 patients, IIA in 1 patient, IIB in 5 patients, IIIA in 2 patients, IIIB in 1 patient. 25 patients (86%) were what is called limited disease. Histological diagnosis was adenocarcinoma in 14 (48.3%) patients, squamous cell carcinoma in 11(37.9%) patients, large cell neuroendocrine carcinoma (LCNEC) in 2 (6.9%) patients, large cell carcinoma in 1 (3.45%) patient, undifferentiated carcinoma in 1 (3.45%) patient. Patients presented postoperative complications in high rate. The complications recorded were delirium in 9 (31.0%) patients, respiratory failure 3 (10.3%) patients, acute renal failure in 2 (6.9%) patients, angina attack in 2 (6.9%) patients, atrial fibrillation in 1(3.5%) patient, cerebral infarction in 1 (3.5%) patient. Although surgical morbidity rate was 62.1%, surgical mortality rate was 0%. 14 patients died until now, 7 of them were recurrent death. Overall patient 1, 3, and 5 years survival rate were 100%, 68.5% and 38.6%, respectively. Postoperative hospital days were 19.5±7.51.　

Conclusion
Although surgical morbidity rate is high and postoperative hospital days is long, surgical results in this study are acceptable and support the value of surgical treatment in super-elderly patients with lung cancer.

Background
Video-assisted thoracoscopic (VATS) lobectomy is being performed more frequently in thoracic centers, but cost is a concern. Earlier studies have shown increased intraoperative but lower postoperative costs over open thoracotomy, but to date there has not been a published cost analyses in North America. The objective of this study is to compare the cost of introducing VATS lobectomy and traditional open lobectomy in a Canadian tertiary care hospital.

Methods
A retrospective cost analysis was done comparing 78 VATS to 149 open lobectomies performed over 32 month period. Intraoperative (disposables, operating time) and postoperative costs (days in intensive care, intermediate care and ward units, days requiring acute pain service (APS), readmission) were compared, as well as hospital stay. A secondary analysis was performed to look at the effect on hospital costs of a learning curve for VATS lobectomy.

Results
The mean intraoperative, postoperative and total costs for VATS and open lobectomy were $4,770 and $2,166 (p-value = 0.01), $3929 and $5,604 (p < 0.0001), and $8,499 and $7,771 (p= 0.3), respectively. Median hospital stay for VATS and open lobectomy were 4 and 5 days (p< 0.0001), respectively. A significant difference in the intraoperative cost of VATS lobectomy was realized after the first 20 cases, with the mean intraoperative cost decreasing from $5095 to $4510 (p = 0.03).

Conclusion
The total costs of VATS and open lobectomy are equivalent. Increased disposables cost and longer operating time account for higher intraoperative cost of VATS; shorter hospital stay and less requirement for APS reduce the VATS postoperative costs. There is a learning curve present when introducing VATS lobectomies to an institution. After 20 cases intraoperative costs reduce significantly with more efficient use of disposables and operating time.

Background
Thoracoscore and the European Society Objective Score (ESOS.01) are two risk scoring systems used to estimate risk of death as part of informed consent, and to allow risk adjusted outcomes to be evaluated. We aimed to evaluate if these are valid tools for use in the United Kingdom (UK) population.

Methods
A multi-centre, prospective study was carried out on patients undergoing lung resection at 6 UK centres. Data were submitted electronically using our online data collection tool. Univariate and multivariate analyses were carried out to determine the factors affecting mortality. A Receiver Operating Characteristic (ROC) analysis was performed in order to determine the ability of the Thoracoscore and ESOS.01 to predict in-hospital mortality.

Results
Data were submitted for 2570 patients. 345 patients were excluded due to incomplete data fields. Of the remaining 2245 patients, the observed in-hospital mortality was 31 patients (1.38%). Mean Thoracoscore was 2.66(SD±3.21). Logistic regression analysis identified gender (p=0.004, hazard ratio 4.786) and co-morbidity score (p=0.005, hazard ratio 3.289) as risk factors for mortality. A sub-analysis was performed using data from 1912 patients. In this group, mean Thoracoscore was 2.55(SD±2.94), mean ESOS.01 was 2.11(SD±1.41), and these were statistically significantly different (p<0.0001). The observed in-hospital mortality was 28 patients (1.46%). The c-index for Thoracoscore was 0.705, and for ESOS.01, 0.739. Furthermore, there was poor correlation between the two scoring systems (r=0.362).

Conclusion
Both Thoracoscore and ESOS.01 overestimated mortality in the UK population. There is a continued need to develop an appropriate risk prediction system for the UK.

Background
Recurrences in patients with completely resected non-small cell lung cancer (NSCLC) rarely occur more than 5 years after operation. Various follow-up programs for postoperative patients are recommended in each guideline. The purpose of this study is to clarify the risk factor of late recurrence and to determine which patients might benefit from routine computed tomography (CT) follow-up more than 5 years after operation.

Methods
Between January 1995 and December 2006, 1,437 consecutive patients with NSCLC underwent pulmonary resections at our institution. Of these, 617 patients remained recurrence-free for 5 years after resection. We retrospectively analyzed the clinicopathological features of these patients. Disease free survival (DFS) was defined as endpoint and was analyzed using Cox proportional hazards model. Variables for univariate analysis were as follows: age, gender, smoking history, carcinoembryonic antigen, operative procedure, pathological type, pathological stage, and pleural lavage cytology (PLC).

Results
At the median follow-up time of 7.5 years, 20 patients (3.2%) developed late recurrence more than 5 years after resection. Distant metastasis occurred in 15 patients and locoregional recurrence occurred in 5 patients (Table 1). There were 3 patients (15%) with positive PLC in late recurrence group and 7 patients (1.2%) in recurrence free group. In univariate analysis, only PLC was significant. In a multivariate analysis, PLC was a significant predictor of late recurrence. The Hazard ratio (HR) for positive PLC in comparison to negative PLC was 5.75 (95% CI 1.16–19.26; p=0.04)Figure 1.

Conclusion
PLC is a strong independent factor for late recurrence. Patients with positive PLC might be good candidates for routine chest CT more than 5 years after resection.

Background
Surgery results in a wide range of unfavorable alterations in body homeostasis which are referred to as surgical stress. Low total antioxidant capacity could be indicative of oxidative damage associated with increased morbidity and mortality. When surgical stress generates oxygen radicals, they are quickly detoxified by antioxidant network, leading to a decrease in the potential of antioxidants which can be measured. Video-assisted thoracoscopic (VATS) and open lobectomy approaches were evaluated to determine if there is any difference in acute phase response and oxidative stress associated with surgery for early stage non-small cell lung cancer (NSCLC).

Methods
Our prospective study included 30 patients who underwent lobectomy for early stage NSCLC: by conventional open thoracotomy approach (n=15) and VATS approach (n=15). We measured changes in plasma total antioxidant capacity (TAC), lactate dehydrogenase (LDH) and C-reactive protein (CRP), fibrinogen, total protein (TP) concentrations and WBC count preoperatively, 24h and 72h postoperatively, on the day of drain removal, and 1 and 9 days after the drain removal. Concentrations of TAC, LDH and TP in pleural fluid were observed 24h, 72h postoperatively and on the day of drain removal.

Results
Changes in TAC, LDH, CRP, fibrinogen, TP and WBC count in plasma for both groups followed similar kinetics. The response of CRP and LDH (p<0.05), as well as WBC count and fibrinogen (0.05

Conclusion
Our results suggest that the VATS approach compared to open thoracotomy approach could be associated with less tissue trauma and surgical stress. We can expect that decreasing intraoperative oxidative stress could dynamically contribute to the improvement of postoperative recovery.

Background
Video-assisted thoracoscopic surgery (VATS) has been applied widely to thoracic surgery such as VATS lobectomy, resection of chest wall/ mediastinal tumors. The appropriate placement of trocar is the keys for these procedures. Many surgeons places the first -port to look inside of the thoracic cavity by thoracoscope, and determine the position of the other ports. However, there are differences in deformation and the thickness of the thorax of individual patients. In this study, we analyzed the adequate surgical approach by analyzing the three dimensional Computed tomography (3D-CT), and develop the prototype system to project the 3D-CT image to the patient body directly to mark the points of surgical approach.

Methods
3D-CT based minimally invasive surgery was designed using 3D-CT volume analyzer Synapse Vincent (Fuji film, Japan). Skin window, skeletal structure, intra-thoracic anatomical images were reconstructed respectively. The designed points of trocar were marked in 3D-CT image of skin window and projected on the patinet’s body by a liquid projector. Positional / magnification power correction was made using the land marks such as sternal notch, bilateral nipples, costal arch, and acromion. The error of the system was evaluated using artificial thorax model. Two chest wall tumor patients and 2 mediastinal tumor patients were enrolled for the clinical study. Each surgical incision was planned based on the system. The system validity was evaluated by 3 surgeons.

Results
The error of liquid projector guided skin marking was within 9.3 ± 2.5mm in the distance of 15 cm the center of projection point using artificial thorax model. 3D-CT based surgical approach were prospectively planned and the position of skin incisions were marked on the patients’ body by the liquid projector which projecting the position of the trocars determined by 3D-CT simulations. In the clinical study, Each skin incision made on the point indicated by the liquid projector were placed in the adequate position.

Conclusion
Direct projection of three dimensional volume analyzed CT images to the surgical field by the portable liquid projector indicated the adequate approaching point on the surgical field for the minimally invasive surgery. This method may present the appropriate surgical approach to thoracic surgeons.

Background
Elderly patients are increasingly diagnosed and treated for non-small cell lung carcinoma (NSCLC). We studied short- and long-term outcomes following pulmonary resection in elderly (≥75 yrs) patients and compared them to younger patients.

Methods
This was a nation-wide study of all patients in Iceland that underwent pulmonary resection for NSCLC between 1991 and 2010. Clinical features, complications and survival were compared between patients older (n=108, 21%) and younger (n=404, 79%) than 75 years.

Results
Preoperative lung function and histology were comparable in both groups, however, coronary artery disease was more common in the elderly group, as were sublobar resections (24% vs. 8%, p<0.001). In the elderly group 91% of patients were diagnosed in TNM-stages I+II compared to 75% in younger patients (p<0.001). Mortality within 30 days was similar in both groups (<1%) as was the rate of minor and major complications. Overall survival at 5 years was comparable for both groups (39% vs. 42%, p=0.28) as was cancer specific survival (55 vs. 47%, p=0,64). In multivariate analysis age over 75 years did not significantly influence survival (HR 1.20, CI: 0.87-1.66, p=0.26).

Conclusion
Early and late outcomes of pulmonary resections in elderly NSCLC-patients were comparable to younger patients. However, selection of patients could have biased the outcome in favour of the elderly patients with lower stages, and more sublobar resections. Surgery seems to be a valid treatment option for NSCLC in the elderly.

Background
Optimal management of stage IIIA-N2 non-small cell lung cancer (NSCLC) is controversial. However, surgery is used increasingly for stage IIIA NSCLC. We believe that surgical outcome of NSCLC patients with clinical N2 and pathological N2 (cN2/pN2) is worst among the NSCLC patients with cN2 disease. Analysis aimed at evaluating survival rates of patients with cN2/pN2 stage, and at studying prognostic factors for long-term survival.

Methods
This is a retrospective study of 72 NSCLC patients with cN2/pN2 stage who underwent surgery with neoadjuvant or adjuvant treatment from 2003 to 2011. Overall survival (OS) and disease-free survival (DFS) were estimated using Kaplan-Meier methods. A multivariate analysis for prognostic factors was performed by the Cox proportional hazards regression model.

Results
The median follow-up time was 24.5 months (range, 1 to 110 months) for 72 NSCLC patients. Neoadjuvant treatment was administered to 32 patients (44.4%), and adjuvant therapy was given to 40 patients (55.6%). Pneumonectomies were performed more frequently in patients who were treated with neoadjuvant therapy (25% vs. 15%). Complete resection was achieved more commonly in patients who underwent surgery followed adjuvant treatment (95% vs. 75%). Five year OS was 40.5% and 3-year DFS was 34.3%. In a multivariate analysis, incomplete resection was prognostic for a worse OS (hazard ratio: 3.07, 95% CI: 1.20 to 7.86). The more advanced pathological T stage was prognostic factor for a worse DFS (hazard ratio: 3.21, 95% CI: 1.42-7.24). Number of metastatic lymph node is important prognostic factor for OS and DFS.

Conclusion
Favorable survival can be achieved in cN2/pN2 NSCLC patients after resection with neoadjuvant therapy or adjuvant therapy. Survival is more favorable for complete resection than incomplete resection.

Background
Patients considered to be at increased risk from surgery may be offered nonsurgical therapies for early stage non-small cell lung cancer (NSCLC). However, video assisted thoracic surgery (VATS) is associated with lower morbidity and thus may permit anatomic resection for patients considered increased risk.

Methods
Our institutional database was queried to find all patients who received lobectomy for early stage NSCLC between 2002-2010. Patients were grouped into cohorts of standard (SR, n=536) or increased risk (IR, n=72) using the ACOSOG Z4099/RTOG 1021 eligibility criteria. Morbidity and mortality were compared based on risk group and surgical method.

Results
Median age was 72 and 67 years for IR and SR respectively. Most patients were stage I (IR: 83.3%; SR: 84.5%). Although IR patients had increased overall and pulmonary complications compared to SR (overall: p=0.0004; pulmonary: p<0.0001), there were no differences between the subset of IR patients who had VATS resections and SR patients resected by either open or VATS techniques (overall: p=0.7697; pulmonary: p=0.3219) [Table 1]. Survival at 5 years was significantly lower for IR patients resected by open techniques (46.2%; p=0.0028) but those resected by VATS (61.2%) had similar survival to SR patients resected by either VATS (65.1%) or open techniques (64.3%; p=0.83) [Figure 1]. There was no significant difference in operative mortality between the IR and SR groups (IR: 1.4%; SR: 0.4%; p=0.2832).

Table 1: Post-operative complications stratified by risk subgroup and surgical method

	Increased risk (%)	Standard Risk (%)	Increased Risk with VATS resection (%)
Overall Complications	33.3	16.2	18.2
Pulmonary Complications	30.6	11.8	18.2

Figure 1 Figure 1: Overall survival following lobectomy for NSCLC, stratified by risk group and surgical method

Conclusion
Surgical morbidity and mortality are reduced in patients at increased risk from lobectomy when resected by VATS offering them equivalent outcomes to standard risk patients.

Background
To assess the feasibility, safety and long-term outcomes of video-assisted thoracic surgery (VATS) lobectomy for the treatment of non-small cell lung cancer (NSCLC) in patients with severe COPD.

Methods
The clinical data of patients with NSCLC and severe COPD (preoperative FEV1% <50%) who underwent VATS lobectomy from January 2000 to January 2011 were retrospectively analyzed to identify their demographic parameters, postoperative complications and outcomes.

Results
The preoperative FEV1/FVC was <70% and FEV1% <50% in all 61 patients in this study, with a mean preoperative FEV1 of 0.99 L (0.54-1.58 L) and mean FEV1% of 38.4% (22-49.82%). All of the 61 patients underwent the VATS lobectomy or sleeve resection plus systemic lymph node dissection. The mean operative time was 218 minutes (120-355 minutes), with a mean intraoperative blood loss of 342 ml (50-1600 ml). None of the patients converted to thoracotomy. Multivariate statistical analysis revealed that age and TNM staging after tumor resection were independent predictive factors for the 5-year survival in those patients (p=0.014 and 0.013).

Conclusion
With preoperative imaging studies, pulmonary function assessment and target positioning, VATS leboectomy can be safely and effectively performed for patients with NSCLC and severe COPD to achieve a satisfying long-term survival outcome.

Background
To determine the incidence of peri-operative complications in non-small cell lung cancer (NSCLC) patients with co-existent chronic obstructive pulmonary disease (COPD) who undergo lung resection via traditional and minimally invasive techniques.

Methods
A retrospective analysis was conducted of 152 NSCLC patients with COPD who underwent thoracoscopic minimally invasive surgery. Particular attention is given to the relationship between disease severity or surgical approach and the incidence of complications.

Results
The prevalence of respiratory and cardiac complications was significantly higher in patients with severe/extremely severe COPD than those with mild to moderate COPD (respiratory compications: 37.3% vs. 20.4%, P=0.022; cardiac complictions: 16.9% vs. 6.5%, P=0.040). Patients who underwent complete-video assisted thoracoscopic surgery (c-VATS) had a significantly lower overall morbidity of adverse reactions than those who had undergone VATS major resection surgery (26.3% vs. 42.1%, P=0.044). Among patients with severe/extremely severe COPD, there was no significant difference in the incidence of any complication between the lobectomy group and wedge resection group (38.8% vs. 70.0%, P=0.072). Overall, the occurrence of adverse reactions was significantly lower in patients who underwent c-VATS than in those who had undergone VATS major resection surgery (34.2% vs. 61.9%, P=0.038).

Conclusion
VATS techniques are suitable for COPD patients and are demonstrated here to lower the incidence of post-operative complications when compared with more invasive approaches. Minimally invasive approaches should be considered in patients with COPD who are deemed high risk for curative surgery by traditional techniques.

Background
Nomogram is a widely used tool for cancer prognosis due to its improved individual prediction of survival through combining various significant prognostic factors. The objective of this study was to develop a clinical nomogram for predicting survival for patient with resected non-small cell lung cancer (NSCLC).

Methods
Based on data from a multi-institutional registry for 6111 patients with resected NSCLC at China between January 2001 and December 2008, we performed univariate and multivariate stepwise Cox regression analyses to identify survival prognostic factors, which were then integrated to build the nomogram. Seventy-five percents of randomly sampled data were used to build the nomogram while the remaining data were used to validate the model. The predictive accuracy and discriminative ability of the nomogram was determined by concordance index (C-index). Risk group stratification within a certain stage was proposed for the nomograms.

Results
Among 26 clinical variables, 13 independent prognostic factors finally entered the nomogram (Figure 1), including age, sex, histology, tumor location, operation type, assess to complete video-assisted thoracoscopic surgery (VATS), primary tumor (T) stage, lymph nodes (N) stage, TN stage, number of dissected lymph nodes, blood loss volume, and complications. The calibration curves for probability of 1, 3, 5, 10-year overall survival (OS) represented good agreement between prediction by nomogram and actual observation in the validation set. The C-index of the nomogram was statistically higher than that of the 7[th] edition TNM stage for predicting survival (0.71 vs 0.66, P=0.01). The stratification into different risk groups allows significant distinction between Kaplan-Meier curves for survival outcomes in each TNM stage respectively (P<0.01 for all stages, Figure 2).Figure 1

Conclusion
We developed a novel and validated clinical nomogram that could provide individual and more accurate predictions for OS of resected NSCLC patients compared with the TNM staging system. This prognostic model may support clinicians and patients in decision making, such as to identify those with higher risk for poor prognosis.

Background
We investigated the prognostic factors in patients with only brain metastasis as the postoperative initial recurrence after resection of NSCLC

Methods
We conducted a retrospective study of patients who had undergone resection for NSCLC between 1992 and 2012 and found 113 had experienced postoperative brain metastasis as initial metastasis. We reviewed these patients retrospectively.

Results
The 1-year survival and 3-year survival rate after the diagnosis of brain metastasis was 39.8% and 19.0 %. On univariate analysis, the significant prognostic factors included gender (3-year survival rate, female versus male; 26.3% versus 15.6%, p = 0.027), pneumonectomy (3-year survival rate, pneumonectomy versus non-pneumonectomy; 5.6% versus 27.4%, p = 0.009), adenocarcinoma (3-year survival rate, adenocarcinoma versus non-adenocarcinoma; 10.7% versus 25.3%, p = 0.016) and interval to brain metastasis after surgery (3-year survival rate, < 1 year versus 1 ~2 year versus >2 year; 14.2% versus 25.3% versus 38.9%, p = 0.049).

Conclusion
In patients with brain metastasis after lung resection for NSLCL, female, non-pneumonectomy, non-adenocarcinoma and longer interval to metastasis after surgery showed favorable positive prognosis.

Background
Incomplete fissure between right upper and lower lobe must be divided when lymph nodes at station 11s are dissected, and an additional maneuver is required to divide the fissure for middle lobectomy. The purpose of this study was to determine whether or not dissection of station 11s lymph nodes is necessary in the case of lung cancers located in the middle lobe.

Methods
Between 2000 and 2012, 1657 patients underwent surgical resection for non-small cell lung cancer (NSCLC). Of these, 112 patients who underwent pulmonary resection greater than lobectomy and systemic lymph node dissection, and who had T1-4N0-2M0 NSCLC in the middle lobe, were analyzed retrospectively. 27 patients had lymph node metastases (N1:13, N2:14).

Results
The distribution of lymph node metastasis is shown in Figure 1a and 1b. Lobectomy was performed in 82 patients, lobectomy plus additional lung resection was performed in 8, bilobectomy was performed in 22, and pneumonectomy was performed in two. Six patients underwent bronchial sleeve resection. The overall 5-year survival rate was 80.2% in patients without lymph node metastasis, 11.3% in N1, and 0% in N2 (p<0.0001). Four patients had metastasis to station 11s, and in all of these patients the tumors were located in the lateral segment (S4), were larger than 3 cm in diameter, and showed adenocarcinoma histologically. Three of these four patients underwent bilobectomy. Figure 1Figure 2

Conclusion
Lymph node metastasis is a significant prognostic factor for primary lung cancer in the middle lobe. Among patients who had lymph node metastasis in middle-lobe NSCLC, 15% had metastasis to station 11s. Therefore, lymph node dissection of station 11s is required for patients with primary lung cancer located in the middle lobe to ensure accurate staging.

Background
Background. Thymomas are the most common tumors of the anterior mediastinum. Clinical manifestations of thymomas depend on their endocrine activity, growth pattern and size. Non-invasive thymomas are mostly asymptomatic. According to different authors, more than 75% of thymomas are combined with myasthenia gravis. Myasthenia gravis affects people of working age, is characterized by a progressive course and often leads to disability. In the last decade, as a result of the active introduction of minimally invasive surgical techniques to the practice, the question of the choice of surgical approach between open access and endoscopic surgery remains to be open.

Methods
Methods. In the period 2002-2012 45 patients with tumor and hyperplasia of thymus were treated in Perm regional clinical hospital. Mean age was 43.9 years. Women of working age prevailed among the patients - 31 (68.8%). During examination hyperplasia of thymus was revealed in 15 patients, thymoma in 30 patients besides 5 of them had invasive malignant tumor growth. Among patients with hyperplasia of thymus (15 patients) the clinic of myasthenia was revealed in 9 cases (60%), 5 of them had generalized form and 4 – local form of myasthenia. In the group of patients with tumor of thymus (30 patients) myasthenia gravis was diagnosed in 26 of them, which amounted to 86.6%. Thus generalized form of myasthenia gravis was observed in 24 patients (92,3%), and its local forms were revealed only in 2 cases.

Results
Results. Surgical treatment was performed in 42 patients, that is 93.3%. In 3 patients surgical treatment wasn’t carried out due to the presence of severe comorbidity. Sternotomy and extended thymectomy was performed in 25 patients, thymectomy through thoracotomy was performed in 4 cases. Since 2008 endoscopy (VATS) started to be used for thymectomy (pic. 2A, pic.2B). It was carried out in 13 patients, but in 3 cases complementary (extra) minithoracotomy was required because of the intraoperatively diagnosed invasion of large vessels. Pic. 2 A Pic. 2 B General postoperative mortality was 4.5%. Two patients after VATS died. They had invasion of the tumor to the mediastinal great vessels and the endoscopic operation required a conversion to thoracotomy because of the massive intraoperational blood loss. The regression of neurological symptoms and the decrease of necessity of the anticholinesterase drugs were revealed in all the patients that were operated.

Conclusion
Conclusions.In spite of the technical capability for extended thymectomy and less traumatic rate of the endoscopic method we consider that a complete longitudinal sternotomy is better in cases of invasion of tumor to the mediastinal great vessels or in suspicion on presence of aberrant thymic tissue in the mediastinal and neck cellular spaces.

Background
Video-assisted thoracoscopic surgery (VATS) lobectomy has been gradually accepted as an alternative surgical approach to open thoracotomy for selected patients with non-small cell lung cancer (NSCLC) over the past 20 years. The aim of this project was to standardize the perioperative management of VATS lobectomy patients through expert consensus and to provide insightful guidance to clinical practice.

Methods
A panel of 55 experts on VATS lobectomy was identified by the Scientific Secretariat and the International Scientific Committee of the ‘20[th] Anniversary of VATS Lobectomy Conference – The Consensus Meeting’. The Delphi methodology consisting of two rounds of voting was implemented to facilitate the development of consensus. Results from the second-round voting formed the basis of the current Consensus Statement. Consensus was defined a priori as more than 50% agreement amongst the panel of experts. Clinical practice was deemed ‘recommended’ if 50-74% of experts reached agreement and ‘highly recommended’ if 75% or more of experts reached agreement.

Results
Fifty VATS lobectomy experts (91%) from 16 countries completed both rounds of standardized questionnaires. No statistically significant differences in the responses between the two rounds of questioning were identified. Consensus was reached on 21 controversial points, outlining the current accepted definition of VATS lobectomy, its indications and contraindications, perioperative clinical management, as well as recommendations for training and future research directions. Figure 1 Figure 2

Conclusion
The present Consensus Statement represents a collective agreement amongst 50 international experts to establish a standardized practice of VATS lobectomy for the thoracic surgical community after 20 years of clinical experience.

Background
The non-small cell lung cancer TNM classification system uses the anatomic extent of lymph node (LN) metastases to define the N category. However, the TNM classification system for breast, gastric, and colorectal cancer has been updated to include number of metastatic lymph nodes (MLNs) in the N staging. In these cancers, the number of MLNs has been shown to be a more effective prognostic factor than the anatomic location of MLNs. Moreover, it has been suggested the ratio of MLNs to total number of LNs examined (lymph node ratio [LNR]) in breast, bladder, gastric, and colorectal cancer is a better prognostic factor. Here, we evaluated the effect of these factors on the disease-free-survival (DFS) of non-small cell lung cancer.

Methods
We retrospectively reviewed 428 patients who underwent with pathological examination of resected LNs from 2001 through 2010. The prognostic value of number of MLNs, LNR, or current pN classification was assessed using a multivariate Cox proportional hazards model for DFS, with sex, age, smoking history, tumor size, histology, histological grade. The number of MLNs and LNR were analyzed as a categorical variable, and the patients were divided into 4 groups by the number of MLNs (n[0]: no MLNs, n[1-3]: 1-3 MLNs, n[4-6]: 4-6 MLNs, and n[≧7]: 7 or more MLNs), or by the LNR (n[none]: 0%, n[low]: 1%-9%, n[moderate]: 10-24%, and n[high]: 25% or higher).

Results
At least one nodal metastasis was found in 100 patients (23%), represented by n[0] in 328 cases, n[1-3 ]in 77 cases, n[4-6] in 15 cases, and n[≧7] in 8 cases. By the LNR, 328, 55, 25, and 20 cases were assigned to n[none], n[low], n[moderate], and n[high] groups, respectively. The 5-year DFS rate of n[0], n[1-3], n[4-6], and n[≧7] groups were 83%, 48%, 24%, and 0%, respectively, and the 5-year DFS rate of n[none], n[low], n[moderate], and n[high] groups were 83%, 51%, 38%, and 9%, respectively. Multivariate analysis showed the number of MLNs and LNR were significant independent prognostic factor, equal to the current pN classification. Hazard ratios for pN1 and pN2 with respect to pN0 were 2.07 and 5.04. In contrast, hazard ratios were 2.70, 4.03, and 14.7 for n[1-3], n[4-6], and n[≧7] with respect to n[0]; and 2.16, 3.62, and 9.95 for, n[low], n[moderate], and n[high] with respect to n[none].

Conclusion
The number of MLN and LNR are strong independent prognostic factor in non-small cell lung cancer. They may add new information to the pN categories of the current TNM classification.

Background
The standard surgical approach for non-small-cell lung cancer is lobectomy with systematic hilar and mediastinal lymph node dissection. The purpose of lymph node dissection is considered to be improvement of prognosis and intraoperative staging. Although improvement of prognosis is controversial, it is clear that intraoperative nodal assessment is important for identifying N2 disease and making postoperative therapeutic decisions． For complete resection (CR), at least three mediastinal nodes including subcarinal nodes and three hilar/ intrapulmonary nodes had to be retrieved. Otherwise It is defined as uncertain resection(UR). The objective of this study is to clarify the difference of prognosis between CR and UR.

Methods
The medical records and the follow-up data of the patients operated for NSCLC(c-stage I to III) between January 2005 and December 2006 in Yokohama City University Hospital and 8 associate hospitals were analyzed retrospectively. Four hundred-eighty-four patients with NSCLC who underwent lung resections (lobectomy or pneumonectomy) with negative surgical margins were included in this study. Complete resection (CR) was performed in 198 patients. And in 286 patients, uncertain resection was done. We compared these 2 groups.

Results
There were no statistically difference between the both groups for age, gender, pathological stage( IA:CR n=69/UR n=153，IB 59/71，IIA 4/12，IIB 27/21，IIIA 36/24，IIIB 3/5), and histology (adenocarcinoma: CR n=122/UR n=185，squamous carcinoma:51/68，large cell carcinoma:15/14，others:14/20 respectively). Five-year disease-free-survival rate in the CR group was 58.1% compared with 63.3% in the UR group. Among patients with p-stage I, the 5-year disease-free-survival rate was significantly lower in UR group (78.1%) than in CR group (88.0%, p=0.027).

Conclusion
Uncertain resection might not be enough for accurate intraoprerative staging to determine pN0 status. However whether the accurate intraoperative staging leads to good prognosis was unclear.

Background
The ideal treatment for N2 disease in NSCLC is a subject of controversy. The treatment policy followed by individual institutes is generally a reflection of physician preference and physical characteristics of the patient population. In view of stronger evidence supporting neoadjuvant chemotherapy and the desire to reserve one modality of treatment it has been the policy in our institution to administer neoadjuvant chemotherapy followed by surgery in all pathologically proven cases of N2 disease. We conducted a retrospective analysis to asses the pathological response of mediastinal lymph nodes to neoadjuvant chemotherapy and the need for adjuvant radiotherapy in this patient population.

Methods
Data from a prospectively maintained database from August 2009 to March 2013 was analyzed. Information regarding number of patients detected to be N2 on mediastinoscopy, patients advised neoadjuvant chemotherapy, number of lymph node stations affected, curative resections, pathological response in mediastinal nodes and adjuvant treatment advised was retrieved. The rate of complete response after neoadjuvant therapy and patients advised postop radiotherapy were calculated as percentages

Results
231 patients underwent mediastinoscopy for operable NSCLC. 36 patients were detected to have N2 lymph nodes of which 29 patients were advised neoadjuvant chemotherapy. The remaining patients were advised definitive chemoradiation in view of significant nodal disease precluding R0 resection. 13/17 patients with single station lymphadenopathy underwent curative resection with 8 (61%)patients achieving complete pathological response in the mediastinal nodes. 8/9 patients with multiple station lymphadenopathy also underwent resection with only 2(25%) patients achieving complete pathological response. 11/21(52%) patients who underwent R0 resection after neoadjuvant therapy were advised post operative radiotherapy in view of residual disease in mediastinal nodes while 6/8 (75%) patients with multistation lymphadenopathy required postoperative radiotherapy

Conclusion
Patients with resectable multistation mediastinal lymphadenopathy on preoperative invasive mediastinal staging have a very high likelyhood of having residual viable disease in the mediastinal nodes even after neoadjuvant chemotherapy. In this subset of patientss radiotherapy should be incorporated in to the treatment strategy at the outset, to achieve mediastinal steriilization. The ideal timing of radiotherapy neoadjuvant or post operative needs more study. In patients with single station lymphadenopathy neoadjuvant chemotherapy with surgery may be an adequate strategy with radiation reserved for those with residual disease.

Background
The occurrence of both emphysema and pulmonary fibrosis in the same patient has received increased attention as the syndrome of combined pulmonary fibrosis and emphysema (CPFE). Patients with CPFE show severely impaired DLCO, hypoxemia at exercise, characteristic computed tomography (CT) imaging feature, and high probability of lung cancer. However, the clinical characteristics of lung cancer patients with CPFE are not well known. The aim of this study is to identify clinical characteristics of completely resected lung cancer with CPFE.

Methods
A total of 559 consecutive patients who underwent complete surgical resection for lung cancer from January 2008 through December 2010 were reviewed. Based on preoperative chest HRCT findings, patients were categorized into three groups: those with normal lung (N) (except for lung cancer), emphysema without pulmonary fibrosis (E), and CPFE. The HRCT inclusion criteria of CPFE is as follows; 1) Presence of emphysema, defined as well-demarcated areas of decreased attenuation in comparison with contiguous normal lung and marginated by a very thin or no wall, and/or multiple bullae with upper zone predominance. 2) Presence of a diffuse parenchymal lung disease with significant pulmonary fibrosis, defined as reticular opacities with peripheral and basal predominance, honeycombing, architectural distortion and/or traction bronchiectasis or bronchiolectasis. Chest HRCT scans were reviewed separately by two thoracic surgeons and one radiologist. The clinical characteristics of patients with CPFE were compared with those of the other groups.

Results
This study cohort included 328 (58.7%) patients in N group, 136 (24.8%) patients in E group, and 95 (17.0%) patient in CPFE group, with median age of 67 years. The 3-year survival rates were 68.4% in CPFE group, 80.2% in E group, and 89.7% in N group (p < 0.001). CPFE group found a positive correlation with each of the following factors compared to N and E groups; > 67 years (p = 0.004), lymph node metastases (p = 0.033), male gender (p < 0.001), tumor size > 3cm (p < 0.001), vascular invasion (p < 0.001), non-adenocarcinoma (p < 0.001), pleural invasion (p < 0.001), elevated preoperative serum CEA level (p < 0.001). The frequency of patients presenting grade 2 or more severe postoperative complication under CTCAE or Clavien-Dindo classification was 28.4% for CPFE group, 24.3% for E group, and 14.9% for N group (p = 0.004), and respiratory complication was higher for CPFE group (22.1%) than N group (5.8%) and E group (11.8%) (p < 0.001).

Conclusion
Resected lung cancer patients with CPFE had some different clinical characteristics in comparison with those with emphysema. Intensive postoperative management and a strict follow-up are required because of higher rate of postoperative complications and aggressive malignant behavior in CPFE patients.

Background
Non-small cell lung cancer (NSCLC) with invasion of mediastinal structures is now classified as stage IIIA or IIIB according to N stages, and has been considered surgically unfavorable. However, in a selected group of these patients, better results have been reported after surgical resection compared to non-surgical group. The aim of this study is to evaluate the role of surgical resection in treatment of mediastinal T4 NSCLC and risk factors that should be considerated.

Methods
Among 2215 patients who underwent surgical intervention for non-small cell lung cancer from Aug 1987 to Dec 2011 in Korea cancer center hospital, 119 patients had an invasion of T4 mediastinal structures. Their medical records in Data Base were reviewed, and they were followed up completely until Apr 2013. Surgical results and prognostic factors of NSCLC invading mediastinal structures were evaluated retrospectively.

Results
Lung cancer was resected completely in 83 patients (69.7%, 83/119). Lobectomy was performed in 25 patients and pneumonectomy in 58. The mediastinal structures invaded by primary tumor were great vessels (44.6%), heart (15.7%), vagus nerve (12.1%), carina (8.4%), esophagus (9.6%), and vertebral body (9.6%). Nodal status was N0 in 16, N1 in 28, and N2 in 39. Neoadjuvant therapy was executed in 11 (13.25%) and adjuvant therapy was added in 53 (63.9%) out of complete resection group (n=83). Complication rate was 30.1% and operative mortality was 8.4% in complete resection group. Median and 5 year overall survival including operative mortality was 20.1 months and 22.7% in complete resection group (n=83), and 6.1months and 0% in exploration only group (n=36, p=.001). Overall 5year survival rates of N2(-) and N2(+) group were 32.8% and 9.3% respectively (p=.002). There was no survival difference between T4 N2(-) group and other IIIA stage group. Mediastinal structure invaded, old age, gender, neoadjuvant and adjuvant chemotherapy showed no significant prognostic difference.

Conclusion
The operative risk of NSCLC invading mediastinal structures was high because of high rate of pneumonectomy and wider range of resection, however it can be acceptable. Long-term result of complete resection was favorable in selected group. Aggressive surgical approach is recommended in well selected patients with good performance and N2(-) in mediastinal T4 group. Stage grouping of T4N2(-) patients in AJCC 7th edition is thought to be adequate when complete resection was likely.

Background
There have been few reports regarding short term survival after lung cancer surgery in Australia. In this study, we analysed the predictors of survival at one year following lung cancer resection in Queensland, the third most populous state in Australia.

Methods
Data on all Queensland residents who were diagnosed with non-small lung cancer (NSCLC) between 2000 and 2010 and who subsequently underwent surgery for lung cancer was obtained from the Queensland Oncology Repository. One year survival following surgery was modelled using multivariate Cox proportional hazards regression controlling for gender, age, comorbidity, anaesthetic score, remoteness of residence, and socioeconomic status.

Results
A total of 2,799 NSCLC patients who underwent resection for lung cancer in 17 hospitals across the state were included in the analysis; the median age was 67 years and 61% were males. Overall one year survival was 88%. In multivariate modelling, independent predictors of death within one year of surgery included male gender (hazard ratio [HR] 1.4, 95% confidence interval [CI] 1.0-2.0, p = 0.04), age (per 10 year increment, HR 1.2, CI 1.1-1.3, p < 0.001), presence of one or more major comorbidities (HR 1.4, CI 1.1-1.8, p = 0.004), and anaesthetic scores of severe disease or worse (HR 1.40, CI 1.1-1.8, p = 0.01). Remoteness of residence and socioeconomic status were insignificant factors in the model.

Conclusion
Demographic and clinical patient characteristics are significant prognostic factors for short term survival following lung cancer surgery. This study further suggests that remoteness and socioeconomic status do not influence the quality of surgical care for lung cancer in Queensland.

Background
Several small studies have reported that some non-small cell lung cancer (NSCLC) patients with isolated adrenal metastasis can achieve long-term survival through an adrenalectomy followed by a surgical resection for primary lung cancer. However, most studies treated such patients as IV stage and suggested that the survival outcome was poor. The choice of treatment approach for such patients is still controversial.

Methods
A search for publications on surgical treatment for primary lung cancer and isolated adrenal metastasis from NSCLC was performed via the MEDLINE and Siencedirect datebase. Studies reporting on survival outcomes were included. When we analyze data to determine which clinical characteristics predict long-term survival, studies not allowing separation of outcomes between groups were excluded. Synchronous metastasis was defined as an adrenal metastasis which occurred before or within 6 months after the primary lung cancer.

Results
There were 11 publications contributing 87 patients (age from 35 to 78 years, 82.1% men and 17.9% women). Median overall survival was 16 months. The 1-year survival, 2-year survival and 5-year survival was 64.0%, 37.7% and 29.2% respectively. Median overall survival (OS) for patients with synchronous metastasis was shorter than those with metachronous metastasis (12 months vs 31 months, P = .017). However, the difference of media overall survival between patients whose pathology of primary lung cancer was adenocarcinoma and squamous carcinoma was not significant (20 months vs 13 months, p = .068). Whether there was mediastinal lymph node metastasis or not was not a prognostic factor because there was no striking difference in media overall survival (p = .496). The patients with ipsilateral adrenal metastasis had the same median overall survival as those with contralateral tumors.

Conclusion
For an isolated adrenal metastasis from NSCLC, part of the patients undergoing surgical treatment for both primary lung cancer and isolated adrenal metastasis have a long survival. Patients with metachronous metastasis predict a better survival.

Background
A body of literature exists describing the evolution of BAC from a subtype of adenocarcinoma of the lung to the currently proposed classification where it is further categorized as adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or lepidic predominant adenocarcinoma (LPA) based on the size of the invasive component of the lesion. The majority of these studies, however, were conducted with Asian populations and very few non-Asian studies on BAC have been published. Our aim was to describe the characteristics of North American patients with BAC, review the management and determine the influence of the epidemiologic difference.

Methods
We retrospectively reviewed all patients with a diagnosis of BAC or adenocarcinoma with BAC features on pathology from February 2000 to June 2012. Patients were categorized according to the IASLC/ATS/ESR classification into those with AIS, MIA or LPA based on the dominant lesion resected. Patients with mucinous BAC were excluded (n=7).

Results
One hundred and forty four patients were evaluated: AIS (23), MIA (18) and LPA (103). Patient demographics were similar between the groups with over 75% being of non-Asian ethnicity. More patients with AIS and MIA were clinical stage IA (table). Lobectomy was performed at comparable frequencies for AIS (48%) and MIA (53%), though it was the predominant resection approach for LPA (70%). The median size of the resected lesion in patients with AIS (1.5cm) and MIA (1.3cm) was significantly smaller than those with LPA (2.5cm), p<0.001. Patients with AIS and MIA had no clinical or pathological nodal involvement, whereas 12% of patients with LPA were found to have positive nodes (pN1: 6%, pN2: 6%). At a median follow-up of 30 months, recurrence rates were – local: AIS 4%, MIA 11% and LPA 2%; regional: LPA 8%; and distant: LPA 10%. Disease-free survival was significantly higher in the AIS (96%) and MIA (89%) groups versus the LPA group (80%). Five year cancer-specific survival was 100% for patients with AIS and MIA dropping to 84% for patients with LPA.

Comparative characteristics between AIS, MIA and LPA

	AIS (N=23)	MIA (N=18)	LPA (N=103)
Age (median)	68	68	69
Female	19 (83%)	15 (83%)	77 (75%)
Non-Asian	19 (83%)	14 (78%)	92 (89%)
Smoker	16 (70%)	13 (72%)	81 (79%)
# Comorbidities (median)	1	1	1
FEV1% (median)	91	89	86
DLCO/VA% (median)	94 [a]	104 [a]	82
Clinical Stage
IA	19 (83%)	16 (89%)	70 (68%)
IB	4 (17%)	2 (11%)	29 (28%)
IIA	0	0	3 (3%)
IIB	0	0	1 (1%)
Pathologic Stage
IA	23 (100%)	18 (100%)	53 (51%)
IB	0	0	31 (30%)
IIA	0	0	7 (7%)
IIB	0	0	4 (4%)
IIIA	0	0	8 (8%)

[a]p < 0.05 vs. LPA

Conclusion
Patients with AIS and MIA have favorable outcomes reflected by the absence of nodal metastases and a 100% 5 year cancer-specific survival compared to patients with LPA. The results of this North American population are consistent with those of published reports based on Asian populations.

Background
This study was performed to assess early and late outcomes of patho-logic N2 disease unexpectedly detected in patients with non-small cell lung cancer undergoing video-assisted thoracic surgery (VATS) lobectomy for clinical stage I.

Methods
We retrospectively reviewed the clinical features of patients with unex-pected N2 non-small cell lung cancer and their early and late outcomes in the VATS lobectomy group versus the open thoracotomy lobectomy group.

Results
The overall survival time for all 358 patients was 33.26±0.90 months. The overall survival time for 117 cases in the VATS lobectomy group was 36.02±1.44 months. The overall survival time for 241 cases in the open thoracotomy lobectomy group was 31.92±1.14 months. The survival rates for patients in the VATS lobec-tomy group were 92.31%, 36.75%, 5.13% at one, three, and five years, respectively. The survival rates for patients in the open thoracotomy lobectomy group were 92.12%, 21.58%, 2.49% at one, three, and five years, respectively. A significant differ-ence was found between the two groups regarding this factor (X[2] =3.88 ,P = 0.049).

Conclusion
VATS lobectomy is feasible and safe to perform on patients with minimal N2 non-small cell lung cancer. Even if lymph node metastasis is unexpect-edly detected during surgery, with rigor ous preoperative evaluation and systematic lymph node dissection, there is no need to convert to open thoracotomy lobectomy.

Background
The role of surgery in the treatment of non-small-cell lung cancer　(NSCLC) with clinically manifested mediastinal lymph node metastasis is controversial. But, the removal of the whole regional lymphatic system together with primary tumor is one of the fundamental rules in oncological surgery.　Systemic nodal dissection（SND） has been accepted by the IASLC to be an important component of intrathoracic staging. Studies addressing the survival benefit of mediastinal lymph node dissection have been inconclusive.　 According to the study of regional lymphatic drainage, we considered reasonable lymphadenectomy contributes the post-operative survival of the patient with NSCLC. And we had devised Systemic extended bilateral mediastinal dissectionand lung resection through a median sternotomy (ND3 operation (Hata’s method)), and reported that ND3 operation can allow for complete dissection of all stations of mediastinal lymph nodes.

Methods
This is a retrospective study of 28１ patients [198 male and 83 female, mean ages 59.5 years (range, 38-75)], underwent ND3 operation due to Left NSCLC, from January 1990 till December 2012. The patients with Lt. NSCLC who are estimated to be able to conventional radical operation and aged 75 year-old or less becomes the adaptation of our ND3 operation. All patients had assessed pathologically stageI-IIIB, underwent R0 operation. The operation were performed through the median sternotomy. As N3αoperation, bilateral mediastinal nodal dissection including bilateral thoracic inlet and lung resection is performed. Lymph node station #1,#2R,#4R,#2L,#4L,#3a,#5,#6,#7,(#8,#9:Left lower NSCLC) were removed.

Results
Postoperative survival rates calculated with Kaplan-Meier method. The clinicopathological data of patients and surgeical outcomes were evaluated. The overall 5-year survival rate in the 28１ patients of left lung primary was 64.1%. Operative mortality in 28１ patients was 3.2%(２００１-２０１２：1.3%). Lymph node metastasis to the mediastinum was confirmed in 89 (31.6%) patients. (pN2 was 50 patients,pN3α was 26 patients, pN3β was 2 patients, pN3γ was 11 patients) Five-year survival rate was 54.5% in cN2(n=47) caces, 49.1% in pN2 cases(n=50), 44.1% in cN3α(n=18), 40.2% in pN3α(n=26), and 53.3% in p-stageIIIA・pN2 (n=46). In these stageIIIA・pN2 caces, no significant difference in survival were found regarding age, sex, tumor location, histologic type, tumor size, clinical N stage (accidental N2 or not), the number of metastatic nodal station (single-station N2 or multi-station N2). There was significant difference in survival regarding recurrence after surgery. ( Five-year survival rate was 42.2% in recurrence cases (n=28), 68.6% in no recurrence cases(n=18)) The patients with N2 disease who have no distant micrometasis can obtain oncological benefit from our ND3 operation.　

Conclusion
N2 and N3α Lt.NSCLC. And better local tumor control than conventional lung operation after complete resection for N2 and N3α disease without leading to increase morbidity. It is important to perform curative operation with complete　dissection of all station of mediastinal lymph nodes.

Background
The aim of the study was efficacy of preoperative pulmonary rehabilitation in patients with thoracic tumors and borderline abnormal spirometry results

Methods
Clinical inclusion criteria were: diagnosis of thoracic tumors and reduced values of FEV1 and FVC predicting postoperative complications. We observed 15 patients (8 women, 7 men) in mean age of 63 (range 46-82) years. Spirometry, six minute walking test distance (6MWT) and maximum metabolic equivalent during exercise on treadmill (MET) were chosen for evaluation of lung function and physical performance during rehabilitation. The tests were performed twice during screening phase to eliminate the factors of learning, and were repeated after first and second week of rehabilitation. Pulmonary rehabilitation included two weeks of training on the treadmill with individually selected speed, physiotherapy exercises and breathing training with using of Triflo

Results
The significant differences were observed during pulmonary rehabilitation: 1). FEV 1 (L): 1,35 ± 0,24 vs 1,58 ± 0,29 (p=0,002) 2). FEV1 (%N): 54,5 ± 13,6 vs 65,5 ± 14,3 (p=0,001) 3). FVC (L): 2,33 ± 0,44 vs 2,76 ± 0,62 (p=0,02) 4). FVC (%N): 75,2 ± 15,5 vs 90,6 ± 12,0 (p=0,006) 5). 6MWT Distance (m): 302 ± 127 vs 359 ± 121 (p=0,0005) 6). MET: 3,20 ± 1,91 vs 3,85 ± 2,27 (p=0,002) Although improvement during rehabilitation, 4 patients were disqualified from lobectomy due to unsatisfactory performance status. Another 3 patients experienced complications during perioperative period. Analysis of pulmonary rehabilitation efficacy showed that 8 patients with favourite outcome, without surgical and cardiovascular complications after thoracic surgery in comparing to others, were characterised by significantly higher improvement in: 1). FEV1 (L): 0,37 vs 0,08 (p=0,02) 2). distance in 6MWT (m): 85 vs 25 (p=0,01)

Conclusion
The increases of FEV1 and 6MWT have important positive predictive value for favourite outcome after thoracic surgery

Background
Routinely performed blood tests may yield important information regarding the risks of post-operative morbidity and survival. Whilst the association between systemic pre-operative inflammatory response and survival in NSCLC chemotherapy patients is recognized, the clinico-pathological correlates in NSCLC surgical patients are less clear.

Methods
NSCLC patients undergoing surgery between 29/8/2007 and 30/3/11 were included. Preoperative blood tests were retrieved from laboratory databases and correlated with prospectively collected data held in our surgical database including clinico-pathological factors, pathological TNM stage and survival. Survival analysis was performed on 17/06/13.

Results
722 patients underwent surgery for suspected NSCLC. In 563 (78.0%) patients (54.2% males, median age 68.5 (range 37.8 - 90.8) years), complete data for all factors enabled subsequent multivariate analysis. At the time of analysis, 377 (60%) were alive and were censored in survival analyses. In univariate analysis, the following factors were identified as poor prognostic factors; serum fibrinogen >4g/dL (p=0.011), haemoglobin <13.1g/dL (p=0.003), platelet count >370x10[9 ]or<140x10[9 ](p=0.006), ALT >63 IU/L or <17 IU/L (p=0.039), total protein >80g/L or <60g/L (p<0.001), albumin >48g/L or <35g/L (p=0.005), globulin >36g/L or <18g/L (p=0.001), cholesterol <5mmol/L (p=0.011). Other factors identified as poor prognostic factors were, age (p<0.001), male gender (p=0.033), nodal stage (p=0.001), tumour size (p=0.001), completeness of resection p=0.025), and histological grade (p=0.008). In multivariate analysis of the factors identified from the blood tests, total protein (HR 2.263 95% CI 1.357-3.775, p=0.002), globulin (HR 1.507 95% CI 1.015-2.238 p=0.042), and haemoglobin (HR 1.462 95% CI 1.091-1.958 p=0.011) Including stage, age and gender in the model, stage (HR 1.286 95% CI 1.164-1.442 p<0.001), age (HR 1.028 95% CI 1.011-1.046 p=0.001), gender (HR 1.419 95% CI 1.048-1.920 p=0.024), total protein (HR 2.503 95% CI 1.465-4.274 p=0.001) and haemoglobin (HR 1.500 95% CI 1.110-2.026 p=0.008) remained independent prognostic factors.

Conclusion
Although survival data are not yet fully mature, pre-operative anaemia and an abnormal serum total protein level are adverse prognostic factors for survival following lung cancer surgery, being independent of other variables including stage, age and gender. Further work is required to determine the clinical implications of these findings.

Background
Carcinoid tumours have been reported in a wide range of organs, but they most commonly involve lungs and gastrointestinal tract. Pulmonary or bronchial carcinoid tumours account for over 25% of all carcinoid tumours and for 2% of all pulmonary neoplasms. Approximately 10-20% of pulmonary carcinoids are atypical, the remaining 80-90% are typical. It was generally accepted that carcinoid tumours were very slow-growing and benign neoplasm with no potential for invasiveness and no tendency to develop metastases.

Methods
This study includes 60 consecutive carcinoid patients referred to the Department of Thoracic Surgery, for surgical treatment, between 1989 to 2011. The study includes all patients treated at the unit during the study period. The inclusion criteria was a histopathologically verified carcinoid tumor. Tumor specimens were obteined at operation.

Results
According to the histological findings, 47 patients (22 male and 25 female patients) had a typical carcinoid tumor, and 13 patients (8 male and 5 female patients) had an atypical carcinoid tumor. All patients were treated with curative surgery: 50 patients had not progressed, 4 had recurrence and in 6 the follow-up was lost. Among patients with typical carcinoid tumor, 37.2% smoked >10 pack-years at the time of diagnosis, while in atypical tumors 69.2% were heavy smokers. The majority of patients in our series presented evidence of bronchial presentation (87.5%), obstructive pneumonitis (78.8%), pleuritic pain (70.7%), pulmonary atelectasis (75.0%), and dyspnea (56.1%). This was followed by cough (75.9%), hemoptysis (41.8%), and a variety of other symptoms/signs, including weakness, nausea, weight loss, night sweats, and neuralgia. The lesions ranged in size from 0.4 to 7 cm, with 35% of the neoplasm having a maximum dimension >3.0 cm. Histological examination of samples showed oncocytic (4 cases), papillary (4 cases) and mixed trabecular/insular/organoid (52 cases) patterns. The growth pattern of carcinoid samples was polypoid (23 cases), nodular (16 cases), hourglass (11cases), stenotic (3 cases), and lobular (1 case).

Conclusion
The goal of this work is not so much to recapitulate lung carcinoids tumors classification but rather to provide an understanding of their clinico-pathological profiles. Although incidence of newly diagnosed patients with carcinoid tumors of the lung is low, the long survival for those with low and intermediate differentiation grade, and the deeper knowledge we now have on molecular processes that governs tumors growth make these tumors a challenging field in Oncology.

Background
Video-assisted thoracoscopic lobectomy plus mediastinal lymph node dissection has been regularly performed for the treatment of patients with non-small cell lung cancer (NSCLC) in high-volume hospitals, owing to its advantage of less trauma and faster recovery. However, it is questioned whether it could be popularized more widely with worry about the technically difficulty and perioperative management. Up to date, no study has discussed the technical and clinical differences based on the number of this minimally invasive surgery performed per year. This study aimed to compare the clinical outcomes of video-assisted thoracoscopic lobectomy plus mediastinal lymph node dissection between a low-volume center (LVC) and a high-volume center (HVC).

Methods
This prospective study was conducted from January 2012 to December 2012 in a LVC and a HVC in the same city. Clinical features and operation characteristics of all patients were collected and compared to determine the differences between the 2 groups.

Results
A total of 511 cases with NSCLC were enrolled in this study. And 469 cases (91.8%) were performed in HVC, while 42 cases（8.2%）in LVC. There was no significant difference found between the two groups in age, gender, body mass index, ASA score, tumor location, histological type and clinical stage. LVC group has longer operation time (131.7±32.4min vs 89.2±39.4min, p=0.000) and more blood loss (125.7±97.1ml vs 82.7±52.6ml, p=0.000), compared with HVC group. However, the other clinical outcomes between LVC and HVC were similar, including the rate of conversion to thoracotomy (4.8% vs 1.9%, p=0.226), number of lymph nodes harvested (12.9±3.7vs 14.1±6.3, p=0.484), postoperative hospital stay (7.6±3.9d vs 6.8±2.7d, p=0.224), total complications (16.7% vs 12.8% , p=0.476) and 30-day mortality (0.0% vs 0.2% , p=1.000).

Conclusion
The study shows that video-assisted thoracoscopic lobectomy plus mediastinal lymph node dissection could also be performed feasibly and safely in LVC with similar clinical outcome, though the long term survival are still necessary to be confirmed with follow up.

Background
Video-assisted thoracoscopic surgery (VATS) lobectomy plus mediastinal lymph node dissection is an innovative technique shown to be minimally invasive and oncologically adequate for the treatment of non-small cell lung cancer (NSCLC), though it is technically difficulty. This study aimed to describe the learning curve for this minimally invasive surgery with guidance by consultant surgeon.

Methods
From September 2011 to March 2013, a total of 46 patients with NSCLC underwent VATS lobectomy plus mediastinal lymph node dissection in our low-volume center. The procedures were guided by experienced consultant surgeons. The patients were divided into three groups. Group A included the first 15 cases. Group B comprised cases No. 16 to 30, and group C included the final 16 cases. The demographic characteristics and the intra- and postoperative variables were collected retrospectively and analyzed.

Results
There was no significant difference found among the three groups in age, gender, body mass index, ASA score, tumor location, histological type and clinical stage. No postoperative death occurred. Two patients required conversion (1 in Group A, 1 in Group B). Compared with group A, a significant decrease in intrathoracic operative time (132±30 vs 185±29 min; P = 0.000), blood loss (128±64 vs 209±117ml; P =0.003), but more retrieved nodes (12.2±3.1 vs 9.3±2.5; P =0.014) was observed in group B, while the postoperative hospital stay was similar (9.9±3.3 vs 11.8±7.0 days; P =0.572). And compared with group B, the last 16 patients (group C) involved significantly less intrathoracic operative time (119±20 vs 132±30 min; P =0.091), less blood loss (92±43 vs 128±6ml; P =0.021), more retrieved nodes (14.3±3.4 vs 12.2±3.1; p=0.040) as well as a shorter postoperative hospital stay (6.8±2.5 vs 9.9±3.3 days; P =0.003). A decline in the overall morbidity from group A to group C (46.7%, 33.3%, 12.5, P = 0.098) was also observed.

Conclusion
This study suggests that at least 30 cases were needed to reach the plateau of VATS lobectomy plus mediastinal lymph node dissection for NSCLC. The guidance of experiened consultant surgeons might be meaningful to reduce the learning curve.

Background
To investigate the correlation of intra-fraction displacement of the mediastinal metastatic lymph nodes with displacement of primary tumor and rates of volume change of lung and heart based on four-dimensional computed tomography (4DCT) for non-small cell lung cancer (NSCLC).

Methods
Twenty-six patients diagnosed as NSCLC with fifty-one mediastinal metastatic lymph nodes were scanned by 4DCT simulation during free breathing. The left group (1L, 2L, 4L, 5 region) and right group (1R, 2R, 4R region) and under group (7, 9 region) mediastinal metastatic lymph nodes and primary tumor, heart, bilateral lungs were contoured on CT images of 10 phases separately. Then the displacement of the lymph nodes and primary tumor at left-right(LR), anterior-posterior(AP) and superior-inferior(SI) direction were measured. And the rates of volume change of lung and heart were acquired on the whole respiratory cycle. Comparison and correlation analysis were performed between displacement of the lymph nodes and the primary tumor as well as the correlation of the displacement with the rates of volume change of lung and heart.

Results
In LR and AP direction, the correlation was significant between the displacement of the left group of lymph nodes and the ipsolateral primary tumor (r=0.965, 0.897; P=0.008, 0.015). It was significant correlation of displacement of the left group of lymph nodes in LR and the right group in AP with rates of volume change of ipsolateral lung (r=0.609, 0.645; P=0.035, 0.024). There were significant correlations between displacement of the left group lymph nodes and rates of volume change of heart in LR, AP and SI directions (r=0.614, 0.897, 0.607; P=0.044, 0.015, 0.037).

Conclusion
There was no difference between the displacements of primary lung tumor and the lymph nodes in three directions during free breathing. The displacement of left primary lung tumor was able to predict displacement of left mediastinal lymph nodes of trachea in the LR and AP directions. Meanwhile, it was reasonable to predict three-dimensional lymph node motion by monitoring the rates of volume change of ipsolateral lung and heart for the mediastinal lymph nodes located in 1L, 2L, 4Land 5 regions.

Background
To determine whether the quality of radiation dose distribution is associated with the tumor response of advanced non-small cell lung cancer (NSCLC) undergoing concurrent chemoradiotherapy (CCRT).

Methods
Thirty one patients with stage IIIA/IIIB NSCLC underwent CCRT with a median dose of 63 Gy (range 40-66 Gy). The chemotherapy combined taxene and alkylating agents. On our actual plans, we drew the planning target volume (PTV) including the electric nodes and tried to make a plan to cover the PTV with at least 95% of prescribed dose. The following CT simulation was done when a cumulative dose was about 36 Gy and the cone-down was undergone at about 40 Gy only including the primary tumor and the bulky nodes. For this retrospective study, each PTV of primary tumor (n=30) and lymph nodes (n=36) was separately re-defined with even margins from gross target volume (GTV) to reduce the variation of target delineation, and the actual plan overlaid the re-defined PTV. For the measurement of tumor response rate during CCRT (RR(mid)) and after CCRT (RR(end)), the GTV on initial simulation was compared with the GTV on following CT simulation and following CT scan after 2 months from end of CCRT, respectively. The relations between the RR(mid), RR(end), dose distribution parameters (D~95~, V~95~, mean tumor dose (MTD) and homogeneity index (HI)), total dose and tumor volume were evaluated by bivariate correlation analysis.

Results
Median overall survival was 15.5 months and 2-year survival 42.3%. For primary tumors and lymph nodes, the dose distribution parameters were favorable (Table). For primary tumors, the relation between dose distribution parameters and tumor response was not significant. The RR(end) was correlated with the GTV on following CT simulation (γ=0.627, p<0.001) and the RR(mid) (γ=0.541, p=0.003). For lymph nodes, the RR(mid) was correlated with the mean tumor dose (γ=0.356, p=0.033).

Mean/SD (%)	D~95~	V~95~	MTD	HI
Tumor Node	97.4/2.5 96.6/6.7	99.7/5.4 100.0/23.5	100.3/1.0 98.6/3.1	2.9/1.4 2.2/1.7

Conclusion
The quality of radiation dose distribution minimally affected the tumor response. Based on the marked association between the RR(mid) and RR(end), further studies of tumor intrinsic factors related to radiation sensitivity will be rewarding.

Background
Australia is one of the most urbanized countries in the world. Patients with cancer in regional Australia have poorer access to oncology services, resulting in lower survival rates for lung cancer[1]. Implementation of SABR lung is focused in metropolitan centres, limiting access for regional patients. The development of new regional radiotherapy centres could improve this, but these centres require support to implement this complex technology. A tripartite collaboration consisting of radiation oncologists, physicists and radiation therapists was formed to enable implementation of SABR.

Methods
The collaboration includes the following 11 radiation oncology departments: Regional hospitals: North Coast Cancer Institute ( Lismore, Port Macquarie and Coff’s Harbour), Newcastle Calvary Mater Hospital, Central Coast Cancer Care Centre ( Gosford) Sydney Metropolitan Hospitals: Westmead and Nepean Hospitals, St George Hospital, Royal North Shore Hospital, Royal Prince Alfred Hospital, Prince of Wales Hospital. The goal was to support centres starting an SABR programme, and facilitate ongoing assessment of outcomes. Multidisciplinary working groups consisting of radiation oncologists, radiation therapists and physicists were formed to cover: Clinical protocol development: existing protocols, including the Dutch (ROSEL), Leeds, RTOG and TROG Chisel protocols were reviewed. Ethically approved SABR protocols for Stage I NSCLC, pulmonary and vertebral metastases were developed with assistance from international and local experts. Planning protocols: development of prescription pages and IMRT checklists for treatment. Physics quality assurance: specification documents for equipment, quality assurance and image verification procedures are being developed. Data collection: a database to archive clinical data and all radiotherapy planning and diagnostic imaging was developed. Ongoing support for regional centres includes: email servers for rapid response to questions, video-conferenced clinical and technical audits of SABR cases. Data collection will facilitate quality assurance and future research.

Results
The tripartite approach has led to the uniform adoption of clinical and technical protocols, and facilitated large-scale data pooling of SABR patient information across NSW. Key drivers of success were: the recognition of the need to data pool, identification of key team members to lead the process who had expertise in trial coordination, database development and implementation of radiotherapy technology, and administrative support from involved departments.

Conclusion
By increasing collaboration between metropolitan and regional radiotherapy centres we have successfully facilitated the safe implementation of SABR lung, increasing accessibility for patients in regional Australia. This model could be used as the basis for a national collaboration, and the development of accreditation and credentialing procedures for Australian departments. 1. Vinod, S. K. et al. . Cancer 116, 686–694 (2010).

Background
Stereotactic ablative body radiotherapy is being implemented in Australia on a wider scale. Significant differences exist between protocols, particularly in the constraint of dose wash. To achieve a high level of dose conformality large numbers of beams, and non-coplanar techniques may be used, resulting in long treatment times. We report on a study comparing volumetric modulated arc therapy (VMAT) to coplanar (CP) and non-coplanar (NCP) conformal radiotherapy (3D-CRT) and intensity modulated radiation therapy (IMRT), and assess whether plans meet RTOG 0915 and TROG CHISEL criteria.

Methods
Eight Pinnacle VMAT, and CP and NCP 3D-CRT and IMRT plans for delivery on Elekta Linacs were assessed for: target coverage (coverage with the prescribed dose), dose conformality (100% and 50% conformity indices, D2cm and high dose outside the PTV) and V20 (volume of lung receiving 20 Gy). Plans aimed to deliver the prescription dose to at least 98% of the volume, with a V20 ≤ 8% . All VMAT plans were single arc with 4° spacing, and all IMRT and 3D-CRT plans used identical beam angles with 10-12 beams. Treatment time was measured by delivering the plans to a phantom. All patients had peripheral tumours and received 48 Gy in 4 fractions.

Results
VMAT achieved equivalent radiotherapy target coverage to 3D-CRT and IMRT (>98.2%). However, 3D-CRT plans had a large volume of surrounding normal tissue receiving a high dose (24.0% and 21.3% of the PTV volume for CP and NCP ) when compared to VMAT and IMRT plans (3.9% and 4-5% of PTV volume). 100% conformity indices were lowest for VMAT and NCP IMRT (1.1 for both) and highest for 3D-CRT (1.5 and 1.4 for CP and NCP). D2cm was also lowest for VMAT and NCP IMRT (26.1 Gy and 25.3 Gy) and highest for 3D-CRT plans (29.4 Gy and 28.7 Gy for CP and NCP). V20 was between 4.4 and 6.1% for all plans. VMAT plans were three times as fast to deliver as NCP IMRT (6min and 6s vs. 19 min 34s) and twice as fast as NCP 3D-CRT plans (12min 35s) . TROG CHISEL criteria: one CP-3D-CRT plan had a major violation on spinal cord. There were no other violations. RTOG 0915 criteria: no major violations were recorded for VMAT plans. CP IMRT plans had 2 major violations and NCP IMRT had 1 major violation. All violations were in the D2cm and 50% CI constraints. Seven CP 3D-CRT had major violations in 50% CI, D2cm and high dose spill, and 4 NCP-3DCRT plans had major violations in high dose spill and 100% CI.

Conclusion
VMAT plans are similar in quality to NCP IMRT, but are faster to deliver due to reduced gantry and couch movements. 3D-CRT plans were unable to deliver equivalent dose conformality, and a larger region of normal tissue was exposed to a high dose when equivalent PTV coverage was achieved. CP 3D-CRT plans do not meet RTOG criteria due to the inability to meet low dose wash constraints, and more complex planning is beneficial in these cases.

Background
Lung tumour motion poses a significant challenge in accurate delivery of radiotherapy. To prevent geographical miss, a generic margin is often added to the CTV to create an internal target volume (ITV). Studies have shown the effectiveness of Active Breathing Coordinator (ABC) (Elekta, Crawley, UK) in reducing this internal margin. For the purpose of this study we wanted to evaluate the feasibility of utilising ABC for our patient population and of defining ITV margin based on breath-hold scans.

Methods
13 patients receiving radical radiotherapy were prospectively recruited. Each patient received a 1 hour training session prior to CT simulation to determine eligibility for the study and provide training for the breath hold procedure. A minimum of fourteen seconds breath-hold was required for patients to be eligible. If eligible, patients were positioned on the CT simulator as per department protocol with the addition of ABC. Standard departmental CT protocol for lung treatment was first performed with contrast. This scan was used to identify the tumour region. A breath-hold scan at normal inspiration and expiration was done in the region of visible tumour volume with the aid of ABC. A radiation oncologist defined ITV based on current departmental protocol and using the ABC scans. To verify accuracy of ABC volume, a 4D Cone Beam CT (4D CBCT) scan was done during week 1 of treatment. To verify the reproducibility of ITV and breath-hold position, a second ABC scan and 4DCBCT were performed mid-way through treatment. The planning ITV and ABC ITV were compared. Variation in tumour position and volume were quantified and compared.

Results
From the 13 patients recruited, only 5 patients were able to tolerate ABC. On average, eligible patients were able to maintain a 20 second breath-hold. The generic ITV margin was larger than the patient specific ABC ITV margin in all cases. A change in centre of volume was noted between simulation ABC GTV and mid treatment ABC GTV. There was an average overall difference of 0.8cm for the 3 dimensional vectors for two eligible patients. There was no correlation between breath hold ability and the patient’s pulmonary function.

Conclusion
ABC was not feasible for the cohort of patients recruited for this study. For the patients who could tolerate ABC scans, the ITV could be individualized and reduced from that based on generic population data.

Background
Magnetic Resonance Imaging (MRI) with its superior soft tissue contrast resolution has the potential to improve Gross Tumour Volume (GTV) delineation of lung cancer. The aim of this study was to assess the inter-observer variability between observers for MRI based GTV delineation for Lung cancer and evaluate whether this factor changed following a GTV delineation workshop with a thoracic radiologist.

Methods
Five radiation oncologists from three different institutions were asked to delineate GTV on 3 patient datasets. Each observer was given a planning CT, PET, T1 and T2 weighted 1.5 T MRI datasets along with patient history and relevant diagnostic test results. Each observer was instructed to delineate a primary GTV and nodal GTV as required on the T1 and T2 weighted MRI datasets (pre workshop contours). A workshop was then conducted. The aim of the workshop was to discuss each case with a thoracic radiologist and for the radiologist to educate each of the observers in how to review thoracic MRI for both T1 and T2 weighted images. Following the workshop each observer delineated a post workshop GTV. Conformity index (CI) was used to evaluate improvement in inter-observer variability between pre and post workshop contours.

Results
Results of two observers are presented here. For patients 1 and 3 slight improvement in CI was noted between pre and post primary and nodal GTV for T1 and T2 weighted datasets. Similarly for patient 3 slight improvements were noted in inter-observer variability for primary GTV for both T1 and T2 weighted images. However there was significant improvement in both T1 and T2 weighted nodal GTV. CI improved from 0.2 to 0.6 and 0-0.6 for T1 and T2 weighted images respectively.

Conclusion
Preliminary results from this study indicate that a radiologist led workshop assisted in improving inter-observer variability for MRI based GTV delineation. Further analysis of all observers is required to assess the significance of the impact of a radiologist led contouring workshop in improving inter-observer variability on MRI delineation of lung cancer.

Background
Although stereotactic body radiotherapy (SBRT) has become a one of the preferred treatment options for patients with stage I non-small cell lung cancer, the patients are not always suitable for SBRT. The purpose of this study was to present the treatment outcomes and prognostic factors for stage I NSCLC treated with SBRT or 3-dimentional conformal radiotherapy (3DCRT).

Methods
The medical records of 77 patients with stage I NSCLC treated in our hospital were retrospectively reviewed. Forty-four patients were treated with SBRT which was delivered a total dose of 48Gy in 4 fractions　for one week. Thirty-three patients were treated with 3DCRT which was delivered a total dose of 60-66Gy in 20-30 fractions. SBRT was done with the real-time tumor-tracking system (RTRT) using 3 to 4 fiducial gold markers. 3DCRT was adapted to the patients who had difficulty of bronchoscopic implantation of fiducial markers, centrally located tumors or low performance status. In dose calculation for the majority of patients, inhomogeneity was corrected by the superposition method. Univariate and multivariate analysis were performed for predictive factors. .

Results
Median follow-up time was 30 months (range, 1 to 94 months). The 3-year local control (LC), disease-free survival (DFS), and overall survival rate (OS) of all patients were 69.2%, 57.1%, and 68.6%, respectively. There was no significant difference between the two groups in 3-year LC (SBRT, 78.6%; 3DCRT, 58.5%; p=0.146) and 3-year OS (SBRT, 66.4%; 3DCRT, 71.1%; p=0.83), but in 3-year DFS SBRT was superior to 3DCRT (SBRT, 66.2%; 3DCRT, 46.3%; p=0.039). Multivariate analysis detected pathological type and patient’s age as significant predictive factors for LC and DFS, respectively. Especially the histologic type of squamous cell carcinoma was detected as an adverse predictive factor for local control. The type of radiotherapy was not detected as a prognostic factor on multivariate analysis. No serious radiation morbidity was observed with either RT method.

Conclusion
Our results suggested that 3DCRT may be a good alternative treatment for patients who are not suitable for SBRT. Well-designed prospective studies investigating the optimal schedule of dose fractionation in early-stage lung cancer are warranted.

Background
To evaluate the prognostic factors associated with overall survival in patients with brain metastasis from non-small cell lung cancer(NSCLC) who received whole-brain radiotherapy (WBRT).

Methods
From September 2002 to December 2010, 257 patients with brain metastasis from NSCLC who received WBRT. Those who had undergone craniotomy or stereotactic radiotherapy before WBRT and those with leptomeningeal metastasis were excluded. The prognostic factors evaluated for overall survival were; gender, neurological deficit, histology, epidermal growth factor receptor (EGFR) mutation status, previous cytotoxic chemotherapy, previous EGFR tyrosine kinase inhibitor (TKI) treatment, RTOG-recursive partitioning analysis (RPA) (age,Karnofsy Performance Scale [KPS], primary tumor control), and diagnosis-specific graded prognostic assessment (DS-GPA)(age, KPS, extra cranial control, number of lesions). All factors with a Pvalue < 0.05 at univariate analysis were entered into a multivariate analysis using Cox regression with confidential interval of 99%.

Results
At the time of analysis, 225 patients (88%) died, 14 patients (5%) were alive, and 18 patients (7%) were lost to follow-up. The median follow-up time was 16.2 months. The median survival time (MST) was 5.6 months and 1-year survival rate was 28.6%. The MST according to gender, neurological deficit, histology, EGFR mutation status, previous chemotherapy, previous EGFR-TKI treatment, RPA class, and DS-GPA were shown in Table 1. In univariate analysis, the significant prognostic factors were; gender (P=0.0002), neurological deficits (P<0.0001), histology (P<0.0001), previous chemotherapy (P=0.0463), EGFR mutation (P=0.0236), RPA class (P<0.0001) and DS-GPA (P=0.0003). In multivariate analysis, RPA class (I and II vs III,KPS>70 vs <70), histology (adenocarcinoma vs non-adenocarcinoma) and previous chemotherapy (none vs present) were found to be significant prognostic factors (Table 2).Figure 1 Figure 2

Conclusion
RPA class I or II (KPS>70), adenocarcinoma and no previous chemotherapy were associated with longer survival. These factors should be taken into account for decision-making in an attempt to find optimal treatment for patients with brain metastasis.

Background
The Vero4DRT is a newly-developed innovative radiotherapy system with two special features. One is a pair of kV x-ray imagers which can monitor a 3-dimensional position of tumor in real time. The other is a gimbaled x-ray head which enables tumor tracking. This study is to evaluate the initial clinical experiences of dynamic tumor tracking irradiation with real-time monitoring for the lung using the Vero4DRT system.

Methods
Eligibility criteria for this study were (1) lung tumor with a diameter of 50 mm or less, (2) respiratory motion of 10 mm or more, (3) performance status of 0–2, and (4) written informed consent. Prior to treatment planning, spherical gold markers were placed around the tumor using a bronchoscope. Gross tumor volume (GTV) for tracking was delineated on a breath-hold CT at end-exhale. Margins for planning target volume (PTV) were determined for each patient considering errors due to the tracking irradiation. Prescription dose was 56 Gy in 4 fractions for T2a lung cancer and metastatic tumor, and 48 Gy in 4 fractions for the others. Dose-volume metrics were compared between the tumor tracking and conventional static irradiation using an in-house developed software. A 6-MV photon beam was delivered to the tumor with the gimbaled x-ray head toward predicted position based on the abdominal wall. During the irradiation, the tumor and the gold markers were monitored with kV imagers and EPID.

Results
The dynamic tumor tracking radiotherapy was successfully performed for 12 patients (10 males and 2 females). A median age was 83 years (range, 60-87 years). Histology was adenocarcinoma in 4 patients, squamous cell carcinoma in 3, non-small cell lung cancer in 1, metastatic tumor in 3, and unconfirmed in 2, respectively. Tumor diameter ranged from 12 to 36 mm (median, 21 mm). Median amplitude of respiratory motion was 16.8 mm (range, 11.3 to 33.5 mm). A mean PTV volume was 42.9 cc for the dynamic tracking, while that was 62.5 cc for the conventional irradiation. The tracking irradiation could reduce normal lung doses by 21.3% in mean. Dose covering 95% volume of GTV was not different between the two irradiation techniques with a mean difference of 0.66%. A mean treatment time per fraction was 37 minutes. The gold markers were well recognized with kV x-ray imagers through the whole treatment fractions. With a median follow-up period of 7.7 months (range, 2.1 – 19.2 months), local tumor was controlled in all patients. One patient experienced grade 2 radiation pneumonitis. No severe toxicity has been observed in any of the patients so far.

Conclusion
Dynamic tumor tracking irradiation with real-time monitoring using the Vero4DRT could reduce normal lung doses without any excess time. Preliminary outcomes were promising.

Background
Lung cancer is the main cause of cancer deaths in the developed world and non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers [1]. Of these with NSCLC, 30-40% have locally advanced or inoperable disease, for which the mainstay of treatment remains thoracic irradiation. Radiation pneumonitis (RP) is one of the major dose limiting toxicities. The aim of this study is to retrospectively analyse the incidence and risk factors for RP in NSCLC patients treated with radiotherapy at Royal Hobart Hospital and to determine suitable dose restriction parameters.

Methods
273 patients with NSCLC who had radiotherapy treatment between 2006 and 2012 were retrospectively reviewed. For each patient, all records were examined for documented evidence of RP using the Common Terminology Criteria for Adverse Events (CTCEA 4.0 [2]) grading criteria at least six months post treatment. In addition, radiation dose, dose per fraction, planning target volume (PTV), total lung volume, mean lung dose, v5, v10, v20 and v30 values were obtained and a comparable biological effective dose (BED) for each treatment was calculated. Previous chemotherapy treatments, smoking status, performance status, use of ACE inhibitors, existing cardiovascular disease, established chronic obstructive pulmonary disease (COPD) and pulmonary function tests were also obtained.

Results
Out of the 273 NSCLC patients treated with radiotherapy at Royal Hobart Hospital, 25 (9%) had stage 1, 15 (5%) had stage 2, 75 (27%) had stage 3 and 129 (47%) had stage 4 NSCLC. 29 (11%) patients had no staging documented. 41 patients (15%) had documented evidence of RP. Of these 41 patients, 24 patients had level 1 RP, 14 patients had level 2, 1 patient had level 3, 1 patient had level 4 and 1 patient had level 5 (death). The mean BED (normalised to 2 Gy per fraction) was 64.9 +/- 21.0 Gy and the mean lung dose was 10.3 +/- 10.50 Gy. Twenty out of the 41 patients had been treated with radical intent and 21 with palliative intent.

Conclusion
In this study, RP has been documented in 15% of patients. More than half of these patients received palliative radiation dose. This could be explained by their advanced disease, poor lung function, and poor performance status. References: 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71–96. 2. Common Terminology Criteria for Adverse Events (CTCEA 4.0), http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm

Background
Esophageal toxicity can be a dose-limiting event in patients with non-small cell lung cancer receiving chemotherapy and radiotherapy necessitating treatment breaks with potential to cause adverse treatment related factors. The objective of this study was to investigate those factors, both clinical and dosimetric, which predict for esophageal toxicity.

Methods
Patients (pts) with non-small cell lung cancer prospectively enrolled into an IRB approved database were retrospectively reviewed. Pts with biopsy-proven non-small cell lung cancer treated with radiotherapy alone, sequential or concurrent chemoradiotherapy had maximal esophageal toxicity scored per CTCAE 4.0 criteria. V5, V10, V20, V30, V40, V50, V60, V70, esophageal hot spot, and dose per fraction were the dosimetric variables and age, sex, race, chemotherapy, and stage were the clinical variables investigated. Data were analyzed using SAS (SAS INC, Cary, NC) Version 9.2 software package. Ordinal maximum reported esophageal toxicity was evaluated using logistic regression. A multivariable regression model was fit using important univariate predictors along with a backwards elimination stepwise regression. Probability of esophageal toxicity as a function of absorbed dose in a partial volume was modeled by the method of Lyman, by converting the dose volume histograms into an equivalent fractional volume receiving the maximum dose in the DVH, using the effective volume method of Kutcher and Burman. The parameters in this model (D50, slope m and volume exponent n) were determined by maximum likelihood estimation.

Results
A total of 100 pts were enrolled between 7/10 and 12/12 into a prospective database and eligible for analysis. Pts were excluded without a complete dose volume histogram data or were stage I disease leaving 71 eligible for analysis, 43 females and 28 males with a median age of 61 (range: 39-85). 14 pts were treated with radiotherapy alone while 23 received sequential treatment and 34 concurrent treatment. The median delivered dose was 66.6 Gy (range: 27.5-66.6) in a median of 1.8 Gy (range: 1.8-3.0) per fraction. Maximal esophageal toxicity was rated as 0: 12pts, 1: 21 pts, 2: 33 pts, and 3: 5pts. Univariate predictors of > grade 2 esophageal toxicity included, V5-V60 and use of concurrent chemotherapy. The maximum likelihood fit of the Lyman model parameters to patients with ≥ 2 esophageal symptoms were n=0.26 m=0.32, TD50=39.1 Gy when the α/β ratio was assumed to be 10 Gy. The maximal likelihood fit of the Lyman model parameters to patients when the α/β ratio was not set were n=0.26, m=0.32 and TD50 39.3 with the α/β calculated at 7.6 Gy. Patients not having chemotherapy had a higher TD50, 46.4 Gy as compared to patients having chemotherapy, TD50=37.1 Gy, p=0.09.

Conclusion
We have shown the TD50 for > grade 2 esophageal toxicity is lower for patients receiving chemotherapy and radiotherapy compared to patients receiving radiotherapy alone. This is first report to show the α/β ratio for esophageal toxicity may be lower than 10. Confirmations of these data are needed in an independent data set.

Background
Radiation induced oesophagitis (RIO) is frequently associated with high dose thoracic radiation therapy (RT). Although RIO is uncommonly life threatening, it is a distressing toxicity associated with pain, decreased oral intake and can significantly impact on patient’s quality of life. The aim of this retrospective analysis was to assess the rates of acute and late RIO and investigate the association of RIO with radiation dosimetrics and neutropenia.

Methods
Criteria for inclusion of patient data included a pathological confirmation of non-small cell lung cancer (NSCLC), treatment with concurrent chemotherapy and radical or high dose palliative RT at our centre between 03/04 and 08/07. Exclusion criteria included previous thoracic RT, RT alone, treatment breaks of > five days, inconsistent radiation dose per fraction and hyper-fractionated RT. Acute and late RIO and neutropenia were scored using the Common Toxicity Criteria for Adverse Events (CTCAE v3.0) criteria. Using Focal (Computerized Medical Systems CMS, St Louis, MO, USA), the outer muscular border of the oesophagus was delineated from the cricoid (superior border) to the gastro-oesophageal junction (inferior border) on CT derived images, using pre-defined soft-tissue window/level settings. Dosimetric data was derived from Xio (CMS) plans (three-dimensional conformal RT (3DCRT) with 6MV photons), including the oesophageal length and volume, maximum and mean doses, percentage of oesophagus receiving 20 to 60 Gy (in 5 Gy increments) and percentage length of oesophagus (whole and partial circumference) receiving 20 to 60 Gy (10 Gy increments). Assessment of potential prognostic factors with respect to acute oesophagitis was done using Wilcoxon rank sum test and Spearman’s correlation. Acute oesophagitis and acute neutropenia reaction were dichotomised as grade 0+1 vs. grade 2+3+4. The association of acute oesophagitis with acute neutropenia was examined using Barnard’s test.

Results
The data of 54 patients were eligible for inclusion in this trial. 48 (89%) patients had acute RIO of at least grade 1 (95% CI [78% to 95%]) and five patients (9%) had late RIO of at least grade 1 (95% CI [4% to 20%]). There was a statistically significant correlation between the grade of acute RIO, oesophagus V20 (r=0.303, p=0.026) and length oesophagus receiving 20Gy (whole circumference) (r=0.319, p=0.019). The mean (SD) maximum dose to the oesophagus was 50.2 Gy (18) (r=0.143, p=0.302) and the mean (SD) mean oesophageal dose was 20.8 Gy (10.8) (r=0.269, p=0.049). The maximum grade of acute oesophagitis was significantly associated with acute neutropenia (p=0.035).

Conclusion
Acute neutropenia, mean oesophageal dose and the volume and length of oesophagus receiving low radiation doses were significantly associated with acute RIO in our patient cohort. No association was demonstrated between RIO and maximum radiation dose.

Background
To evaluate the feasibility of stereotactic ablative radiotherapy (SABR) for Non-Small Cell Lung Cancer (NSCLC), especially when tumor tracking system is used.

Methods
From August 2010 to March 2013, 66 patients (44 male, 22 female, mean age 70, range 55-86 years) with node-negative NSCLC were treated. CyberKnife ver. 8.5 and Multiplan ver. 3.2 were used. Mostly, 60 Gy in 3-5 fraction was applied for GTV (CT lung window –W2000 L700) + 3 mm margin, in case of tumor dimension smaller than 1 cm, 30-33 Gy in one fraction was delivered. XSight Lung (XLT) for real-time tumor tracking or Xsight Spine (XST) for internal target volume (ITV) were used. Volume of GTV, treatment time, toxicity, 1-year year local control (LC), free survival (PFS) and 2-year overal survival (OS) were analyzed.

Results
Of the 66 patients, XLT and XST were used in 83% and 17%, respectively. Median tumor volume was 18 ml (range 2-137 ml). Mean treatment time of one fraction was 56 minutes (range 20-90 minutes). Acute toxicity was mild with no need for therapeutic intervention. We have noticed only radiological signs of late pneumonitis and/or fibrosis with no clinical manifestation. One year LC, PFS and OS were 95%, 85% and 92%, respectively. Two years OS was 75%. Figure 1 Fig. 1. Overall survival Figure 2 Fig. 2. Dose distribution, tumoricidal 60Gy isodose (orange), significant lung tissue sparing - 7Gy isodose (dark blue)

Conclusion
CyberKnife´s SABR of NSCLC is feasible and our image-guidance protocol allows to use high number of online tumor tracking to spare as much lung tissue as possible. This results in excellent overall survival and minimal toxicity in patients that were not candidates for surgery. Longer follow-up is necessary for mature data.

Methods
The cost-effectiveness analysis was performed using a previously developed decision model that simulates the disease progression of individual lung cancer patients until they are deceased or have reached a pre-specified time-horizon of 3 years. Simulated patients move from the start of radiotherapy treatment to the absorbing state of death, potentially visiting the intermediate health states ‘local recurrence’ and ‘metastasis’. Transition rates in the model were estimated by multi-state statistical modelling and include the impact of patient and tumour features on disease progression. Data for model quantification was available for 200 NSCLC patients with inoperable stage I-IIIB, provided by the Maastro Clinic. Resource use estimates, costs and utilities were obtained from the data of the Maastro Clinic, the literature and Dutch guidelines. Primary outcomes were the difference in life years, quality adjusted life years and costs and the incremental cost-effectiveness and cost-utility ratio (ICER and ICUR) of PET-CT versus CT based radiotherapy planning. Model outcomes were obtained from averaging the outcome for 50 000 simulated patients. A probabilistic sensitivity analysis was done as well as a number of scenario analyses.

Results
The incremental costs of PET-CT based planning were €581 (95% CI: €-4474 – €6064) for 0,42 incremental life years (95% CI: 0,20 – 0,62) and 0,33 quality adjusted life years gained (95% CI: 0,16 – 0,54) (figure 1). The base-case scenario resulted in an ICER of €1370 per life year gained and an ICUR of €1761 per quality adjusted life year gained. The probabilistic analysis gave a 35% probability that PET-CT based planning improves health outcomes at reduced costs and a 65% probability that PET-CT based planning is more effective at slightly higher costs.Figure 1 Figure 1. Results of probabilistic sensitivity analyses showing incremental costs and incremental life years for PET-CT-based radiotherapy treatment planning compared to CT-based radiotherapy treatment planning.

Background
To evaluate and compare outcomes of two fractionation scheme of thoracic radiotherapy(once-daily vs twice daily), concurrently with cisplatin and etoposide(EP) in limited disease small cell lung cancer(LD-SCLC).

Methods
LD-SCLC patients treated with definitive chemoradiotherapy at Seoul St. Mary’s Hospital were reviewed. Total 51 patients received radiotherapy concurrently with EP, after initially starting 1~2 cycle of EP. Once-daily group (33 patients) irradiated total 50~56 Gy in 28~32 fractions, and twice-daily group (18 patients) irradiated 45 Gy in 30 fractions. Subsquently, additional 1~2 cycles of EP was given. After CRT, 38 patients who get remission in thorax, received prophylactic cranial irradiation(PCI). PCI consisted of total 24~30 Gy over a 2-weeks period.

Results
Median follow-up duration is 47.5 months. 2-year overall survival(OS) rate of all patient was 61.2 % (median 27.4 months). 2-year OS rate was higher (83.9%) in once-daily group, compared with twice-daily group (53.1%), but there were no statistically significant difference (p=0.071). After CRT, 47 patients (93%) had partial to complete remission, but 30 patients were eventually progressed or recurred. Main failure pattern was distant failure (46.7%). The trend of higher loco-regional failure (50%) was detected in twice-daily group compared with once-daily group (30%), but there was no statistically difference between two groups (p=0.055). 7 brain metastasis were detected in once-daily group and 2 in twice-daily group, but there was no statistically difference between two groups (p=0.184). In subgroup analysis of 38 PCI patients, brain metastasis rate were not statistically differ from two groups (once-daily; p=0.171, twice-daily; p=0.146). There were also no significant differences of 2-year progression free survival(PFS), loco-regional disease free survival(LRDFS) and distant metastasis free survival(DMFS) between groups(PFS; p=0.541, LRDFS; p=0.238, DMFS; p=0.792), and no statistically differences in acute toxicities(non-hematologic p=0.128, hematologic p=0.277).

Conclusion
Our results showed no statistically significant differences between two groups, although showing relatively higher OS rate and lower brain metastasis in twice-daily group favoring twice-daily radiation scheme and the trend of higher loco-regional failure in twice-daily group. It could be clues for considering thoracic radiation dose escalation above 45 Gy in twice-daily scheme to lower loco-regional failure. Small patients number and short follow up duration of twice-daily group could be the factor and limitation of the results causing these low statistical power. Further long term evaluation and analysis of comparing two groups could be important in that points.

Background
Stereotactic body radiation therapy (SBRT) is becoming a standard of care in medically inoperable early stage non-small cell lung cancer (NSCLC), and is increasingly used in some solitary lung metastases. Lung toxicity remains however the main concern of this technique. This study aims to evaluate pulmonary function and lung membrane diffusion modifications after lung SBRT.

Methods
Retrospective analysis of pulmonary function tests (PFTs) in 40 patients undergoing SBRT for NSCLC (N=30) or lung solitary metastases (N=10), between 2009 and 2010. SBRT doses ranged between 48-60 Gy in 5-13 fractions. Normal lung volume receiving ≥ 30 Gy (V30), ≥ 20 Gy (V20), ≥ 5 Gy (V5) and mean lung dose (MLD) were calculated. Forced Expiratory Volume in 1 Second (FEV1), the ratio FEV 1 and Forced Vital Capacity (FEV1/FVC), and diffusing capacity of the lung for carbon monoxide (DLCO) were measured 1 month before SBRT, 6 weeks, then 4 and 6 months after SBRT. Respiratory symptoms were assessed according to CTCAE v4.0. Pre-and post-treatment values were compared using t Student test

Results
Median follow-up was 7 months (3-32 months), and median age was 70.6 years (51-86 years). Grade II-III chronic obstructive pulmonary disease was observed in 7 patients. Mean tumours diameter was 19.2 mm (7-53 mm), and 72% of lesions were peripheral. Mean planned target volume (PTV) and normal lung volume were 44 cc (3-162 cc), and 36149 cc (993-5146 cc) respectively. The mean values of MLD, V30, V20, and V5 were 4.7 Gy, 3.5%, 6.5%, 17.8% respectively. The mean decrease was 6.8% for FEV1, and 11.3% for FEV1/FVC. Mean DLCO remained unchanged with values of 48% predicted, and no significant respiratory symptoms were observed. The complete response rate was 90%, and 2 years progression free survival and overall survival were 72 and 76% respectively.

Conclusion
SBRT did not impair DLCO, and the changes of forced expiratory volumes were not significant, without significant clinical repercussion, even in COPD patients. These data are similar to those observed in several studies, and confirm the safety of the dose fractionation delivered to the current patients. Further studies are needed to evaluate the impact of SBRT on membrane diffusion. We are presently evaluating, the diffusing capacity for nitric oxide, a potentially more sensitive surrogate that could unmask subtle diffusion troubles and allow more accurate lung function monitoring.

Background
Prophylactic cranial irradiation (PCI) improves survival in limited-stage small cell lung cancer (LS-SCLC). However, PCI is not always delivered to these patients, possibly due to concerns about neurocognitive effects. Efforts to reduce neurotoxicity of PCI, such as hippocampal-sparing radiation, may mitigate these concerns. Little is known about the utilization rate of PCI and the reasons it is not delivered. Therefore, we reviewed the experience with LS-SCLC at a large academic institution to determine the rate of PCI use and factors associated with the lack of use.

Methods
We retrospectively reviewed all patients with LS-SCLC treated at our institution between 2000 and 2012. Receipt of PCI was recorded, as well as information about clinical presentation and initial treatment. In patients who did not receive PCI, we reviewed clinical notes from both medical and radiation oncologists to determine the reason. Overall survival (OS) and brain metastasis-free survival (BMFS) were estimated using the Kaplan-Meier technique. Pearson’s chi-squared test was used to evaluate factors associated with PCI use.

Results
We identified 229 patients treated with thoracic radiotherapy (TRT) for LS-SCLC at our institution. Median followup was 15.1 months. Of these, 119 (52.0%) did not receive PCI. Thirty-three patients (27.7%) had progressive disease or concern for progression after initial therapy and therefore did not receive PCI. The next most common causes for no PCI were patient refusal (n=25, 21%) or deemed medically unfit by an oncologist (n=25, 21%). In 20 patients (16.8%), the reason for lack of PCI could not be ascertained. Other infrequent causes were patient death or lack of followup (n=8), age (n=3), and prior radiotherapy to the head (n=2). Patients who did not get PCI were significantly older (p<0.001) and had worse performance status at initial presentation (p<0.001). Patients who received sequential rather than concurrent chemoradiation, or who received once-daily rather than twice-daily TRT, were also significantly less likely to receive PCI (p<0.001). Patients who did not receive PCI had significantly worse OS (median 17 vs. 30 months, p=0.01) and BMFS (71% vs. 91% at 1 year, p=0.02) than those who did.

Conclusion
Even at a major academic center, fewer than half of patients with LS-SCLC ultimately receive PCI. Patients receiving PCI had better intracranial control. They also had better OS, but this is likely also attributable to other clinical and treatment characteristics. Younger and fitter patients, as well as those receiving optimal TRT, are significantly more likely to undergo PCI. The most common reason for lack of PCI is progression of disease after initial therapy, which is clinically appropriate. However, a significant number of patients are appropriate for PCI yet refuse therapy, generally due to concerns about toxicity. PCI is withheld from an equivalent number of patients due to oncologist concerns about ability to tolerate therapy. This indicates that efforts to reduce neurotoxicity of PCI, such as hippocampal-sparing radiation, may impact a significant number of patients with LS-SCLC and expand the application of this survival-enhancing intervention.

Background
This study was conducted to identify prognostic factors in resected NSCLC T3N0, and to determine what factors were related to postoperative radiotherapy (RT) outcome.

Methods
Non-small-cell lung cancer (NSCLC) T3N0 patients (n=102) who underwent resection between January 1990 to October 2009 at four hospitals were enrolled. The median tumor size was 5 cm (range 1-15). Postoperative chemotherapy (CT) and radiotherapy (RT) were given in 51.0% and 55.9% of cases, respectively. The median follow-up was 43.7 months.

Results
Forty two patients (41.2%) experienced recurrence. Large tumor size (> 7 cm) and costal pleural invasion were associated with a higher relapse rate (p=0.015 and p=0.077, respectively). The 5-year overall survival (OS) was 46.6%. Tumor size and pleural invasion were significant prognostic factors for overall survival (p=0.003 and p=0.044, respectively). Patients with costal pleural invasion tended to have worse survival than others (5-year OS: 42.8% vs. 48.9%, p=0.117). CT and RT did not affect OS (p=0.122 and p=0.584). The patients that had moderate to large tumors (≥ 3 cm) combined with costal pleural invasion had decreased OS after postoperative RT (p=0.046) compared with others.

Conclusion
In postoperative T3N0M0 patients, tumor size is an independent prognostic factor and predicts survival. The primary tumor site also tends to be related to treatment outcomes. Pleural invasion was associated with lower survival. PORT should be avoided in moderate to large size (≥3 cm) tumors with costal parietal pleura invasion.

Background
Radiographic lung changes after Stereotactic Body Radiotherapy (SBRT) for non-small cell lung cancer (NSCLC) are difficult to interpret. The reliability of previous scoring systems and their relationship to local failure has not been assessed. The purpose of this study was to design a synoptic radiographic scale for characterizing late radiographic changes after SBRT and to determine the inter-rater reliability of the scale.

Methods
A Recurrence Scale (RS) was developed among lung radiation oncology/SBRT experts at a single institution, and a Synoptic Radiographic Scale (SRS) was designed in collaboration with an expert thoracic radiologist. For the RS, the suspicion for local recurrence on CT images was scored on a 5 point scale: 1) complete response, no recurrence; 2) fibrosis, not suspicious for recurrence; 3) fibrosis/mass, indeterminate for recurrence; 4) fibrosis/mass, suspicious for recurrence and 5) biopsy proven recurrence. On the SRS, CT changes were scored as ‘increasing’, ‘stable’, ‘decreasing’, ‘no change’ or ‘obscured’, along five dimensions: changes in the primary tumor site, involved lobe, consolidation, ground-glass opacity, and volume loss. Early stage NSCLC patients treated with SBRT at the institution with a minimum follow-up of 6 months were included. Serial post-treatment CT images at 12, 18, 24, 36, and 48 months were presented to the expert group (up to 6) who scored both scales in a blinded fashion. Krippendorff's alpha (KA) was used to assess inter-rater reliability. The association between RS score and known local failure was compared using Fisher’s Exact Test. The association between ‘growing tumor’ on the SRS and known local failure was compared using Fisher’s Exact Test.

Results
79 patients were scored; 7 of them had documented local failures. Experts did 11243 scorings in total, ranging from 2351 at 6 months to 480 at 48 months. For the RS, the KA was 0.27, 0.36, 0.23 and 0.45 at 12, 24, 36 and 48 months respectively. For the SRS, KA was 0.22, 0.14 and 0.11 for the treated tumor at 12, 24 and 48 months and 0.33, 0.36 and 0.22 for consolidation at 12, 24 and 36 months. The tumor was scored as obscured in 40% of patients by 24 months. Of patients with local failure, 71% were at least once scored as ‘suspicious for recurrence’ by at least one rater, compared to 28% in patients without failure (p = 0.03). 86% of patients with failure were scored at least once as increased opacity in tumor site by at least one of raters, compared to 35% in patients without failure (p = 0.01).

Conclusion
The RS has a significant relationship with local failure, and there is fair inter-agreement among experts on the suspicion of recurrence following SBRT. The SRS has low inter-rater reliability. Among its categories, only an increase in the opacity of treated tumor site is significantly related to failure. With future refinement of SRS categories, it can be a useful tool to standardize post-SBRT radiology reporting.

Background
Stereotactic ablative Radiotherapy (SABR) is a safe and effective treatment modality for primary or metastatic lung cancer. Despite its no great difference in response by pathology according to known publications, there is few report for pulmonary oligometastasis (PM) from hepatocellular carcinoma (HCC). We conducted this study to evaluate treatment outcomes of the SABR for PM from HCC under primary control.

Methods
We reviewed the records of patients with PM from HCC who received SABR for metastatic lesion from January 2006 to December 2011. Thirty-seven patients were included for analysis and total number of metastatic lesions was 52. SABR dose was 45, 48 or 60Gy in 3 or 4 fractions. Patients were evaluated by chest and liver dynamic CT scan at 1 and 3 months after completion of SBRT and followed up regularly thereafter.

Results
Median follow-up time was 19.9 months (range 6.1-33.6). The complete response (CR) rate was 38.0%. The local progression free survival (LPFS) rate was 92.1% and 88.7% at 1 and 3 years, and mean LPFS time was 69.9±3.6 months. Survival rates at 1 and 3 years were 52.1% and 33.5% in DFS, 67.6% and 35.5% in OS, respectively. Median survival time was 13.6 months (95% CI; 3.9-23.4) in DFS and 19.9 months (95% CI; 6.1-33.6) in OS. Only the initial treatment modality of primary HCC was significant factor in OS from univariate and multivariate analysis, and hazard ratio was 2.70 (95% CI: 1.04-6.96, p=0.040). There was no significant SABR-related acute or chronic complication.

Conclusion
SABR for pulmonary oligometastasis from HCC showed good local tumor control equal to the result in primary lung cancer. Patient with pulmonary oligometastasis from HCC can be treated without complication by SABR and assured good quality of life

Background
Stereotactic ablative body radiotherapy (SABR) can achieve high local control rates in patients with Stage I non-small cell lung cancer (NSCLC) and pulmonary metastases. We report the outcomes of patients treated at Royal North Shore Hospital between January 2010 and January 2012.

Methods
Patients were discussed in a multi-disciplinary meeting and were medically inoperable, or refused surgery. Lesions were divided into central or peripheral zone lesions. Peripheral lesions received 48 Gy in 4 fractions, and central lesions 50 Gy in 5 fractions. Minimum coverage was 98% of the target to receive the prescribed dose. Treatment was delivered on a Varian Clinac with 7-10 field IMRT. Toxicity was graded according to CTC-AE version 4.0.

Results
Patient Characteristics Thirty-four lesions were treated in 27 patients. Eleven patients (41%) had Stage I NSCLC, 15 (56%) pulmonary metastases, and 1 patient presented with synchronous primary NSCLC and brain metastases. The commonest primary site for pulmonary metastases was colorectal cancer (n=6). The median age was 72 years. Sixty-seven percent (n=18) of patients were medically inoperable with the other patients refusing surgery. Radiotherapy Dosimetry Eighteen tumours received 48 Gy in 4 fractions, and 12 tumours received 50 Gy in 5 fractions. Three patients with multiple lesions received 18 Gy in 1 fraction to their third lesion. One patient, with a tumour adjacent to the brachial plexus, received 49 Gy in 7 fractions. Survival With a median follow-up period of 24 months, 4 patients with pulmonary metastases and 3 patients with primary NSCLC have died. In patients with pumonary metastases the first site of failure was distant in 5 patients and pulmonary in 5 patients, with 1 in-field recurrence. In NSCLC patients there were 4 pulmonary recurrences, 3 of these were in-field. Two patients have developed metastatic disease. The median time to first event was 18 months in NSCLC patients and 7 months in patients with pulmonary metastases. Figure 1 Toxicity Two patients developed grade 2 pneumonitis. Grade 1 chest, dyspnoea or cough were seen in 7 patients. No other toxicity was seen.

Conclusion
Our data support the increasing use of SABR lung as an effective and non-toxic treatment. However, there were a number of local recurrences in NSCLC patients occurring after 15 months, supporting the need for long-term follow-up. In patients with pulmonary metastases the median time to further disease progression was 7 months- more rigorous patient selection may help to identify patients in whom SABR lung will be most beneficial.

Background
Radiation esophagitis grade III caused by chemo-radiation for small-cell lung cancer is a burden for patients and thus of concern to radiation oncologists. Neutropenia and radiation dose to the esophagus are known treatment factors influencing the rate of esophagitis during treatment, but currently the only factors that can be discussed with the patient at diagnosis are the choice of concurrent versus sequential chemo-radiation and the radiation dose fractionation schedule. In order to build predictive models to more accurately tailor treatment and advise patients on treatment options, more prognostic factors known at the moment of diagnosis are needed.

Methods
Analysis of all patients in our prospective database with stage I-III SCLC referred for concurrent chemo-radiotherapy between 5-2004 and 1-2012. All patients were PET-staged and received 45 Gy in 1.5 Gy fractions twice daily to the tumour and PET-or pathologically proven positive lymph nodes. Chemotherapy consisted of carboplatin-etoposide given concurrently with radiotherapy. All pathological lymph node regions were noted for each patient. Based on the Mountain Dressler atlas, the lymph node regions closest to the oesophagus were designated ``high risk`` regions for esophagitis, namely: 1R, 1L, 3P, 4L, 7, 8 and 9. Toxicity was scored according to CTC AE 3.0. Univariate analysis was done using the Chi-square test, reporting for p-value the Fischer exact Test for small numbers of events. Multivariate analysis was done using logistic regression. .

Results
170 patients were included in the present analysis. Thirty-seven (20%) patients developed grade III esophagitis. In univariate analysis the number of nodal regions (0, 1-4, ≥5) (p=0.02) and the number of high risk nodal regions (0, 1-2, ≥3) (p=0.001) had a significant effect on the risk of grade III esophagitis whereas the location of the primary tumour or having a T4 tumour did not. In multivariate analysis including age, gender and T4 tumour, the number of pathological nodal stations lost significance. In the multivariate analysis using age, gender, T4 tumour and the ``high risk`` count (0, 1-2, ≥3 areas) having nodes in ≥3 high risk areas was the only significant factor (p=0.002), with a hazard ratio (HR) of 7.4 for developing oesophagitis grade III (95% CI for HR: 2.2-25.2). The absolute rates of esophagitis grade III were: 5/51 (10%), 19/91 (21%), 12/28 (43%) for patients with respectively 0, 1-2 and ≥3 pathological high risk nodal areas.

Conclusion
In this series of stage I-III small cell lung cancer treated with radical chemo-radiation, the strongest predictor for esophagitis grade III known at diagnosis is the presence of nodal disease in ``high-risk regions`` 1R, 1L, 3P, 4L, 7, 8 and 9. Analysis of the correlation of this finding with the dose to the esophagus (Dmax/ Dmean) is ongoing and will also be presented at the conference.

Background
Precise definition of the target volume is one of crucial factors in the management of early stage non-small cell lung cancer (NSCLC) with stereotactic body radiation therapy (SBRT). Recent studies have shown that individualized planning margins are required to account for the variability and unpredictability of lung tumor motion. The spatial and temporal information on tumor motion can be derived with respiration-correlated 4DCT scans. In this study, we investigated how these uncertainties may be individually minimized for SBRT of NSCLC.

Methods
Twelve patients with early stage NSCLC who were undergoing SBRT were imaged with free-breathing 3-dimensional computed tomography (3DCT) and 10-phase 4-dimensional CT (4DCT) for delineating gross tumor volume (GTV)3D and ITV10Phase (ITV2). The maximum intensity projection (MIP) CT was also calculated from 10-phase 4DCT for contouring ITVMIP (ITV1). Then, ITVCOMB (ITV3), and ITV10Phase+GTV3D (ITV4), were generated by combining ITVMIP, ITV0% phase and ITV50% phase, ITV10phase and GTV3D, respectively. All 5 volumes (GTV3D and ITV1 to ITV4) were delineated in the same lung window by the same radiation oncologist.

Results
The mean (range) tumor motion (RSI, RAP, RML, and R3D ) were 6 mm (2-11 mm), 3 mm (1-4 mm), 4 mm (0-6 mm), and 7 mm (3-12 mm), respectively. The trend of volume variation was GTV3D < ITV1 < ITV2 < ITV3 ≈ ITV4 . The means ± SDs of these volumes were 10 ± 7 cc, 10 ±8 cc, 12 ± 7 cc, 11 ± 8 cc, and 12 ± 8 cc, respectively. All comparisons between the target volumes showed statistical significance ( P<0.05), except for ITV3 and ITV4 (P= 0.732). The volume of the combining ITVMIP, ITV0% phase and ITV50% phase was closed to ITV10phase plus GTV3D.

Conclusion
Uncertainties in individualized ITVs for SBRT of early stage NSCLC could effectively be minimized by combining information from free-breathing 3DCT and 10-phase 4DCT. If these images cannot be efficiently contoured, a combination of ITVMIP, ITV0% phase and ITV50% phase could be an effective alternative.

Background
Preservation of lung function is critical for patients undergoing radiotherapy for thoracic malignancies, especially in patients with compromised lung function at diagnosis. Physical properties of proton radiotherapy may permit selection of beam pathways that avoid functional lung while providing adequate tumor coverage. We demonstrate the potential for proton radiotherapy avoidance of functional lung volumes defined on SPECT/CT perfusion and ventilation imaging.

Methods
SPECT/CT imaging was performed with [99m]Tc-MAA for lung perfusion and [99m]Tc-DTPA for lung ventilation assessment. SPECT/CT images were co-registered to treatment planning CT images and avoidance structures representing perfused or ventilated lung regions were defined using a gradient search algorithm in MIM 6.0. Photon and proton radiotherapy plans were calculated to spare lung avoidance structures in Pinnacle 9.0 and XiO 4.8, respectively, and dose-volume parameters in total lung and functional lung avoidance structures were compared.

Results
A representative thoracic cancer patient with compromised and spatially heterogeneous lung function is reported on (Fig. 1). Relative to the photon plan, the proton plan provided superior total lung dose sparing (V~5~=9% vs. 25%, mean dose = 1.6 Gy vs 4.5 Gy; Fig. 2) while achieving similar tumor coverage. Additionally, superior sparing of functional lung was achieved by protons in perfused regions (V~5~<1% vs. 22%, mean dose = 0.1 Gy vs 3.1 Gy) and ventilated regions (V~5~=1% vs. 33%, mean dose = 0.1 Gy vs 4.5 Gy). Figure 1 Figure 2

Conclusion
Functional avoidance treatment planning for thoracic patients receiving radiotherapy has been demonstrated with SPECT/CT imaging. In particular, proton radiotherapy may provide strong advantages in this paradigm. Future investigation will focus on regional dose-response modeling and radiotherapy targeting strategies for functional avoidance.

Background
Internal margin was a margin to compensate for expected physiologic movements and variations in size, shape, position of the target during therapy. The purpose of our study was to identify the particular tumor characteristics to determine suitable internal margin.

Methods
43 patients with 44 lung tumors who underwent 4DCT based radiotherapy were included. GTVs on 10 respiratory phases were contoured by single radiation oncologist. Internal gross tumor volume (IGTV) was obtained by combining the GTVs at ten phases of the respiratory cycle. No separate margins were used to account for microscopic tumor extension. Additional isotropic setup margin of 3mm to derived the PTV-4D from the IGTV. In order to encompass PTV-4D, there were four expansion approaches to derive PTVs by adding uniform expansion in step of 1mm in each three direction: (1)PTV(lowest), derived from a margin to the GTV whose phase corresponding to the movement of lowest; (2) PTV(highest), derived from GTV at peak positon of the breathing cycle with an appropriate margin; (3) PTV(20%), derived from GTV on phase 20% with a suitable margin; (4) PTV(20%80%), derived from the composite GTV on phase 20% and 80% with a fit margin. The GTV centroid motions were collected. The association between margin expansion and tumor motion was analyzed. Using multivariate logistic regression, image-based risk factors for the presence of narrow margin (≤8mm) in four expansion approach were identified, and a prediction model was developed based on these factor.

Results
For the cases of GTV centroid motion less than 3mm, an isotropic margin of 10mm from any GTV can fully covered PTV-4D (IGTV+3mm). For the cases of GTV centroid motion exceeding 5mm, the dominant directions for GTV and margin expansion were not always in accordance. The former was almost all in SI, while the latter may be affected by tumor shape heavily. Even an irregular tumor in very small mobility, probably needed a large margin expansion. A model of three steps to screen the tumors with margin less than 8mm: As an initial step, tumors with fSI >0.5 and fAP >0.6 were filter out. In a second step, the small tumors (<45 cm[3]) with fSI 0.4-0.5 and fAP 0.5-0.6 were kicked out. In a third step, the irregular tumors were eliminated. (fSI or fAP was the relative fractional location in the lung in AP or SI direction. fSI equaled to 0 when tumor located in the apex of the lung ,and equaled to 1 when tumor in the lowest of the lung. fAP was the same, equaling to 0 meant front edge of the lung and equaling to 1 meant at the back. )

Conclusion
In selected patient group, fewer internal margins could be applied. Individual internal margin is necessary for high-mobility tumors. More cases were needed to confirm our model.

Background
To investigate volume changes in single photon emission computed tomography (SPECT) ventilation (V) and perfusion (Q) - weighted functional lung (FL), and dose-volume histogram of functional lung (FDVH) from V or Q SPECT and factors associated with radiation-induced lung toxicity (RILT) during the course of radiotherapy (RT) in patients with non-small-cell lung cancer (NSCLC).

Methods
Seventy NSCLC patients treated with definitive RT were enrolled prospectively. V and Q SPECT-computed tomography (CT) were performed prior to and during RT at approximately 45 Gy. FL was created from the whole lung (WL) in V and Q SPECT using a threshold of 30% of the maximum uptake of normal lung. Patients had dose calculated on CT scan to provide a standard dose-volume histogram (DVH). V and Q SPECT scans provided FDVH and functional dosimetric parameters. From the FDVH; the percent of FL receiving from 5 to 60 Gy and the mean doses of functional lungs (FMLD) were computed. Differences of volume and volume change of WL and FL between pre-RT and during-RT were compared. Differences of functional and standard dosimetric parameters between the patients with and without RILT were compared. Clinical fibrosis and pneumonitis were graded according to Common Terminology Criteria for Adverse Events version 3.0, grade 2 and above were considered to be clinically significant.

Results
The accumulative incidence of ≥ grade 2 clinical fibrosis and pneumonitis were 23.4%. The volume of lung measured by using CT or V and Q SPECT did not change significantly during radiotherapy (All p>0.05). The difference of lung volume between standard CT or V and Q SPECT was not significant in patients with RILT and without RILT (All p>0.05). However, the volume of V and Q both functional lung changed -12.4% (95% CI, -29.6% to 4.9%) in patients with RILT and 13.3% (95% CI, 2.7% to 23.9%) in patients without RILT during RT (p=0.02). The difference of mean lung dose (MLD) measured by using CT or V and Q SPECT was not significant in patients with RILT and without RILT (All p>0.05). However, the MLD was significantly higher in patients with RILT in during-RT than pre-RT CT (17.1 Gy and 15.4 Gy, p=0.01); the FMLD was significantly higher in patients with RILT in during-RT than pre-RT V and Q SPECT (16.7 Gy and 13.0 Gy, p=0.03). The V20-V55 by using during-RT CT were higher in the patients with RILT than without RILT (V20:29.0% and 22.0%, p=0.06; V25: 26.4 % and 19.0 % p=0.04; V30: 23.7 % and 16.6 %, p=0.03; V35: 21.1 % and 14.6 %, p=0.03; V40: 18.9 % and 12.6 %, p=0.02; V45: 16.4 % and 10.9 %, p=0.03; V50: 14.3 % and 9.1 % p=0.03; V55: 11.6 % and 7.5 %, p=0.04). The V5-V60 by using during-RT V and Q SPECT was higher in patients with RILT than without RILT, but not statistically significant.

Conclusion
Functional DVH metrics of V and Q SPECT and during-RT CT based DVH may be more significant in their association with RILT than pre-CT. However, these results need to be validated.

Background
The Age-adjusted Charlson Comorbidity Index (ACCI) was originally developed as a tool to predict survival for a wide range of patients based on their co-morbidities. As a growing proportion of Stage I non-small cell lung cancer (NSCLC) patients are treated with radiotherapy alone, in part due to extensive comorbidities, we hypothesize that a) ACCI is a useful prognostic tool for this understudied group of patients and b) the advent of stereotactic ablative radiotherapy (SABR) has lead to a redistribution of patients such that more patients of the poorest class are now treated, with similar or better survival.

Methods
A single institution, ethics-approved database with outcome data for 406 Stage I NSCLC patients treated with curative radiotherapy alone from 2001 to 2011 was queried. 283 patients were treated with conventional radiotherapy and 123 with SABR. Conventional doses ranged from 50-60Gy over 15-30 fractions, SABR 48-60Gy over 3-8 fractions. For each patient the ACCI score was retrospectively calculated and then arbitrarily stratified into 3 groups based on score ( ≤3, 4-5, ≥6) (higher score indicates higher number of comorbidities). Log rank test and Kaplan-Meier survival analyses was performed and the relationship between ACCI and survival was assessed using proportional hazards analysis.

Results
Median follow up was 26.4 months (22.8 months in the SABR group). The median patient age at treatment was 75 (range 41 to 92) for the entire cohort and for the SABR subset 74 (range 54-89). Percentage of patients by ACCI grouping was 22% (≤3), 48% (4-5) and 30% (≥6) for the entire cohort and for the SABR subset was 21% (≤3), 59% (4-5) and 20% (≥6) (p> 0.05). The median overall survival (OS) from time of diagnosis was 39.6 months (95% CI 34.8-44.4) and by ACCI groupings (≤3, 4-5, ≥6) was 51.6, 39.6 and 30 months (log rank test p=0.023) with hazard ratios for survival of 1.00, 1.45 (p = 0.049) and 1.73 (p = 0.0067) respectively. In the subset of patients treated with SABR, median OS was 46.8 months however there is lack of power to demonstrate any OS difference between ACCI groups.

Conclusion
The ACCI is predictive of overall survival in medically inoperable Stage I NSCLC patients irradiated with curative intent. The benefit of radiation is evident in even the poorest of patients (CMI ≥6) with a median survival of 30 months exceeding what one would expect without any treatment at all. Further follow up will be required to comment on any increased benefit with SABR.

Background
Induction therapy for non-small cell lung cancer has given promising results in a select group of patients, but no prognosis of these patients makes the future uncertain. Using a technique revild EGFR mutations and expression of ALK translocation with follow targeting induction chemotherapy increased the possibility of this technique, even in previously unresectable patients

Methods
From 2008 to 2013, 78 patients with potentially unresectable NSCLC was held from 2 to 4 courses of standard (platinabased) induction chemotherapy and 5 patients of targeted chemotherapy with the identification of mutations in EGFR or ALC traslocation. Subsequently assessed resectability of the tumor and in the case of regression of tumors - lung resection

Results
In the first group (standard) was 29 patients with adenocarcinoma. Tumor regression was achieved in 3 (10%), partial response in 5 (17%), the rest had stable or progress desease. Toxicity grade 3-4 was observed in 45% of patients. Resectability was achieved in 5 patients (17%). In the second group (target), complete response was achieved in 60% of cases, partial in 1 (20%) and stabilization in 1 patient (20%). Toxicity of chemotherapy is not more than grade 3. Resectability rate was 80%. In operated patients - time to progression was 9 months in the (standard) and 1 year 4 months in the second group (target). Median survival was 11 months versus 18 months.

Conclusion
Using the induction of targeted therapy in patients with previously unresectable NSCLC is promising, since the best results were found tolerability, objective response rate and better survival rates of patients.

Background
newly diagnosed patients with Non-small cell lung carcinoma (NSCLC) presented with advanced stages IIIA and IIIB were randomized to receive 3 protocols of neoadjuvant chemotherapy aiming at determining of both (pathologic response, and local-regional control) before undergoing surgery according to the response.

Methods
85 enrolled Patients treated from Jan 2009 till Dec 2011 with neoadjuvant chemotherapy. 75 pts males and 10 pts females.48 patientsts (56,5% ) had less than 60 years. 58 patientsts (68,2%) had Squamous cell carcinoma subtype (SCC) , while the other 28 patients (31,8%) had (Non SCC). All patients underwent pretreatment evaluation at AL BAYROUNI University cancer center. Some patients (32%) had complete mediastinoscopy staging, and all were believed to be poor candidates for up-front surgery because of the bulky disease. Different chemotherapeutic protocols were employed (Gemcitabine/cisplatin)(Docetaxel/cisplatine) and( vinorelbine/cisplatine). Study end points included resectability, pathologic response, local-regional control,and the best chemotherapy regimen . An exploratory comparison between pathologic response and both histology and age was performed. On the other hand,An exploratory comparison between the three different regimens of chemotherapy was also done.

Results
Of the 85 patients, 26 (30,5%) were deemed surgical candidates after induction therapy however 7 patients of which (8,2%) refused surgery, were 27/85 males and 2/10 females had good downstaging P value (0.0001) and (0.0003) respectively with T downstaging 20/85 (23.5%), and N downstaging 18/85 (21.2%) but T+N downstaging was seen in only 9 patients (10.6%)P value (0,0001).The response to induction chemotherapy was 30/85 patients (35.2%) with one patient only (1.2%)` in a complete response . furthermore, 26/85 patients were in objective response (30.5%) and while 3 patients (3,5%) had a stable disease.Regarding responders' age, there were 18/48 patients (37%) aged 60 years and less, and only 8/37 patients (21.6%) were over 60 years.Depending on response to histology, 21/58 patients (34.5%) of SCC had got a response to preoperative chemotherapy, P value (0,0015), while 10/27 patients (37%) had no SCC,P value ( 0,0295).50/85 patients (58.8%) treated by (Gemcitabine / Cisplatin) protocol with 15 responders( 30%) P value (0,0197 ),24/85 patients (28.2%) treated with (Vinorlbine / Cisplatin) protocol with 11 responders (45.8%) P value(0.0125) . And finally,11/85 of pts (12,9%) treated by (docetaxel / cisplatin) protocol showed four responders (36.4%) P value ( 0,0332 )

Conclusion
The preoperative chemotherapy gives a good response for a possible surgery in patients with stages IIIA and IIIB NSCLC. this response was better in patients under 60 years , P value ( 0,0055 ). With response was better seen in Vinorelbine and Cisplatin arm . However , regarding histology based response, there was no preference.the former results lead us to use neoadjuvant chemotherapy in locally advanced NSCLC,but we still need a larger trials to reach the best protocol.

Background
Half of non-small cell lung cancers are diagnosed in locally-advanced stages (LA-NSCLC), and warrant multidisciplinary treatments. Inductive and adjuvant therapies obtain the same survival benefit after surgery, but the first ones also provide prognostic information. Mediastinal downstaging and pathologic complete response preclude a better outcome. However, the value of minor pathologic features has been scarcely analyzed. We report the impact of lymphovascular invasion (LVI) and tumor necrosis (TN) on the prognosis of resected LA-NSCLC after inductive chemotherapy (iCT) or chemoradiation (iCRT).

Methods
We retrospectively reviewed 50 resected LA-NSCLC treated with iCT or iCRT in our center from October-2004 to June-2012. Three patients died due to early surgery complications and were not analyzed. The impact of ILV, TN, pneumonitis, extracapsular (EI) and pleural invasion (PI) on disease free (DFS) and overall survival (OS) was analyzed in the remaining patients

Results
Our series included 42 men and 5 women aged between 47-82 years. Non-specified NSCLC was diagnosed in nine, adenocarcinoma in two, and squamous carcinoma in 36. Thirty-seven received cisplatin-based and 10 carboplatin-based chemotherapy. Concurrent radiation was administered in 21. Pneumonectomy was performed in 15, lobectomy in 26, and segmentectomy in 6. The presence of pneumonitis, EI, or PI in the resected specimens did not impact on the outcome of the patients. However, DFS and OS clearly worsened when LVI (34.1 vs. 14.1 months, p= 0.01; and 43 vs. 29, p= 0.005; respectively), and absence of TN (31.6 vs. 24.3, p= 0.045; and 42 vs. 33, p= 0.041, respectively) were found. Radiation, cis- or carboplatin administration, and treatment length did not modify LVI and TN incidence

Conclusion
Minor pathologic features as LVI and TN significantly impact on the prognosis of resected LA-NSCLC after iCT or iCRT. Implications of that should be further analyzed. Figure 1. DFS and OS Kaplan-Meier curves according to the presence of LVI. Figure 1

Background
Half of non-small cell lung cancers are diagnosed a locally advanced stage (LA-NSCLC) and are treated by combining chemotherapy, radiation, and surgery (S). However, many patients are not able to receive complete multidisciplinary therapies due to previous respiratory dysfunctions. We report the feasibility and efficacy of inductive chemotherapy (iCT) or chemoradiation (iCRT) followed by S or consolidative radiation (RT) in LA-NSCLC patients with normal (NRF) and reduced respiratory function (RRF)

Methods
We retrospectively reviewed 100 LA-NSCLC ECOG-0-2 patients treated with iCT or iCRT followed by S or RT in our center between October-2004 and June-2012. No patient was excluded to receive treatment due to RRF, but all those without initial determination of basal forced expiratory volume in the first second (FEV1) were not analyzed. Patients were classified into two groups according to initial FEV1: 1) NRF FEV1≥ 60%, and 2) RRF FEV1< 60%. A comparison of toxicity, compliance, treatment modality, and outcome between these groups was performed

Results
Seventy-two patients initially presented NRF, and 28 RRF. Seventy (97.2%) patients with NRF completed curative treatments (20 iCRT+S; 20 iCRT+RT; 19 iCT+S; and 11 iCT+RT). Twenty-six patients (92.8%) with RRF completed curative treatments (3 iCRT+S; 14 iCRT+RT; 3 iCT+S; and 6 iCT+RT). The rest of them progressed during inductive treatment and did not receive curative approaches. Any patient interrupted the treatment due to toxicity. Resection rate was lower among patients with RRF (55.7% vs. 23%, p= 0.004), but tolerance to S was similar to those with NRF (p= 0.72). RT was applied in 44.2% and 76.9% of patients with NRF and RRF, respectively. Incidence of grade 3-4 toxicities was similar in both groups of patients (13.9% vs. 11%; p= 0.72). There were no significant differences in disease free survival (16 vs. 21.8 months, p= 0.689), but overall survival paradoxically trended to be better in patients with RRF (27.4 vs. 37.3 months, p= 0.066)

Conclusion
RRF does not necessarily contraindicate a multidisciplinary curative approach for LA-NSCLC. In our series, iCT and iCRT were followed by S in 23% of patients with RRF, and by RT in 77%. Outcome of patients with RRF receiving an intentionally curative treatment was at least as good as that of patients with NRF. Figure 1. Kaplan-Meier DFS and OS curves according to initial FEV1. Figure 1

Background
Lung cancer is the most frequent cause of cancer-related death worldwide. Brain metastasis is an important issue because its incidence of 20-40% in non small cell lung cancer (NSCLC) patients and association of significant mortality and morbidity. There are several therapeutic modalities for CNS lesions such as whole brain radiotherapy (WBRT), stereotactic radiosurgery (SRS) and metastasectomy. It is well known that conventional chemotherapy is not effective for brain metastasis because of blood brain barrier (BBB). On the other hand, tyrosine kinase inhibitors (TKI) have showed efficacy in patients with brain metastasis from activating epithelial growth factor receptor (EGFR) mutant NSCLC. We assumed that SRS for brain metastasis could be delayed for the patients with activating EGFR mutation on taking gefitinib or erlotinib.

Methods
We retrospectively identified patients with brain metastasis from NSCLC harboring a sensitizing mutation of EGFR who were treated with SRS for the brain metastasis combined with TKI. EGFR mutations in exons 18, 19, 20 and 21 were analyzed by direct sequencing. SRS treatment was indicated for brain metastasis less than 6 lesions and not exceeding 4cm each of them. The patients in SRS first group were treated by SRS for brain metastasis, and then TKI was administrated. The other patients were treated with TKI before SRS and they were assigned to TKI first group. Progression free survival time was compared with each group.

Results
Forty-three patients were eligible (SRS first: 29, TKI first 14). Twenty-nine patients of them (67.4%) were women, median age was 56 (range 36-78). Most of them were adenocarinoma, except 1 squamous cell carcinoma and 1 NSCLC NOS. All patients have activating EGFR mutations, 2 patients had also T790M mutation known as TKI resistant mutation. TKI was used as 2[nd] line treatment for twenty five patients (58.1%). WBRT and brain metastasectomy were additionally employed for 9 and 6 patients. Although eight patients complained headache after SRS, it was self-limited. The median duration of TKI treatment were not different from each group (13.4 months in SRS first group, 14.7 months in TKI first group, p=0.986) As a result, the median progression free survival time was prolonged in SRS first group than in the TKI first group (12.6 months versus 2.3 months, p<0.001). And there was no significant adverse effect related with SRS in both groups.

Conclusion
It is better to consider SRS for brain metastasis as soon as possible, even if TKI is effective for patients with brain metastasis from activating EGFR mutant NSCLC. Also, the combined treatment of TKI with SRS was well tolerated by all patients in this study.

Background
Annually 63,000 approximately new patients of lung cancer are diagnosed in India, with two-third of them in advanced stage at presentation.. Radiotherapy with concurrent chemotherapy has shown survival benefit and is the standard of care for this group of patients in western literature.

Methods
One hundred seventy four consecutive patients Of Non Small cell Lung cancer (NSCLC) were treated with radical (chemo)-radiotherapy at Tata Memorial hospital from January 2008 to December 2012, . Detailed study of patient, tumour and treatment related factors were performed. Outcomes in the form of progression free survival (PFS) and overall survival (OS) at two years were calculated. Follow-up and analysis of data was performed in February 2013. Univariate and multivariate analysis was performed to see the impact of patient related factors like age, smoking, KPS, presence of comorbidity, tumour related factors including T stage, N stage and stage group and treatment related factors chemotherapy timing , total dose, duration of radiotherapy and response to treatment on PFS and OS.

Results
Of 174 patients, Males were 154(88.5%) with median age of 58 years (range 30-84 years) and 121(69.5%) were smokers. Median KPS was 80 (range 60-100) and 59 patients had significant comorbidity. Cough was the commonest presenting symptom (28%) followed by chest pain (27%) and haemoptysis (24.7%). Most common histology was squamous carcinoma 47.1% followed by adenocarcinoma in 41.4%. Stage III (A+B) constituted 90.2% of the patients. All patients received thoracic radiotherapy using 3D Conformal technique using 6/15 MV photons to a median dose of 60Gy (range 4-66Gy) over a median duration of 44 days(2-85 days) .Of 174 patients 166 patients (95.4%) completed the planned treatment. Median GTV and PTV volumes were 132 cc (16.7-741cc) and 538 cc(56-5680 cc) respectively. The median lung-PTV volume was 2599cc and V20 Gy was 23.79 %. Out of 174 patients 76% patients received concurrent chemotherapy, while 11.5% received sequential chemotherapy also. Response assessment using WHO criteria was done at 2 months post treatment, 63 (36.1%) had complete response whereas 64patients (36.8%) had partial response or stable disease and 7 patients(3.4%) had progressive disease . At last follow-up of with mean follow up 9.3 months (range 0-37 months), 32 patients had loco-regional recurrence and 33 patients had distant metastases. Acute pneumonitis grade I and II was observed in 87(50%) and 13(7.4%), acute oesophagitis grade II and III was seen in 56(32.1%) and 7(4%) patients respectively as per RTOG grading. Median PFS and OS were 20 months and 23 months with the estimated 2 year PFS & OS was 35% & 47.4%respectvely.On Univariate analysis, complete response to treatment (p=0.000) had favourable impact on PFS whereas smoking (p=0.009), advanced T stage (p=0.002) and less than complete response (p=0.000) had negative impact on OS.

Conclusion
Even under resource limited conditions patients who were treated with an intensive (chemo) radiation with supportive care had an acceptable treatment compliance and comparable treatment outcomes. , Patients with advanced stage and smokers did worse. Patients treated with concurrent chemoradiation and complete responders had better OS.

Background
The study was aimed to evaluate association between number of chemotherapy (CT) cycles and prognosis in stage IIIB non-small cell lung cancer (NSCLC) patients treated with thoracic radiotherapy (TRT) and concurrent CV CT protocol given in 1-3 cycles according to the toxicity or patient preference.

Methods
A total of 475 18-70 years old stage IIIB NSCLC patients, who received 60-66 Gy TRT concurrently with at least 1 cycle CV (q21) regimen between January 2007 and December 2011 were retrospectively evaluated. Primary end point was overall survival (OS), and secondary end points were progression-free survival (PFS) and locoreginal PFS (LRPFS).

Results
Median follow-up time was 21.7 months (range 17-24.1). Histolopathologic subtype was squamous cell in 51.8%, and adenocarcinoma in 48.2% of cases. Treatment-related mortality was reported in 6 (1.3%) patients. There was no grade-4 non-hematological toxicity but grade-4 hematological toxicity was observed in 59 (12.4%) patients. Respective grade-3 non-hematological and hematological toxicity rates were 14.5% (n=69) and 24.6% (n=117), with leukopenia (9.3%) and esophagitis (17.3%) being the most common toxicities. Median OS, PFS and LRPFS for whole group were 20.7 (%95 CI: 19.4-22.0), 9.9 (%95 CI: 9.9-10.4) and 13.4 months (%95 CI: 12.6-14.2), respectively. Comparative median OS, PFS and LRPFS results for groups receiving 1 (n=44), 2 (n=68) and 3 (n=363) cycles of chemotherapy were 13.2 vs. 19.3 vs. 21.9 months ( p≤0.001), 6.1 vs. 9.7 vs. 10.5 months (p≤0.001), 6.6 vs. 12.8 vs.14.2 months ( p≤0.001), respectively. For all survival parameters, there were significant difference between patients receiving 1 cycle and 2 or 3 cycles of chemotherapy (p<0.05 for each) but there was no difference between those receiving 2 and 3 cycles of chemotherapy (p≥0.05 for each). T-satge (T1-2 vs T3-4) and N-stage (N2 vs N3) were the other factors influencing OS (p<0.05 for each) on univariate analyses. However, only T-stage (p=0.004) and Number of chemotherapy cycles (p≤0.001) retained prognostic significance on multivariate analyses.

Conclusion
Our results revealed that CV protocol was a relatively well-tolerated chemotherapy regimen given concurrently with TRT in stage IIIB NSCLC cases, and number of chemotherapy cycles than could be given during TRT course was an important prognostic indicator in such patients. Although results of randomized trials are needed, findings of superior survival in cases receiving 2 or 3 cycles of chemotherapy during TRT and similar survival results between 2 and 3 cycles suggest that 2 cycles of chemotherapy may potentially reduce toxicity rates, which results in increased treatment tolerability and quality of life, without compromising efficacy of treatment.

Background
The standard of care for patients with a good performance status and inoperable stage III non-small cell lung cancer (NSCLC) is concurrent chemoradiotherapy (1–3). Carboplatin and paclitaxel is an alternative to the cisplatin-based doublets that have been used traditionally (4). Early-phase studies with small numbers have been reported (5–7) but a randomised phase 3 comparison has not been performed. The objective of this study was to assess outcomes in a large cohort of patients treated with curative-intent radiotherapy and concurrent carboplatin and paclitaxel chemotherapy.

Methods
Consecutive patients between March 2004 and May 2012 with stage III NSCLC undergoing curative-intent 3D-conformal radiotherapy to 60-66 Gy in 30-33 daily fractions with concurrent weekly carboplatin (45mg/m[2]) and paclitaxel (AUC=2) were identified from a prospective database. Consolidation chemotherapy was not given. Individual medical charts, radiology and laboratory investigations were reviewed retrospectively. Baseline clinico-pathologic, treatment, outcome and toxicity details were recorded. A minimum follow-up of three months after completion of treatment was required unless death occurred sooner. Median follow-up and survival times were calculated from date of first contact using the Kaplan-Meier method.

Results
One hundred and sixteen patients were identified and baseline characteristics are provided in Table 1. Nine patients were excluded as the chemotherapy regimen changed after at least one cycle and the remaining 107 patients were analysed. Median follow-up was 43.5 months. Imaging at three months post-treatment demonstrated a complete response in 4 (4%) patients and a partial response in 68 (64%) patients. Nine (8%) patients had already died. Median progression free survival and median survival were 15 and 22 months, respectively. Locoregional control was 53%. Failure at any site occurred in 75 (70%) patients. Isolated distant failure occurred in 24 (22%) patients with 9 in the brain. Adjustments to chemotherapy dose or number of planned cycles were required in 29 (27%) patients. Fifty two (49%) patients were admitted during treatment. Seven patients experienced an acute hypersensitivity reaction to paclitaxel. Grade 3/4 neutropenia, thrombocytopenia, nephrotoxicity, oesophagitis and pneumonitis were observed in 15%, 1%, 3%, 11% and 9% of patients, respectively. There was 1 episode of fatal radiation pneumonitis.

Table 1. Patient characteristics. ECOG= Eastern Co-operative Group; FEV-1= forced expiratory volume in one second; PET= positron emission tomography; NOS = not otherwise specified.
Total number of patients (%)	116	(100)
Age, years
Median	65
Range	32-80
Sex: number (%)
Male	78	(67)
Female	38	(33)
Performance status: number (%)
ECOG 0	45	(39)
ECOG 1	62	(53)
ECOG 2	7	(6)
Unknown	2	(2)
Smoker: number (%)
Never	3	(3)
Current or ex-smoker (≥10 pack-years)	111	(96)
Unknown	2	(2)
Smoking history, pack-years
Mean	53.4
Range	0-250
FEV-1, litres
Mean	2.05
Range	0.62-4.25
Loss of weight: number (%)
None	98	(84)
≥10%	18	(16)
Stage: number (%)
IIIA	75	(65)
IIIB	41	(35)
PET-staged before treatment: number (%)
Yes	113	(97)
No	3	(3)
Histology: number (%)
Adenocarcioma	43	(37)
Squamous cell carcinoma	45	(39)
Carcinoma NOS	22	(19)
Other	3	(3)
Unknown	3	(3)

Conclusion
This review of a large, single institution series of patients with inoperable Stage III NSCLC treated with curative intent demonstrates that the concurrent administration of carboplatin and paclitaxel with radiotherapy is feasible. Survival and toxicity outcomes compare favourably to those reported with concurrent cisplatin and etoposide (4).