Virtual Library

Background
AUY922 is an HSP90 inhibitor that degrades client onco-proteins including mutant EGFR. Preclinical studies utilizing cell lines and xenografts harboring EGFR T790M demonstrate that HSP90 inhibition is effective in models of AR. This phase II study combines AUY922 and E for the treatment of patients with EGFR-mutant lung cancer and RECIST-progression on EGFR TKIs.

Methods
Eligible patients had EGFR mutations and developed AR (per Jackman, JCO 2010) after treatment with EGFR TKIs. Patients underwent tumor biopsies after developing AR and prior to study entry. Tumor tissue from re-biopsy was analyzed for EGFR T790M and other mechanisms of resistance. Patients received AUY922 70 mg/m[2 ]IV weekly and E 150 mg oral daily in 28-day cycles. Response assessment was done at 4 weeks (wks), 8 wks, and every 8 wks thereafter. The primary objective was overall response rate (ORR, CR+PR) at 8 wks. A Simon mini-max design determined sample size (stage I: 16 pts (≥2 responses needed to proceed to stage II), stage II: 9 pts; α=10%, β=10%, p0=10%, p1=30%).

Results
The trial has completed accrual, and twenty-five patients have been treated (18 women, median age 59 (range 42-76)). The median time on EGFR TKI prior to the development of AR was 11 mo (range 3-26 mo). Ten patients (40%) had EGFR T790M identified by tumor re-biopsy. In the 25 patients evaluable for response, ORR was 4/25 (16%, 95% CI 6-35%). Three of four patients with PR had EGFR T790M. An additional four patients had stable disease for at least 8 weeks. To date, four patients were on study drug for ≥ 4 cycles, and four patients currently remain on study. Adverse events reported in ≥ 20% of patients were diarrhea, fatigue, myalgias, nausea, mucositis, and night blindness. Sixty-eight percent (17/25) experienced night blindness (grade 1-2 only), and three patients came off study due to eye-related toxicity. Grade 3 toxicities included elevated liver function tests, diarrhea, fatigue, constipation and anemia.

Conclusion
AUY922 and E is an active, well-tolerated regimen for patients with EGFR-mutant lung cancer. Visual disturbances, particularly night blindness, were common, but resolved with drug discontinuation. AUY922 and erlotinib demonstrate activity as combination therapy for patients with EGFR mutant lung cancers and AR to EGFR TKI. Activity is not limited to patients with EGFR T790M. Supported by Novartis, Inc.

Background
Dacomitinib is an oral, irreversible small molecule inhibitor of all active members of the HER (human epidermal growth factor receptor) family of tyrosine kinases: EGFR (HER1), HER2 and HER4. Dacomitinib has shown superior activity to the reversible EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib in preclinical studies of lung cancer cell lines with sensitive and resistant EGFR mutations, and superiority to gefitinib in cell-line models with a HER2 insertion mutation or amplified HER2. As part of dacomitinib’s phase II testing, we studied a cohort of patients with HER2-mutant or -amplified lung cancers.

Methods
As a cohort of a larger phase II study, we enrolled patients who had stage IIIB/IV lung cancers and either HER2 mutations or HER2 amplification ([centromere of chromosome 17]; ratio >2), any number of prior systemic chemotherapies, but no prior HER2-targeted treatment. Dacomitinib was administered at 45 mg once daily continuously, or 30 mg if the patient had no prior systemic therapy, with the option to escalate to 45 mg. Patients were evaluated every 28 days. Endpoints included progression-free survival (PFS) rate at 4 months (PFS4m), PFS, objective response rate by RECIST, duration of response, overall survival (OS), and toxicity.

Results
30 patients with HER2-mutant (n=26) or HER2-amplified lung cancers (n=4) were enrolled. Characteristics: 15 female; 18 never smokers (60%); 11 (37%) former smokers. 25 received a 45 mg starting dose; 5 patients received 30 mg. 10 patients had received ≥3 prior systemic therapies. 73% of patients had a PFS event. PFS4m overall was 27% (95% CI: 11%–46%; HER2-mutant subgroup: 21% [95% CI: 6%–43%]). Median overall PFS was 3 months (95% CI: 2–4; HER2-mutant subgroup: 3 months [95% CI: 2–4]). Of 25 patients in the HER2-mutant subgroup evaluable for response, 3 (12%; 95% CI: 3%–31%) experienced a partial response, all with 9 base-pair insertions in HER2 exon 20. The partial response durations were 3+, 11, and 11+ months. The preliminary estimate of median OS was 10 months (95% CI: 7–21; HER2-mutant subgroup: 10 months [95% CI: 7–21]). Among the 4 patients with HER2 amplified lung cancers, no partial responses were seen and the PFS ranged from 1–5 months. Of 29 patients evaluable for toxicity, the most common treatment-related adverse events were diarrhea (90%; grade 3/4: 21%/3%), dermatitis (72%; grade 3/4: 3%/0), fatigue (52%; grade 3/4: 3%/0), and dry skin (48%; grade 3/4: 0/0). 10% of patients discontinued treatment due to adverse events.

Conclusion
Dacomitinib demonstrated an overall 12% objective response rate in patients with HER2-mutant lung cancers. All 3 responding patients had 9 base-pair HER2 exon 20 insertions. No responses were seen in the 4 patients with HER2-amplified lung cancers. Dacomitinib was well tolerated with manageable toxicities, consistent with the class effects of EGFR TKIs.

Background
The immune checkpoint receptor programmed death-1 (PD-1) negatively regulates T-cell activation upon interaction with its ligands, PD-L1 and PD-L2. In a Phase 1 dose-escalation/cohort expansion study (CA209-003; NCT00730639), nivolumab, a fully human PD-1 receptor blocking antibody, delivered durable responses in patients with solid tumors, including advanced NSCLC. Immunohistochemistry (IHC) analysis of tumor samples from this study suggested an association between pre-treatment tumor PD-L1 expression and clinical response to nivolumab in patients with melanoma (Grosso JF J Clin Oncol. 2013;31(suppl):abs 3016; Topalian SL NEJM 2012;366:2443-54). Here we investigate the association between PD-L1 expression by IHC and response to nivolumab in patients with NSCLC, and patient response with pre-/post-dose absolute lymphocyte counts (ALC) and selected lymphocyte cell subsets.

Methods
129 patients with NSCLC from the CA209-003 trial received nivolumab between 2008 and 2012 (1–10 mg/kg IV every 2 weeks) during dose escalation and/or cohort expansion. Archived formalin-fixed paraffin-embedded pre-treatment tumor tissue and pre-treatment and on-treatment peripheral whole blood samples were analyzed to explore potential pharmacodynamic/predictive biomarkers associated with nivolumab therapy. Pre-treatment tumor PD-L1 expression was evaluated by IHC using an automated assay developed by Dako based on a sensitive and specific anti-PD-L1 monoclonal antibody (28-8). Tumors were defined as PD-L1 positive (PD-L1+) when ≥5% of the tumor cells had membrane staining at any intensity. Lymphocyte subsets in the periphery were measured using flow cytometry.

Results
Tumor membrane PD-L1 expression was measured in 63 patients with NSCLC (29 squamous; 34 non-squamous). 31/63 (49%) NSCLC biopsies were PD-L1+. There was no apparent association between PD-L1 protein expression and NSCLC histology: for squamous and non-squamous tumors, 52% (15/29) and 47% (16/34) were PD-L1+, respective. Objective response rates for PD-L1+ and PD-L1- NSCLC patients with non-squamous and squamous histology are shown in the Table. Objective responses were observed in patients with squamous and non-squamous NSCLC who were negative for PD-L1 expression. Since increases in on-treatment ALC and activated T-cell phenotypes have been shown to positively associate with favorable clinical outcomes in ipilimumab monotherapy (Ku GY Cancer 2010;116:1767-75; Carthon BC Clin Cancer Res 2010;16:2861-71), results from an analysis correlating patient response with pre-/post-dose ALC and T-cell populations in patients with NSCLC receiving nivolumab will be presented.

Table. Patient response according to PD-L1 expression status in patients with NSCLC

Tumor type	PD-L1 expression status	Objective response rate, n/N (%)
NSCLC (all patients)	+	5/31 (16.1)
	–	4/32 (12.5)
NSCLC (squamous)	+	2/15 (13.3)
	–	3/14 (21.4)
NSCLC (non-squamous)	+	3/16 (18.8)
	–	1/18 (5.6)

Conclusion
Further evaluation of PD-L1 as a molecular marker of nivolumab therapy is required. Association of PD-L1 protein expression with clinical outcome is currently being prospectively assessed in ongoing Phase 3 trials. Clinical Trial Registration Number: NCT00730639

Abstract not provided

Background
Pemetrexed combined with cisplatin is an approved first-line treatment regimen for patients with advanced/metastatic nonsquamous non-small cell lung cancer (NSCLC). New targets are needed to further improve first-line therapy outcomes. Cixutumumab, a fully human IgG1 monoclonal antibody, specifically blocks the insulin-like growth factor-type 1 receptor, inhibiting its activation and signal transduction. Early studies have reported clinical efficacy and safety with cixutumumab. However, the clinical benefit of adding cixutumumab to conventional chemotherapy is yet to be established. This study assessed whether pemetrexed and cisplatin combined with cixutumumab was superior to pemetrexed and cisplatin as first-line therapy.

Methods
This open-label, multicenter, randomized, phase II study (N=172) enrolled patients ≥18 years of age with stage IV nonsquamous NSCLC and ECOG performance status 0–1. Patients were randomized (1:1) to receive cixutumumab 20 mg/kg combined with pemetrexed 500 mg/m[2] and cisplatin 75 mg/m[2] (cixutumumab arm; n=87) or pemetrexed and cisplatin (control arm; n=85) every 21 days up to 6 cycles of induction therapy. Patients eligible for maintenance therapy received pemetrexed and cixutumumab (cixutumumab arm) or pemetrexed (control arm). The primary endpoint was progression-free survival (PFS) based on radiographic assessments. To test for superiority (1-sided significance level 20%; study power 80%), a median PFS of 7.16 months in the cixutumumab arm (HR cixutumumab/control=0.74) was expected. Secondary endpoints included objective response rate (ORR), duration of response, and overall survival (OS). Adverse events (AEs) were assessed using CTCAE version 4.0. Between-arm comparisons of unstratified data are presented.

Results
Baseline patient and disease characteristics were similar between arms in the intent-to-treat population. The mean age of the population was 59 years (range, 32 to 83). Dose intensity for all treatments was ≥90% during both study phases. Median PFS was 5.45 months (95% CI, 3.88–6.05) vs. 5.22 months (95% CI, 4.24–6.74) in the cixutumumab and control arms, respectively (HR 1.15, 95% CI, 0.81–1.61; P=0.440). ORR did not significantly differ between treatments (37.9% cixutumumab vs. 30.6% control; P=0.338); however, the median duration of response was numerically greater in the cixutumumab arm (4.9 months; 95% CI, 4.17–6.28) than in the control arm (3.91 months; 95% CI, 2.92–6.41), although differences were not significant (HR 0.74, 95% CI, 0.40–1.38; P=0.340). Median OS was 10.68 months (95% CI, 8.74–not evaluable) in cixutumumab vs. 10.38 months (95% CI, 7.43–14.39) in control patients (HR 0.85, 95% CI, 0.56–1.30; P=0.450). Common AEs reported in ≥10% of patients were nausea, hyperglycemia, fatigue, vomiting, and anemia. A greater proportion of patients in the cixutumumab arm (74.1%) had grade 3/4 AEs than patients in the control arm (61.7%). Grade 3/4 hyperglycemia occurred at a higher rate in the cixutumumab arm than the control arm (11.8% vs. 1.2%). One possibly cixutumumab-related death occurred during the study.

Conclusion
Superior PFS was not achieved in nonsquamous NSCLC patients when cixutumumab was added to the pemetrexed and cisplatin treatment regimen, and no significant improvement for any other endpoint was observed. Pemetrexed combined with cisplatin and cixutumumab was tolerable, with no new safety concerns reported.

Background
Smoothened (SMO) is a 7-membrane spanning receptor involved in the hedgehog signaling pathway. In the absence of Patched inhibition, SMO accumulates and inhibits proteolytic cleavage of transcription factors. We previously identified a lung cancer patient with SMO mutation (Patient A, Table 1) and successfully treated him with erivedge, a hedgehog inhibitor. We therefore sought to determine the incidence of SMO mutations in The Cancer Genome Atlas (TCGA) lung cohorts, identify additional NSCLC patients with SMO mutations, and initiate therapy with hedgehog inhibition as proof-of-concept.

Methods
TCGA databases for lung adenocarcinoma (n=230) and squamous cell carcinoma (n=178) were interrogated for SMO mutations and hedgehog pathway dyregulation. Mutations were determined by whole exome sequencing. Copy number was assessed by GISTIC 2.0 (scores of 2 considered high level amplification). The lung SMO mutation patients were undergoing treatment at M.D. Anderson Cancer Center Thoracic Clinic for metastatic/refractory disease. Mutations in hotspot regions of 46 cancer-related genes including SMO was performed as part of their clinical diagnostic evaluation (Ion AmpliSeq Cancer Panel; Life Technologies, CA).

Results
In TCGA lung adenocarcinomas, alterations in SMO (mutation, amplification, mRNA overexpression) were observed in 12.2% of tumors. The incidence of SMO mutations was 2.6% and SMO gene amplifications 5%. SMO mutations and amplifications strongly correlated with sonic hedgehog gene dysregulation (p<0.0001). In TCGA squamous cell, SMO was altered in 10.1% of tumors, primarily via mRNA upregulation. Only 1 SMO missense mutation was identified in the Lung SCC cohort (D209Y). We identified 3 NSCLC patients with SMO mutations (Table 1) by the 46-gene panel. Patient A was treated with erivedge as he had a concomitant localized basal cell carcinoma (BCC) with a significant reduction in tumor burden. He continues to respond to therapy after 14 weeks. It is possible that Patient A’s NSCLC-SCC was misidentified and that this was metastatic BCC or that this is a germline variant. Germ-line mutation analysis is underway. However, the precise SMO mutation in Patient A was also identified in a lung adenocarcinoma Patient C (Table 1). Two additional SMO-mutated patients have just initiated erivedge and updates on their status will be provided at WLCC.

Table 1

Patient	Biopsy site	SMO mutation	Reported Histology	Duration of Erivedge Therapy	Response to Erivedge
A	Lung	Codon 641, exon 11 (CCT to GCT) p. Pro641Ala	NSCLC SCC	14 weeks	PR
A	Skin lesion	Codon 641, exon 11 (CCT to GCT) p. Pro641Ala	BCC	14 weeks	CR
B	AP window lymph node	Codon 525, exon 9 (ATG to TTG) p.Met525Leu	NSCLC Adenoca	pending	pending
C	Axillary lymph node	Codon 641, exon 11 (CCT to GCT) p.Pro241Ala	NSCLC Adenoca	pending	pending

Conclusion
SMO mutations and pathway alterations occur in NSCLC and may be an actionable target with hedgehog inhibitors; a clinical trial is under development. Screening lung SCC tumors for SMO mutations is recommended to prevent misdiagnosis of metastatic BCC. Additional analysis of hedgehog signaling pathway alterations is underway and will subsequently be reported.

Background
MARQUEE, a Phase 3 study which investigated the role of tivantinib, a c-MET inhibitor, in previously treated non-squamous NSCLC, collected EGFR and KRAS genotype on >90% of randomized patients, and MET expression was determined for 42%. In the ITT population, addition of tivantinib to erlotinib significantly improved PFS and ORR but did not show benefit in OS. Additional efficacy analyses in the pre-defined molecular subgroups are presented.

Methods
Patients with locally advanced or metastatic non-squamous, EGFR inhibitor naive NSCLC previously treated with 1 or 2 lines of systemic therapy, including a platinum-doublet, were stratified by number of prior therapies, sex, smoking history, and EGFR and KRAS mutation status, then randomized to oral tivantinib (360 mg twice daily) + erlotinib (150 mg once daily) or placebo + erlotinib until disease progression. Primary endpoint was OS with one interim analysis for futility/superiority. MET was assessed centrally by IHC using CONFIRM (SP44) antibody. Based upon a stability study, tumor tissue must have been sectioned within 90 days prior to MET immunostaining to be considered reliable. MET High was pre-specified as ≥50% of tumor cells staining with 2+ or 3+ intensity.

Results
From 1/2011 to 7/2012, 1048 patients were randomized to tivantinib + erlotinib (TE, n=526) or placebo + erlotinib (PE, n=522). Baseline characteristics were median age = 62 years (range, 24-89), prior therapies = 1 (66%) or 2 (34%), ECOG performance status = 0 (32%) or 1 (68%), EGFR mutant (10.4%), and KRAS mutant (27.1%). In 9/2012, the data monitoring committee recommended trial discontinuation because the pre-planned interim analysis of OS crossed the futility boundary. At the 12/2012 data cutoff, median OS was 8.5 months and 7.8 months for TE and PE, respectively (hazard ratio [HR] = 0.98; 95% CI, 0.84-1.15; p = 0.81). Median PFS was 3.6 months and 1.9 months, respectively (HR = 0.74; 95% CI, 0.62-0.89; p < 0.0001). Overall response rate (ORR) improved to 10.3% for TE compared with 6.5% for PE (p < 0.05). MET expression was obtained for 445 patients. In the pre-specified, MET High subgroup (n = 211), median OS improved to 9.3 months for TE vs 5.9 months for PE (HR = 0.70; 95% CI, 0.49-1.01; p = 0.03). In the MET Low subgroup (n = 234), median OS was 8.5 months for TE and 7.7 months for PE (HR=.90, 95% CI, 0.64-1.26, p=.53). OS did not differ between treatments in KRAS wildtype (n=702), KRAS mutant (n=284), and EGFR wildtype (n=937) subgroups; OS was immature for the EGFR mutant (n=109) subgroup at the cut-off time. Consistent with ITT, PFS was increased with TE vs PE across all molecular subgroups. Common adverse events (TE vs PE, respectively) included rash (33.1% vs 37.3%), diarrhea (34.6% vs 41.0%), and asthenia/fatigue (43.5% vs 38.1%), which occurred at similar rates between treatments; neutropenia (Grade 3/4: 10.0% vs 1.0%) was more common with TE.

Conclusion
Tivantinib significantly improved PFS and OS in the prospectively defined MET High subgroup. Further investigation of tivantinib in MET High selected, non-squamous NSCLC is warranted.

Background
The VEGF pathway has become an important therapeutic target in lung cancer. In Phase III trials, blocking VEGF using Bevacizumab (Avastin[®]) has yielded improved progression-free and overall survival in NSCLC patients when combined with standard chemotherapy, demonstrating the use of VEGF as a target for therapy. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC.

Methods
NSCLC cells (H460, H647, A549 and SKMES-1) were screened for expression of VEGF and its receptors at the mRNA and protein levels. The effect of recombinant VEGF (100ng/ml) and its blockade using neutralising antibodies (1µg/ml) on lung tumour cell proliferation (BrdU) and cell cycle (FACS) was examined. Phosphorylation of Akt and Erk1/2 proteins was examined by High Content Analysis (HCA) and confocal microscopy. The effects of silencing VEGF (100nM siVEGF)) on cell proliferation and survival signalling were assessed using BrdU and Western blot analysis, respectively. The role of NP1 in cell proliferation (BrdU), apoptosis (HCA) and cell survival signalling was also examined. A NP1 stable transfected H460 cell line (NP1-negative) was generated and NP1 overexpression verified at the mRNA (RT-PCR) and protein (Western Blot) levels relative to empty vector controls (EVC). Cell proliferation, pAkt and pErk1/2 levels were assessed in response to recombinant VEGF and VEGF antibodies. Tumour growth studies were carried out in female Balb/c nude mice following subcutaneous injection of NP1-overexpressing cells (n=8) and EVC control cells (n=8). The role of epigenetic modifications in the regulation of VEGF and its receptors was also examined using the histone deacetylase (HDAC) inhibitors, Trichostatin-A (TSA) and Virinostat (SAHA).

Results
VEGF increased proliferation of NP1-expressing NSCLC cells (H647, A549 and SKMES-1). Blocking VEGF inhibited VEGF-mediated proliferation and induced growth arrest in the G0/G1 phase of the cell cycle. Phosphorylation of Akt and Erk1/2 proteins was significantly upregulated in response to VEGF, while antibodies to VEGF inhibited this effect. VEGF siRNA significantly inhibited lung tumour cell proliferation and decreased phosphorylation of pAkt and pErk1/2. NP1 blockade significantly inhibited proliferation and apoptosis of NP1-expressing NSCLC cells, with no effect on the NP1-negative cell line, H460. Cell proliferation was significantly decreased in response to NP1 siRNA. Stable transfection of H460 cells with NP1 pcDNA significantly increased proliferation relative to EVC cells. This effect was further increased in NP1 stable transfectants upon the addition of VEGF, while neutralising antibodies to VEGF inhibited this effect. Expression levels of pAkt protein was significantly increased following treatment of cells with VEGF, with little or no effect on the pErk1/2 pathway. Tumour growth was significantly increased in mice injected with NP1-overexpressing cells (p=0.0052). HDAC inhibitors increased VEGFR1 and VEGFR2 and downregulated NP1 and NP2 expression. Significant inhibition in proliferation of NP1-positive lung cancer cells was associated with a decrease in the NP1 receptor at the mRNA and protein levels.

Conclusion
We demonstrate that VEGF is an autocrine survival factor for NSCLC cells, mediating its effects through the Neuropilin-1 receptor. Combining anti-VEGF therapies with HDAC inhibitors may offer potential as a promising avenue for future clinical research in the treatment of NSCLC.

Background
KRAS mutations are one of the most common driver mutations in non-small cell lung cancer (NSCLC) and efficient therapeutic stratergies for oncogenic KRAS-driven NSCLC are urgently needed. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the present study, we assessed clinicopathological and biological significance of EREG expression in NSCLC.

Methods
Seventy-eight lung cancer cell lines (23 small cell lung cancers [SCLCs] and 35 NSCLCs), five noncancerous bronchial epithelial cell lines and 174 surgical specimens from NSCLC patients (136 adenocarcinomas and 38 squamous cell carcinomas) were used for EREG expression analysis by real-time RT-PCR methods. In vitro cell growth was evaluated by MTT assay, colony-formation assay in liquid culture and soft agar assay. Apoptosis was evaluated by the DNA fragment detection method and the annexin-V-fluorescein staining method. The Kaplan-Meier method was used for analysis of disease-free survival (DFS) and overall survival (OS) and log-rank test was used for survival differences. Cox proportional hazards model was used to identify independent prognostic factors for PFS and OS.

Results
EREG is predominantly expressed in NSCLC lines harboring KRAS, BRAF or EGFR mutations whereas most SCLC lines lack EREG expression. Small interfering RNAs (siRNAs) targeting against these mutations resulted in down-regulation of EREG expression in NSCLC cells. EREG expression was significantly reduced by treatments with the inhibitors of MEK or ERK in EREG-overexpressing NSCLC cell lines, irrespective of mutation status of KRAS, BRAF and EGFR. EREG expression significantly correlated with KRAS copy number in KRAS-mutant NSCLC cell lines whereas EREG expression significantly correlated with EGFR copy number in NSCLC cell lines with wild-type KRAS/BRAF/EGFR. In the analysis of surgical specimens from NSCLC patients, EREG was predominantly expressed in lung adenocarcinomas. In a subgroup of adenocarcinomas, EREG expression was significantly higher in the tumors from elderly patients (≥70-year-old), males and smokers and was higher in the tumors with pleural involvement-, lymphatic permeation- or vascular invasion-positive. EREG was highly expressed in lung adenocarcinomas with KRAS mutation compared to those with EGFR mutation or wild-type EGFR/KRAS. Lung adenocarcinoma patients with high EREG expression had significantly shorter DFS and OS compared to those with low EREG expression. When the patients were divided into four groups according to EREG expression levels and KRAS mutation status, DFS and OS were significantly shorter in the patients with KRAS-mutant/EREG-high than those with wild-type KRAS/EREG-low. Cox regression analysis demonstrated that elevated EREG expression was an unfavorable prognostic factor. siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis in KRAS-mutant and EREG-overexpressed lung adenocarcinoma cells.

Conclusion
Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and unfavorable prognosis in lung adenocarcinoma patients, and EREG could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Background
We previously developed a 76-gene signature of epithelial-to-mesenchymal transition (EMT) that predicted resistance to EGFR and PI3K inhibition in non-small cell lung cancer (NSCLC). This analysis also identified Axl, a receptor tyrosine kinase, as a novel target for mesenchymal lung cancers. Here, we conducted an integrated molecular analysis of EMT in resected, treatment-naïve tumors from three clinical cohorts, including the Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC), with particular focus on Axl as a potential target in mesenchymal NSCLC.

Methods
Using our 76-gene EMT signature, TCGA patient tumors (230 LUAD, 178 LUSC) and a large MDACC cohort of resected tumors (n=279) were assigned an “EMT score.” Expression of >160 total and phosphoproteins were measured in the tumors by reverse phase protein array (RPPA). Proteomic profiles and other molecular markers (including mutation status, miRNA expression, and copy number) were correlated with EMT scores and Axl expression levels.

Results
The EMT score, derived from our EMT signature, identified NSCLC tumors with mesenchymal gene expression signatures (average 23% of tumors across all cohorts, range 14-34%). In both LUAD and LUSC, EMT scores were highly correlated with (1) expression levels of the miR200 family, a group of miRNAs previously known to regulate EMT (p-values <0.001 by Pearson correlation) and (2) levels of proteins central to EMT (e.g., E-cadherin, alpha-catenin, beta-catenin, claudin-7, fibronectin; p<0.001 for all). Mesenchymal tumors also had lower expression of TTF1 in LUAD (p=0.0002) and lower p63 in LUSC (p=0.003). Although pEGFR levels were higher in epithelial LUAD tumors (p=0.01), the frequency of EGFR mutations was not significantly higher in this group. EMT score was not associated with smoking status. Consistent with our previous findings in cell lines and patients with advanced NSCLC (BATTLE trial), protein expression of the receptor tyrosine kinase Axl was significantly higher in tumors with mesenchymal signatures (high EMT scores) and with low E-cadherin protein expression (p<0.005 for both). The inverse correlation between tumor E-cadherin and Axl expression was confirmed in an independent group of NSCLC cases by immunohistochemistry. Although a small number of Axl mutations were observed (<3% of tumors), few occurred in the kinase domain and their biological significance is unknown. Other potential therapeutic targets expressed at higher levels in mesenchymal lung cancers included PKC-alpha, NFKB, and FGFR1.

Conclusion
The EMT gene expression signature performed well in the TCGA LUAD, TCGA LUSC, and MDACC cohorts, correlating strongly with established markers of EMT on other data platforms (miRNA and protein). We observed strong protein expression of the receptor tyrosine kinase Axl (as well as other targets) among mesenchymal tumors, supporting further investigation of AXL as a potential EMT target and into the mechanism of its overexpression in NSCLC.

Background
Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas(SqCC), is the massive degradation of the extracellular matrix (ECM); and the remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan sinthases (HAS), E-cadherin adhesion molecules, and the transformer growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation.

Methods
We examined HAases proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and nonsmokers.Fifty-six patients, mean age 64 years, who underwent lobectomy for AD (n = 31) and SqCC (n = 25) were included in the study.

Results
HAS1, 2 and 3, and Hyal 1 and 3 were significantly more expressed by tumor cells than normal and stroma cells (P<0.01) . E-cadherin and TGF-β immunostaining indices were significantly increased in the tumour cells compared to stroma cells (p<0.01). When stratified according to histologic types, HAS3 and Hyal 1 immunoreactivity was significantly increased in tumour cells of AD (p = 0.01) and stroma of SqCC (p = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS 3 immunoreactivity in tumour cells (p<0.01). Stroma cells of SqCC from non smokers patients presented a significant association with HAS3 (p<0.01). A positive association was found between TGF-β in tumor cells and HAS2 in stroma cells (R = 0.40, p= 0.03), Hyal 3 and HAS1 in stroma cells (R = 0.58 p = 0.02), Hyal 1 in tumor cells and HAS1 in stroma cells (R: 0.44, p = 0.01), Hyal 3 and HAS1 in tumor cells (R: 0.35, p = 0.01). A negative association was found between TGF-β and HAS3 (R = -0.54, p = 0.004), and TGF- β and Hyal 1 (R = -0.40, p = 0.03). A Cox model analysis show low risk of death associated with female patients (R=0.11) age < 69 yrs (R=0.02), I and II stage (R=0.18 and 0.00, respectively), and high risk of death associated to HAS2 < 14.6% (R=8.69) and HAS3 > 7.4%..

Conclusion
HAase,HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. An inverse relationship between epithelial and stroma biomarker expression provides a different spectrum of aggressiveness. While an overexpression of HAS1 and HAS3 indicates aggressive subtypes of SqCC and AD, an overexpression of TGF-β and E-cadherin, indicates a protective role of the ECM in avoiding invasion by tumor cells in both histological types. HAasesin resected AD and SqCC were strongly related to the prognosis,Therefore, our findings suggest that strategies aimed at preventing high HAS3 and Hyal1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis. Proof of this idea will require further study in a randomized, prospective clinical trial.

Background
Many studies have reported the presence of human papilloma virus (HPV) primary oncoproteins in lung cancer patients. Their detection depends on histological and geographical patterns and seems to be associated with the response obtained to EGFR inhibitors.

Methods
Information regarding 84 patients suffering lung adenocarcinomas and EGFR mutations and another 48 patients lacking them (including 7 KRAS carriers) was explored for the presence of HPV16 in paraffin-embedded tumour tissue using INNO-LiPA PCR-based assays. The results were correlated with clinical characteristics and multiple outcomes, including response rate, progression-free survival (PFS) and overall survival (OS).

Results
Mean age was 59.9 years (+/- 12.2) and HPV16 infection positivity was 39% (N=52). HPV was predominant in females (N=42; p=0.032), no differences being found regarding histological pattern (p=0.72) or having a background of smoking (p=0.54). 62% of the patients had EGFR exon 19 deletions and 22.6% the L858R mutation. Changes in exon 19 were positively related to the presence of HPV16 (p=0.043), differently to the exon 21 mutation (p=0.3). Overall response rate to tyrosine -kinase inhibitors in EGFR mutation carriers’ was 65%, stable disease was 31% and clinical benefit 86.5%. Positive differences were found for response according to HPV virus status (p=0.03). PFS rate was greater in patients who were EGFR+/HPV+ compared to the EGFR+/HPV- population (p=0.014). Likewise, OS was longer for the EGFR+/HPV+ population compared to the EGFR+/HPV- population (34 months versus 24 months; p=0.0001). OS was also longer for HPV+ patients in the absence of EGFR mutations (p=0.001). The presence of HPV also discriminated OS in the small cohort of KRAS+ patients.

Conclusion
The present study has documented a high HPV positivity rate in Hispanic patients suffering lung adenocarcinoma. The presence of viral DNA can thus be presumed to be a positive prognostic factor for EGFR and KRAS mutated patients, thereby leading to considering infection as a dominant part of carcinogenesis amongst non-smokers in Latin America.

Background
Intra-tumor pressure (ITP) was reported to be one of prognostic factors for head and neck cancer and hepatocellular carcinoma. It was reported that ITP of rectal cancer fell after Bevacizumab treatment. We measured ITP of lung cancer and investigated relationship between ITP and clinicopathological factors of lung cancer.

Methods
After institutional review board approval, we measured ITP in the center of the tumor after resection of lung cancer using Ultra-Miniature Mikro-Tip Pressure Transducer Catheters (SPR1000, Millar). Lung cancer showing pure ground glass opacity (GGO) appearance in CT was excluded from this study. Between September 2009 and January 2013, 219 lung cancer patients, 142 male and 77 female, entered the study. Mean age of patients was 70 ± 10 year old (range: 39 to 87). The histologic types of lung cancer were adenocarcinoma (AD) in 148 patients, squamous cell carcinoma (SqCC) in 52 patients, adenosquamous carcinoma (AdSq) in 10 patients, and others. The clinical (c) T stages were T1a in 69 patients, T1b in 64 patients, T2a in 60 patients, T2b in 11 patients, T3 in 14 patients, and T4 in one patient. The cN stages were N0 in 187 patients, N1 in 22 patients, N2 in 8 patients, and N3 in 2 patients. The cStages were IA in 122 patients, IB-IIIB in 97 patients. The pathological (p) T stages were T1a in 84 patients, T1b in 45 patients, T2a in 67 patients, T2b in 9 patients, T3 in 9 patients, and T4 in 4 patients. The pN stages were N0 in 183 patients, N1 in 21 patients, N2 in 14 patients. The pStages were IA in 117 patients, IB in 53 patients, IIA in 19 patients, IIB in 6 patients, IIIA in 21 patients, and IIIB in 2 patients. Mean maximal diameter of the tumors on CT was 2.7±1.4cm (range: 0.8 to 7.9). Of these tumors, 59 showed part-solid and 147 showed solid appearance on CT.

Results
Mean ITP was 8.7 ± 6.5 mmHg（range: 0 to 37.2）. Mean ITPs according to histological type were as follows: 8mmHg in AD, 10mmHg in SqCC, 7mmHg in AdSq. Mean maximal standardized uptake value (SUVmax) in fluoro-deoxy glucose-positron emission tomography was 4.6±3.5 (range: 0.3 to 15.1). ITP correlated to maximal diameter of tumor on CT (r=0.312) and pT factor (r=0.336). The ITP in pN0 (8mmHg) was significantly lower than that in pN(+) (12mmHg) (p=0.005). The ITP in pStage IA patients (6mmHg) was significantly lower than that in other stages patients (11mmHg) (p=0.001). The ITP of tumors showing solid appearance (10mmHg) was significantly higher than that of tumors showing part-solid (5mmHg). While ITP correlated to SUVmax (r=0.402), lymphatic vessel invasion (r=0.269), blood vessel invasion (r=0.293), pleural invasion (r=0.287), tumor grade (r=0.238), MIB-1 index (r=0.292), did not correlate to microvessel density (CD34) .

Conclusion
ITP showed correlation to tumor aggressiveness factors of lung cancer.

Background
Heat shock protein 90 (Hsp90) maintains the stability and activity of numerous signaling proteins involved in cancer metastasis. Interim results of the GALAXY-1 trial (NCT01348126) showed an improvement in overall survival in patients with non-small cell lung cancer (NSCLC) treated with an Hsp90 inhibitor, ganetespib (G), plus docetaxel (D) compared to D alone. We evaluated the antimetastatic activity of ganetespib in preclinical models, and compared time to appearance of new lesions in patients with radiologic progression in the two arms of the GALAXY-1 trial.

Methods
Preclinical effects of ganetespib were investigated on the: (1) migration and invasion of cancer cells via scratch and Boyden chamber assays, (2) expression of metastatic factors using protein array and gene profiling, (3) architecture of NSCLC xenografts by IHC, and (4) metastasis to the lung in tail vein and orthotopic breast cancer mouse models assessed by bioluminescence and histology. GALAXY-1 is a Synta sponsored randomized, international open-label study of D with or without G in patients with advanced lung adenocarcinoma, one prior systemic therapy, and ECOG PS 0/1. D was given at 75 mg/m2 on Day 1 of a three-week cycle in both arms. In the combination arm, G was given at 150 mg/m2 on days 1 and 15. Time to appearance of new lesions (TTNL) was measured using serial computed tomography scans, starting from randomization and until a new metastatic lesion was reported. Patient enrollment completed in November 2012.

Results
Ganetespib blocked the directional migration of NSCLC cells in a monolayer of cancer cells, significantly reduced their invasion into type I collagen and induced the degradation of metastasis drivers including HIF-1α, activated FAK and MET. In mouse models, ganetespib significantly reduced tumor angiogenesis and proliferation in NSCLC xenografts; significantly blocked (>8X, p<0.005) the development of lung cancer metastases in a tail vein model; and significantly reduced multi-organ metastasis in an orthotopic model and blocked extramedullary hematopoiesis induced by the primary tumor. In GALAXY-1 trial patients, the population that exhibited the strongest survival improvement (diagnosis of advanced disease >6 months, N=175), median TTNL increased from 6.9 months (D) to 11.3 months (G+D), with hazard ratio 0.5 (p=0.0053) at time of abstract submission.

Conclusion
Ganetespib induces the degradation of key drivers of metastasis, resulting in the reduction of cancer cell migration and invasion in vitro and tumor angiogenesis and new lesion formation in vivo. Preliminary data from the GALAXY-1 study suggests that ganetespib treatment may reduce the risk of emergence of new metastatic lesions by 50% in advanced NSCLC patients.

Background
Osteopontin (OPN) is a ubiquitous extracellular protein associated with a wide range of normal and pathologic functions. It is a central regulator of the malignant phenotype in non-small cell lung cancer (NSCLC) through binding of cell surface receptors, but underlying mechanisms are poorly understood. Among OPN’s critical roles in NSCLC progression, is preventing apoptosis related to oxidative stress by activating alternative survival pathways. While OPN’s central RGD domain is considered important to this function, we hypothesize that exon 4, in the amino terminus, which is present in OPN’s A and B isoforms, but not in the C isoform, is essential to this process. We sought to determine the impact of inibition of binding between NSCLC cells and OPN exon 4 on apoptosis and apoptotic protein expression in NSCLC.

Methods
A 16 amino acid peptide mimicking the central sequence of OPN exon 4 and a scrambled sham were constructed. Competitive binding assays had previously determined that the OPN exon 4 peptide binds the NSCLC cell surface and inhibits OPN binding. Two NSCLC cell lines with wt p53, A549 (moderate endogenous OPN) and H460 (high endogenous OPN), were placed in serum-free media with OPN exon 4 peptide or scrambled peptide for 48 hours. Cells were then evaluated by TUNEL assay or harvested, lysed and protein expression measured using Human Apoptosis Array (R&D, Minneapolis, MN).

Results
Exon 4 peptide treatment resulted in significant increases in apoptosis in both cell lines (Fig). A similar pattern of change in apoptotic protein expression was seen in both cell lines, with significant increases in Bax, cleaved Caspase-3, CytC, Hsp32, Pon2, Cdnk1, and p53-pS15, p53-pS46, and p53-pS392, while Survivin and Claspin expression were significantly decreased (Fig). Notably no significant change was seen in Bclx, Bcl2, and pro-Caspase 3 expression. Scrambled peptide had no effect on apoptosis or protein expression. Figure 1

Conclusion
Inhibition of binding between OPN exon 4 and NSCLC cells significantly increased the rate of apoptosis and expression of proteins which modulate apoptosis associated with oxidative stress, including several key phosphorylated p53 variants. These data implicate OPN exon 4 interactions in NSCLC progression and resistance mechanisms and may explain the importance of OPN’s A and B isoforms as opposed to isoform C in NSCLC pathogenesis.

Background
Background and Aim of Study: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Erlotinib (tyrosine kinase inhibitor, TKI) is widely used as a specific treatment targeting epidermal growth factor receptor (EGFR), while crizotinib serves as a c-MET and anaplastic lymphoma kinase (ALK) inhibitor. Autophagy is a regulated cellular catabolic process in response to stress. This study aimed to investigate whether autophagy could confer acquired resistance to TKI in NSCLC.

Methods
Methods: Two NSCLC cell lines (HCC827 and HCC4006) with EGFR mutations (exon 19 deletions) and two NSCLC cell lines (H1993 and H2228) with c-MET amplification or ALK rearrangement were selected. Cell proliferation (MTT) and annexin-V binding assays were performed to determine cell proliferation and apoptotic cell death upon TKI treatment respectively. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot. Acidic vesicular organelle (AVO) formation was shown by acridine orange staining. Autophagy inhibitor (chloroquine) was used to study the functional significance of TKI-induced autophagy.

Results
Results: MTT assay confirmed that all cell lines were sensitive to corresponding TKI after 72 hours incubation (IC~50~ <0.5 μM). Erlotinib or crizotinib increased LC3II expression, p62 degradation and formation of AVO, compatible with induction of autophagy. Combination of 10 μM chloroquine (autophagy inhibitor) with 0.2 μM erlotinib or crizotinib for 48 hours increased apoptotic events compared to single treatment (Table 1)

Table 1. Apoptotic events in NSCLC cell lines after different treatments.

Cell line	Chloroquine + Erlotinib	Erlotinib	Chloroquine	p-value
HCC827	52.3 ± 2.8%	21.2 ± 4.2%	13.0 ± 2.5%	<0.01
HCC4006	49.3 ± 4.8%	9.4 ± 2.4%	9.0 ± 1.8%	<0.01
	Chloroquine + Crizotinib	Crizotinib	Chloroquine
H1993	42.6 ± 12.0%	17.3 ± 5.1%	10.5 ± 5.4%	<0.01
H2228	34.9 ± 10.1%	12.2 ± 2.6%	7.6 ± 1.2%	<0.01

Conclusion
Conclusions: TKI induced both apoptosis and autophagy in EGFR-mutated, c-MET-amplified and ALK-rearranged NSCLC cell lines. Inhibition of autophagy with chloroquine enhanced TKI-induced cell death. Autophagy may serve as a protective mechanism in NSCLC upon treatment with TKI.

Background
Recently, the personalized treatments for non-small cell lung cancer (NSCLC) have progressed with the understanding of the biology and the molecular mechanisms of the tumor. Treatment strategy is determined based on the biomarker profiles examined with the primary tumor tissue or the metastatic lymph nodes. However, because tumor cells acquire the metastatic phenotype through the process of clonal evolution occurring during the tumor progression, there could be the heterogeneity of the biomarker profiles between the primary tumor and the metastatic lesions. The aim of this study was to investigate the heterogeneity of the biomarkers between the primary tumor and the metastatic lymph nodes, and to evaluate the clinical significance of its discordancy.

Methods
Seventy-two patients of NSCLC with lymph node metastases who underwent complete resection with systematic lymph node dissection between 2004 and 2010 were studied. The study was conducted with the approval of the institutional ethics committee. Immunohistochemical analysis of ERCC1 and VEGF and the mutation analysis of EGFR were performed with the paraffin-embedded tissue of both the primary tumor (PT) and the metastatic lymph nodes (LN).

Results
There were 47 male and 25 female, with the median age of 69.4 years. Thirty-eight patients had adenocarcinoma, 23 had squamous cell carcinoma, and 11 had other histologic types of NSCLC. Twenty-nine patients had N1 disease, and 43 had N2. Median observation period was 35.7 months. The 5-year overall survival rate of 72 patients was 38.0%, with 53.2% in N1 cases and 28.8% in N2 cases. ERCC1 was positive in 41.7% of PT and 58.3% of LN. There were 34 cases (47.2%) with the discordant expression of ERCC1. VEGF was positive in 66.7% of both PT and LN, whereas 32 cases (44.4%) were discordant. EGFR mutation was detected in 31.9% of PT and 18.1% of LN. There were 10 cases (13.9%) with discordant mutation status. All 10 cases had mutant EGFR in PT, whereas EGFR mutation was not found in LN. Of the 49 patients with EGFR mutation-negative in PT, none showed mutations in LN. There were no differences in relapse-free survival and overall survival among the biomarker profile groups. Among patients who had negative ERCC1 in PT or LN, overall survival was significantly better in patients who received platinum-based chemotherapy perioperatively compared with that in patients who did not. However, there were no differences in patients with positive ERCC1 in PT or LN. Seventeen patients who had mutant EGFR in PT were treated with gefitinib. The responses were CR in 4, PR in 7, SD in 2, and PD in 4, with the response rate of 64.7% and the disease control rate of 76.5%. All the nine patients who had mutant EGFR in both PT and LN showed disease control, whereas 4 out of 8 patients who had mutant-negative EGFR in LN showed PD.

Conclusion
It should be noted that discordancy of biomarker profiles between PT and LN was not a few. Understanding the heterogeneity of biomarkers would be essential for the establishing the personalized treatments for NSCLC.

Background
The urokinase plasminogen activator (uPA) system (uPAS) has been shown to play a significant multifunctional role in tumour progression including angiogenesis, adhesion and migration. Increased levels of urokinase plasminogen activator (uPA) and its receptor uPAR (CD87) strongly correlate with poor prognosis and a poor clinical outcome. It has been shown previously that a subpopulation of uPAR-positive cells in Small Cell Lung Cancer (SCLC) cell lines demonstrate significant drug resistance to traditional chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU) and etoposide. The uPAS is regulated by NF-κB which has been shown to be constitutively activated in several cancer types including non-small cell lung cancer (NSCLC). Furthermore, we have shown NF-κB to be involved in the development of resistance to cisplatin in NSCLC. This project focuses on determining the role of the uPA system in the invasive phenotype of cisplatin resistant NSCLC cells.

Methods
Expression of NF-κB (p65) in parent and resistant NSCLC cell lines was quantified by qPCR, western blot and high content screening (HCS). The expression profiles of NFκB target genes were quantified using a Roche custom NFκB RTPCR array. Gene “hits” with a fold change >2 between parent and cisplatin resistant cells were validated by qPCR analysis. The upregulation of the urokinase-type plasminogen activator (uPA) in cisplatin resistant cells was determined by western blot. The effect of uPA inhibition on cell migration and invasion, using the monoclonal anti-uPAR antibody ATN-658, is being determined using the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform.

Results
Gene expression data, from the NFκB target gene array identified a panel of genes including; PLAU (gene for uPA), RIPK and NLRP12 amongst others that were over-expressed in H460 cisplatin resistant cell lines compared to the isogenic parent cell line. uPA overexpression at the protein level was confirmed in a panel of cisplatin resistant cells compared to parent cell lines. The effect of ATN-658 on the inhibition of cell migration and invasion in cisplatin sensitive and resistant cell lines will be presented.

Conclusion
Overexpression of uPA across a panel of cisplatin resistant NSCLC cell lines highlights its significance as a marker of resistance. Targeting the uPA system may be exploited in cisplatin resistant NSCLC to inhibit cell migration and invasion.

Background
Malignant pleural mesothelioma (MPM) is a rare cancer affecting the pleura and is commonly caused by prior exposure to asbestos. Treatment of MPM is difficult with limited options. The current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed), yet most patients die within 24 months of diagnosis. There is therfore an unmet need to identify new therapeutic approaches for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of many KDMs occurs in many cancers, and these proteins play important roles in tumorigenesis. One such family, the KDM6/JMJD3 family, was investigated for changes in expression in MPM and to determine if this family could represent a novel candidate target(s) for intervention in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM6 family members (KDM6A/UTX and KDM6B/JMJD3) 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 KDM6B/JMJD3, (GSK-J4) on cellular proliferation and gene expression were examined.

Results
We show that the expression of the KDM6 family is detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of KDM6A/UTX was very significantly (p<0.001) and KDM6B/JMJD3 was significantly elevated (p<0.05) in malignant MPM compared to benign pleura. When separated across histological subtype KDM6A/UTX was most significantly elevated in the Sarcomatoid subtype (p<0.001), while only KDM6B/JMJD3 was significantly elevated (p<0.05) in the Biphasic subset. Treatment of REN/ NCI-H226 cells with the KDM6B/JMJD3 inhibitor GSK-J4 caused significant inhibition of cellular proliferation, with the REN cell line being more sensitive than NCI-H226. The effects of GSK-J4 on gene expression were examined on a panel of genes associated with Tumor-Invasion/Metastasis and on pro-inflammatory cytokines.

Conclusion
The KDM6 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. We continue to assess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

Background
Cisplatin-based chemotherapy is the standard therapy used to many cancers. However, its efficacy is largely limited by the acquired drug resistance. So far, little is known about the RNA expression changes in the cisplatin resistant cancers. Identification the RNAs related to the cisplatin resistance may provide precise clues for cancer therapy.

Methods
Expression profiling of 7 cancer cell lines that performed using oligonucleotide microarray analysis got from GEO database. Bioinformatic analyses such as the Gene Oncology and KEGG pathway were used to identify genes and pathways specifically associated with cisplatin resistance. Signaling transduction network was established to identify the core genes in regulating cancer cell cisplatin resistance.

Results
A number of genes were differentially expressed in 7 groups of cancer cell lines. They mainly participated in 85 GO terms and 11 pathways in common. All differential gene interactions in the Signal-Net were analyzed. CTNNB1, PLCG2 and SRC were the most significantly altered.Figure 1

Conclusion
Bioinformatics may help excavate and analyze large amounts of data in microarrays by means of rigorous experimental planning, scientific statistical analysis and collection of complete data about cancer cell cisplatin resistance. In the present study, a novel differential gene expression pattern was constructed and advanced study will provide new targets for diagnosis and mechanism of cancer cisplatin resistance.

Background
EZH2, a polycomb group protein, has histone methyltransferase activity, and is involved in malignant transformation of several cancers. We have previously shown that an EZH2 inhibitor, DZNep, inhibits growth of non-small cell lung cancer (NSCLC) cell lines via G1 arrest and apoptosis. Recent studies have shown that EZH2-mediated gene silencing requires a recruitment of the histone deacetylase (HDAC) activity. Co-treatment with DZNep and an HDAC inhibitor has been shown to induce apoptosis synergistically in AML, ovarian cancers and renal cancers. However, the effect of combined therapy with DZNep and an HDAC inhibitor on NSCLC cells has not been reported.

Methods
We evaluated the effect of combined therapy with DZNep and an HDAC inhibitor, SAHA, on four human NSCLC cell lines, H1299, H1975, A549 and PC-3. Cell proliferation of untreated or drug-treated cells was measured by MTT assay. Percentage of apoptotic cells was measured using FITC-conjugated annexin V with a flow cytometer. Western blot analysis was performed on total cell lysates.

Results
Co-treatment with DZNep and SAHA inhibited cell proliferation synergistically, and reduced EZH2 expression and histone H3 lysine 27 trimethylation more effectively compared with each agent alone. The co-treatment greatly induced accumulation of p27[ Kip1] and decrease in cyclin A expression. Flow cytometry analysis demonstrated that the apoptotic fraction was increased in an additive or synergistic manner by the combination therapy. These effects were more evident in H1975 and PC-3 cells with EGFR mutation, in which expression of EGFR and phosphorylation of EGFR, AKT and ERK1/2 were markedly decreased by the co-treatment.

Conclusion
Combined epigenetic therapy with an EZH2 inhibitor and an HDAC inhibitor may represent an effective strategy for NSCLCs.

Background
Breathing demands our lungs to be soft and elastic. In contrast, lung tumors exhibit an abnormal tissue hardening concomitantly with an altered lung architecture and function, which brings the mechanical microenvironment of the tumor closer to that of muscle or bone. Carcinoma-associated fibroblasts (TAFs) within the tumor stroma are known to be conspirators of tumor progression. It remains unknown whether tissue hardening is sufficient to drive abnormal abundance of activated fibroblasts in the stroma of the different NSCLC subtypes. We aimed to determine the role of abnormal tumor tissue hardening in the overabundance of TAFs in different NSCLC subtypes

Methods
To address this question, we cultured the human lung fibroblast cell line CCD-19Lu or primary human lung fibroblasts on collagen-coated polyacrylamide gels exhibiting normal (soft) or tumour (stiff)-like substrates in the absence (0%) or presence (0.1%) of serum for 5 days. Primary cells were isolated from both tumor-free regions (referred to as control fibroblasts) or tumors of NSCLC patients diagnosed with either Adenocarcinoma (ADC, n=5) or Squamous Cell Carcinoma (SCC, n=5) histological subtypes.

Results
We observed a significantly higher density of CCD-19Lu fibroblasts in tumor-like gels compared to normal-like gels regardless the presence of serum. Similar results were obtained in both primary control fibroblasts and SCC-TAFs. In contrast, tumor-like gels induced a weaker density increase in primary ADC-TAFs. The increased fibroblast density in the tumor-like gels was associated with increased survival rather than proliferation, as revealed by a downregulation of caspase-3 and a rise in FAK and AKT phosphorylation. To further understand the apoptotic effect on stiffer substrates, we examined the expression of the mechanoreceptor integrin beta-1, responsible of sensing stiffness signals from the ECM. We found a significantly higher expression of integrin beta-1 in SCC compared to control and ADC primary fibroblasts .

Conclusion
Our results indicate that tumor extracellular hardening alone is sufficient to induce an increased density in activated lung fibroblasts, a well known conspirators of tumor progression, by activating pro-survival FAK-AKT pathways. Collectively these findings underline the major role of tissue hardening in the abnormal abundance of TAFs in NSCLC, particularly in the SCC subtype. These data warrants the development of novel targeted therapies against the pro-survival effects of tissue hardening.

Background
Cisplatin based doublet chemotherapy is the mainstay of non small cell lung cancer (NSCLC) treatment with an initial objective response rate of approximately 40-50%. However, intrinsic and acquired resistance to cisplatin constitutes a major clinical obstacle in lung cancer management and has yet to be fully understood. Inflammatory mediators may play an important role in the development of cisplatin resistance, such as those regulated by NF-κB. We have previously demonstrated that levels of NF-κB are increased in cisplatin resistant cells compared with sensitive Parent cells. We are currently assessing a number of NF-κB regulated targets in cisplatin resistant cell line models, using DHMEQ, a specific NF-κB inhibitor. DHMEQ treatment results in greater cell death in the cisplatin resistant cells compared with Parent. This study will elucidate the efficacy of DHMEQ to overcome cisplatin resistance and identify novel targets within the NF-κB pathway that may improve therapeutic strategies for NSCLC patients.

Methods
NF-κB downstream targets and signalling mediators were examined using NF-κB signalling and target pathway qPCR arrays (168 genes) in the H460 CisR and Parent cell line model. Targets identified are currently undergoing validation using qPCR and western blot. Biological and functional relevance of these targets in the development of cisplatin resistance will be examined further using DHMEQ and siRNA knockdown strategies. In addition, a xenograft murine model will be utilised to assess the effect of DHMEQ alone and in combination with cisplatin on tumour growth in vivo.

Results
Data from qPCR arrays have demonstrated that a number of genes are differentially regulated between the CisR and Parent cell lines. These include genes which activate the NF-κB signalling cascade (TLR3, TLR4), regulators of the pathway (BIRC3, CASP1), transcription factors (Myc) and NF-κB responsive genes (TNF, CXCL8). A number of these genes will be modulated to determine their involvement in cisplatin resistance. In addition, DHMEQ is being used in combination studies to determine, whether it can re-sensitise cells to cisplatin therapy. At present a dosing study is ongoing to establish the effect of DHMEQ on xenograft tumours derived from Parent and CisR cells. The results of which will be presented.

Conclusion
Preliminary data indicates that NF-κB and a number of its downstream targets are deregulated in cisplatin resistant cells. This project aims to validate the role of these NF-κB regulated genes in cisplatin resistant NSCLC. It will also determine whether DHMEQ may be a novel targeted agent for the treatment of NSCLC. The data obtained in this study will ultimately benefit patients by providing insights into novel druggable targets and new clinical strategies to re-sensitise patients to cisplatin therapy.

Background
Model of metastatic spreading of extrapulmonary malignancy into mediastinal lymhnodes.

Methods
Subcutaneous administration of 0,4 ml of the ink (compound from black carbon dispersed on colloid casein solution i.e. colloidal carbon / KOH-I-NOOR Hardtmuth Ltd., Praha, CZ) diluted with sterile saline solution 1:3 was performed under i.m. general anesthesia (diazepam, xylazine, ketamine) in 42 outbred Wistar rats (An-Lab Praha, CZ) divided into 7 groups according the application sites: retroperitoneum, testis / ovary, hind leg, front leg, thoracic wall, pleural cavity. On seventh day after the administration all rats were euthanatized, the thoracic wall was opend and mediastinal lymphnodes were detected macroscopically, taken out, and preserved in 18% formalin. Paraplast fixation and the hematoxyline-and-eosine staining were used for histological examination by 40-times and 100-times magnification. The samples with monocyte- macrophage system cells containing colloidal carbon (CC) in the mediastinal lymphnodes (MLNs) were considered positive. Two statistical methods were used to determine the probability of model cell transport and retention. First is based on random error theory and consists of mean values, standard deviations and confidence intervals. Second analysis calculated the confidence intervals of probabilities using F-distribution. Programs Matlab 2012 (MathWorks) and Microsoft Office 2010 Excel (Microsoft Corp.) were used for the statistical analysis. FIG 1 The sites of ink administration 1 2 3 4 5 6 7 1 - thoracic wall, 2 - front leg, 3 - pleural cavity, 4 - retroperitoneum, 5 – testis, 6 - hind leg, 7- ovary

Results
Macrophages containing carbon particles were found in MLNs of all groups of animals, at least in one animal of each tested group. Positive lymphnodes were found in 4/6 (66.7%) of animals after paraovarial, paratesticular and pleural cavity application and in 2/6 (33.3%) with retroperitoneal and distal part of hind leg application. In animals injected in distal part of front leg and in thoracic wall it was 1/6 (16.7%) with positive nodes. Statistical analysis showed significant retain of CC administered into different parts of the periphery of the body on the 5% significance level.

Conclusion
The transport of CC with the monocyte macrophage system cells from very different parts of the body to the mediastinal lymph nodes of Wistar rats and their persistence there after seven days has been proved on the 5% significance level. That indicates the relation between mediastinal lymphnodes and alien loading of the body.

Background
CD151 is a member of the tetraspanin superfamily. It is expressed on the surface of a variety of cell types and is involved in cellular processes such as cell motility, adhesion, proliferation, differentiation and signal transduction. CD151 is a positive regulator of tumor progression; metastasis, tumor cell growth and survival. It is over-expressed in various cancers, including non-small cell lung cancers (NSCLCs). The 5-year survival rate for NSCLC patients with CD151 gene-positive tumors has been shown to be lower than that of those with CD151 gene-negative tumors. Most CD151 studies to date have compared the cellular functions in the presence or absence of CD151 via over-expression and knockdown techniques respectively. However, the endogenous mechanisms by which CD151 protein is up-regulated in cancer cells remain unknown. The epidermal growth factor (EGF) is a distinct ligand for EGF receptors (EGFRs). Over-expression of EGFRs has been observed in more than 60% of NSCLCs. EGF is secreted by NSCLCs, causing the hyperactivity of EGFRs and the activation of its downstream signaling pathways to promote cell proliferation. In this study, we investigated the impact of EGF stimulation on CD151 protein expression in NSCLC cell proliferation.

Methods
A549 cells (a NSCLC cell line) were stimulated with increasing concentrations of EGF for 48hr and the CD151 protein abundance was measured via immunoblotting. To examine whether the effect of EGF on CD151 expression was a class-effect of growth factors, we also stimulated A549 cells with various stimulants (IL-1b, TNF-a, TGF-b1 and VEGF). To determine whether the effect of EGF on CD151 protein expression in NSCLC cells was cell-type selective, we examined the effects of EGF stimulation on CD151 protein abundance in other NSCLCs (H358, H1975), colon cancer (SW620) and breast cancer (MDA-MB-231) cell lines. Subsequently, silencing of CD151 via siRNA was carried out to investigate the effect of CD151 on EGF-stimulated A549 cell proliferation. Cell number was measured via trypan blue exclusion after 72hr of EGF stimulation, and phospho-ERK1/2 levels were examined via immunoblotting after 6hr of EGF stimulation.

Results
In A549 cells, EGF induced significant up-regulation of CD151 protein expression and this effect was not a class effect of growth factors. The up-regulation of CD151 protein by EGF was observed in more than one type of NSCLCs and in other cancer cell types. Silencing of CD151 down-regulated EGF-stimulated increase in A549 cell number and this effect was dependent on ERK1/2 phosphorylation.

Conclusion
This is the first study to show that EGF up-regulates CD151 protein expression to promote NSCLC cell proliferation, possibly via the MAPK (ERK1/2) pathway.

Background
MLPA® is a robust method that detects aberrant copy numbers in one simple PCR reaction by using one primer-pair. The SALSA® MLPA® kits P171, P172-B1 and P173 (MRC-Holland, Amsterdam, Netherlands) containing 42, 42 and 43 probes respectively for genes that often have an increased copy number in one or more types of tumours were used.

Methods
Tumor samples from 42 lung adenocarcinoma of never-smokers were studied (17 with EGFR L858R mutation, 9 with EGFR exon 19 deletions, and 16 with wild type EGFR). Normal lung parenchyma from the same individual was used as internal control. The normalization with reference controls and data analysis was done according to the MLPA® protocol and Coffalyser developed at MRC-Holland.

Results
Significant copy number gains in > 15% cases (incidence rates indicated in brackets) were reported here. For tumours with EGFR L858R mutation, significant gains were detected in EGFR (60%), RNF139 (50%), TERT (28%), NFKBIE (22%), UCKL1 (23%), MYC (18%), NTRK1 (17%), RUNX1 (17%) and SERPINB9 (17%). For tumours with EGFR exon 19 mutations, significant gains were detected in PSMB4 (50%), EGFR (45%), UCKL1 (23%), MYC (23%), MET (23%), CENPF (23%), CYP27B1 (23%), CDK4 (23%) and ERBB4 (23%). For tumours with wild-type EGFR, significant gains were detected in STK6 (25%), IRS2 (20%) and MYC (20%). Therefore, gains in MYC copy numbers were common in all 3 groups. Gains in EGFR and UCKL1 were common in the 2 groups with EGFR mutations. Gains in STK6 were common to L858R mutation and wild-type EGFR groups.

Conclusion
The 3 groups of adenocarcinoma in never-smokers with respect to the absence, presence and types of EGFR mutation showed quite different patterns of aberrant copy numbers in other cancer-associated genes. This in turn reflected the distinctively different patterns of genetic instability.

Background
Recently developed molecular targeting drugs are highly effective against various cancers, however the duration of response is limited. To overcome this problem, researchers have sought to find the resistance mechanism and identify novel targets. The majority of the drugs are related to receptors tyrosine kinase (RTKs), and recently, G-protein coupled receptor (GPCR) has also been reported to play a critical role in cancer biology. We thus attempted to identify the target GPCR for the treatment of lung cancer cells.

Methods
We analyzed 124 patients with non-small-cell-lung cancer who had undergone surgery from January 2008 through November 2011. Expression levels of GPCR mRNA in the tumor tissues and the adjacent normal tissues were examined by comparative genomic analysis. We then sought to find the relationship between clinical features and the GPCRs that showed differential expression levels between the two types of tissues. In addition, we examined the protein levels of the GPCRs by immunohistochemistry.

Results
We identified 3 GPCRs and 1 related molecule. Of the 4 molecules, serotonin receptor 2B (HTR2B) was expressed higher in tumor tissues than in normal tissues. HTR2B was expressed statistically higher in the tumor tissues of female, adenocarcinoma, and non-smokers (p=0.012. 0.001, 0.045, respectively), and tended to be expressed higher in patients who harbored EGFR mutation (p=0.086). No statistically significant differences were observed in relapse-free survival. Immunohistochemistry demonstrated that HTR2B was expressed especially in the invasive front of the tumor.

Conclusion
Differential expression of HTR2B between cancer cells and normal tissues and its invasive potential suggest that further investigation into this molecule is needed.

Background
Paclitaxel is one of the key drugs used in chemotherapy for the non-small cell lung cancer (NSCLC). Anti-microtubule agents, such as paclitaxel, stabilizes the microtubule polymer and prevents their breakdown. Data from the metastatic study suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to paclitaxel. To clarify whether TUBB3 is a true predictive marker for chemotherapy with paclitaxel, chemosensitivity was examined using an in vitro drug sensitivity assay.

Methods
Initially, 12 specimens were obtained to analyze the dose-response curve and to measure the median effective dose 50 (ED50) in the histoculture drug response assay (HDRA). The HDRA was perfomed for paclitaxel at several concentrations (minimum 0 μg/ml ,maximal 256μg/ml), in order to analyze the dose-response curves for individual patients. The value that was obtained from HDRA were directly applied for non-linear least square analysis using a formula of simplified dose-response curve. Subsequently, 41 surgically resected NSCLC specimens were applied to the HDRA and inhibition ratio at the concentration of 25μg/ml paclitaxel (IR25) was measured. H-scores were calculated by immunohistochemical staining. The patients comprised 26 male patients and 15 female patients. Mean age was 72±6.8. The specimens examined were 24 adenocarcinomas 17 squamous cell carcinomas.

Results
The mean (±SD) slope factor, ED50 and maximal response was 11.7±6.5, 24.6±7.73 μg/ml and 87.4±6.15% respectively. And the mean H-score was 50; (20-140). Among 12 specimens whose dose-response curve was obtained, the ED50 was higher than 25μg/ml in 5 (Resistant group) and lower in 7 (Sensitive group). The median H-score was significantly (p=0.0076) higher in Resistant group (240) than in Sensitive group (10). The mean IR25 was 53.8±26.6%. The median H-score in the specimens with IR25 above 50% (60, n=15) was significantly (p=0.0337) higher than that in specimens with IR25 under 50% (35, n=26).

Conclusion
Tumors with high TUBB3 levels exhibited chemoresistance to paclitaxel than tumors with low TUBB3 levels. HDRA revealed that TUBB3 expression is a true predictive factor for the response of paclitaxel in NSCLC.

Background
Lung cancer easily metastasizes to multiple organs, such as bone, lung, brain, liver, and adrenal gland. The frequency of the distant metastasis usually appears to be dependent on the volume of each target organ. However, metastasis to the adrenal gland is frequently observed in spite of its small volume. We hypothesized that some organotropic mechanisms exist in lung cancer adrenal metastasis. We applied microarray analysis to find the gene expression influencing organotropism of lung cancer adrenal metastasis.

Methods
Human lung adenocarcinoma cell lines PC-14, A549, and VMRC-LCD were used. Cell were cultured for 7 days on the fresh tissue slice of athymic mouse (Balb/c-nu/nu) adrenal gland. After that, proliferated cancer cells were collected from the surface of adrenal gland and cultured in the flask again. This process was repeated up to 5 times. This procedure was also applied to the other organs, which were lung, kidney, bone, and muscle, at the same time. Then the conditioned cells from each organ were obtained. Microarray analysis was applied to these cells including original cells in order to detect specific gene alteration in each organ. Expressions of 28869 genes were evaluated in each cell using microarray analysis. Gene expressions of conditioned cells obtained from each organ were compared with original cells. The genes with expression altered 1.5 fold or more were regarded as significant. Adrenal gland specific alterations were checked using unpaired t-test. The values P<0.05 were regarded as significant.

Results
Adrenal gland metastasis specific alteration was observed in 76 genes. There were 59 genes with increased expressions and 17 genes with decreased expressions. The same statistical analysis was applied to lung, liver, kidney, bone, and muscle, too. We detected 22 genes as lung metastasis specific alterations, 212 with liver, 25 with kidney, 141 with bone, and 27 with muscle. The most change in adrenal grand was increased expression of calbindin-D28k. Calbindin-D28k has anti-apoptotic properties. These properties may suppress steroid induced apoptosis and promote adrenal metastasis in lung cancer. In vitro evaluation revealed that proliferation of original PC-14 cells was inhibited by 1,4, 16, 64 µg/ml of dexamethasone in the dose-dependent manner. On the other hand, proliferation of PC-14 cells obtained from adrenal grand was not inhibited by dexamethasone in all concentrations.

Conclusion
Microarray analysis was applied to find the gene expression influencing organotropism of lung cancer adrenal metastasis. Our study results detected 76 genes as the candidates. Calbindin-D28k seemed to regulate lung cancer adrenal metastasis.

Background
Sex difference is an important factor to differentiate the clinical characteristics of lung cancers. Sex hormones derived signaling should be involved in the etiology of lung cancers. The most important factor may be estrogen that is suspected carcinogen, since strong epidemiological evidence associates the hormone to breast, endometrial, and uterine cancers. We have been studied the involvement of ATBF1 that is a large transcription factor playing an important role as a tumor suppressor in various cancers. Intriguing fact is that estrogen at lower levels increases the expression of ATBF1, but at higher levels decreases ATBF1. The response of ATBF1 is explained by the negative feedback through the estrogen-responsive proteasome system. This is the first study to reveal the expression of ATBF1 in lung cancers and explain the clinical characteristic of lung cancer differentiated by sex.

Methods
We prepared a cell line array from 17 lung cancer cell lines, which was consisted of twelve adenocarcinomas, three squamous cell carcinomas, one large cell carcinoma and one undifferentiated cancer. We generated five antibodies against ATBF1 (MB34-2, MB39-1, D1-120, MB44-2, MB47-2) at distinct part of the protein from N-terminal to C-terminal. We also analyzed the expression of p53, p21, ATM, psATM, estrogen receptors (ER), progesterone receptor (PR) , thyroid transcription factor 1 (TTF-1) and CEA by immunohistochemical analysis. The intensity of each factor was graded (score: 0-3) from weak expression (score: 0) to strong expression (score: 3). The intensity was scored in comparison with the intensity of ATB1 for other factors.

Results
Positive rates of ATBF1 with each antibody (MB34-2, MB39-1, D1-120, MB44-2, MB47-2) were 88%, 100%, 100%, 76% and 71%. Positive rates of p53, p21, ATM, psATM, ER, PR, TTF-1 and CEA were 53%, 41%, 47%, 41%, 65%, 42%, 29% and 76%, respectively. There was no significant difference in the expression pattern of each factor between adenocarcinoma and squamous cell carcinoma but TTF-1. We divided lung cancer cells into two groups by expression pattern of ATBF1. Wide range expression (W) group is characterized by the positive expression with all antibody whereas the limited expression (L) group that is characterized by limited number of positive with these antibodies. The expression rate of ER was significantly low in W group (45%, 5/11) in contrast to L group (100%, 6/6) (p=0.025).

Conclusion
The higher expression of ER, the lower and limited expression of ATBF1. The observation may be relevant to the breaking down mechanism of ATBF1 through estrogen-ER signaling discovered in breast cancer. The protein stability of ATBF1 should be an important factor for the prognosis of lung cancer distinguished by the sex.

Background
Microvasculature and microenvironment play important roles in proliferation, metastasis and prognosis in human lung adenocarcinoma, which might be altered by many anti-angiogenic drugs and cause “vessel normalization”. Epigallocatechin-3-gallate (EGCG), a natural anti-angiogenesis agent refined from green tea, was defined to have multiple effects on angiogenesis factors. So we hypothesizing that EGCG might cause “vessel normalization”, and in addition combined chemotherapy exert a synergistic effect in the tumor vessel normalization window caused by EGCG.

Methods
Build nude mice xenograft tumor model(A549 cell line). Randomly divided them into three groups (treated with saline, EGCG, bevacizumab). Test following indexes at day of 0, 2, 4, 6, 9, 12: Vessel structure: MVD, MPI; vessel GBM; Transmission-electron-microscope of microvessles; Vessel functional: perfusion function, vessel permeability; Microenvironment effect: IFP, PO2. Test cisplatin concentration in tumor tissues with different combination of EGCG and cisplatin. Treated mice with saline, cisplatin, EGCG, EGCG+cisplatin on day0 and EGCG+cisplatin on day5 and record growth delay.

Results
EGCG treated group undergoing a persisting decrease of MVD, a gradual decrease of MPI, a transient elevation of vessel perfusion function, permeability and PO2, transient decrease of IFP in tumor tissue. Full-dose cisplatin at day5 had a concentration significantly higher than Full-dose at day0 and half-dose at d5. Statistical analysis shows EGCG and cisplatin had synergistic effect as a combined anti-tumor chemotherapy. Combined treatment groups had significantly lower xenograft tumor growth rates than other three groups, and tumor growth rate in combining cisplatin on day5 was significantly lower than on day0.

Conclusion
EGCG causes vessel normalization in human lung adenocarcinoma tumor, the window is between Day 4 to Day 9. Combined therapy in this window period can escalate drug concentration in local tumor tissue, and leads to anti-tumor synergistic effect, providing a new strategy for EGCG applying as a complementary chemotherapy drug.

Background
Extracellular adenosine induces apoptosis in a variety of cancer cells via diverse signaling pathways. The present study investigated the mechanism underlying adenosine-induced apoptosis in A549 human lung cancer cells.

Methods
MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, real-time RTPCR, Western blotting, monitoring of mitochondrial membrane potentials, and assay of caspase-3, -8, and -9 activities were carried out in A549 cells, and the siRNA to silence the A~3 ~adenosine receptortargeted gene was constructed.

Results
Extracellular adenosine induces A549 cell apoptosis in a concentration(0.01-10 mM)-dependent manner, and the effect was inhibited by the A~3~ adenosine receptor inhibitor MRS1191 or knocking-down A~3~ adenosine receptor. Like adenosine, the A~3~ adenosine receptor agonist 2-Cl-IB-MECA also induced A549 cell apoptosis. Adenosine increased expression of mRNAs for Puma, Bax, and Bad, disrupted mitochondrial membrane potentials, and activated caspase-3 and -9 in A549 cells, and those adenosine effects were also suppressed by knocking-down A~3~ adenosine receptor.

Conclusion
Adenosine induces A549 cell apoptosis by upregulating expression of Bax, Bad, and Puma, to disrupt mitochondrial membrane potentials and to activate caspase-9 followed by the effector caspase-3, via A~3~ adenosine receptor.

Background
Malignant mesothelioma (MM) is an aggressive, fatal tumour of the pleura or peritoneum and strongly related to asbestos exposure. Malignant pleural mesothelioma (MPM) is the most common and occurs with a latency of up to 40 years. The mechanism of MM carcinogenesis is not well understood and the heterogeneity of the tumour is considered to be a major barrier to successful therapy. Several studies have identified changes in the expression and activities of defined cell signalling pathways in mesothelial and stromal cells, but the relationship between different cell types in the process of tumorigenesis has not been studied.

Methods
To examine the pro-oncogenic role(s) of different cell populations, the effect of primary fibroblasts from human mesotheliomas and activated macrophages on cellular signalling in normal untransformed mesothelial cells was monitored using imaging and immunoblotting techniques.

Results
Although the subcellular location of damage-associated molecular pattern (DAMP) protein HMGB1 remained nuclear in normal mesothelial cells co-cultured or treated with conditioned medium, the activation levels of growth modulating signalling pathways were altered in these cells. The proliferation and migration rates of normal mesothelial cells were also affected by paracrine signalling from activated fibroblasts and macrophages.

Conclusion
Thus, non-mesothelial cells instigate alterations in cellular signalling in mesothelial cells. Further integral examination of the aberrant signalling pathways, especially at early stages of neoplasia, will provide new insights into the mechanisms underlying malignant mesothelioma carcinogenesis.

Background
With the development of whole genome and transcriptome sequencing technologies, long noncoding RNAs (lncRNAs) have received increased attention. Multiple studies indicate that lncRNAs act not only as the intermediary between DNA and protein but also as important protagonists of cellular functions. LncRNAs can regulate gene expression in many ways, including chromosome remodeling, transcription and post-transcriptional processing. Moreover, the dysregulation of lncRNAs has increasingly been linked to many human diseases, especially in cancers. Here, we reviewed the rapidly advancing field of lncRNAs and described the relationship between the dysregulation of lncRNAs and human diseases, highlighting the specific roles of lncRNAs in human diseases.

Methods
not applicable

Results
not applicable

Conclusion
Continuing advances in transcriptomics indicate that lncRNAs fulfill important functions in the regulation of gene expression. With this updated view of molecular biology, the central dogma may be re-written. In this review, we have described some examples of lncRNAs involved in disease-associated processes (such as cancer initiation and progression), as well as highlighted the diverse mechanisms of lncRNAs. As with the dysregulation of miRNAs, dysregulation of lncRNAs is becoming recognized as a hallmark feature of many types of diseases. Importantly, cancer-associated lncRNAs may serve as diagnostic or predictive biomarkers of cancer and also provide a new therapeutic strategy of selectively silencing cancer-associated lncRNAs. However, compared with coding RNA and miRNA, there are still significant gaps in our current understanding of lncRNA function. Further studies are needed to elucidate the networks of proteins, miRNAs and lncRNAs.

Background
MicroRNAs (miRs) play important roles in the development and progression of human cancers. MiR-146a down-regulates epidermal growth factor receptor and the nuclear factor-κB regulatory kinase interleukin-1 receptor-associated kinase 1 genes that play important roles in lung carcinogenesis. This study was conducted to evaluate the association between rs2910164C>G, a functional polymorphism in the pre-miR-146a, and lung cancer risk.

Methods
The rs2910164C>G genotypes were determined in 1,094 patients with lung cancer and 1,100 healthy controls who were frequency matched for age and gender.

Results
The rs2910164 CG or GG genotype was associated with a significantly decreased risk for lung cancer compared to that of the CC genotype (adjusted odds ratio = 0.80, 95% confidence interval = 0.66-0.96, P = 0.02). When subjects were stratified according to smoking exposure (never, light and heavy smokers), the effect of the rs2910164C>G genotype on lung cancer risk was significant only in never smokers (adjusted odds ratio = 0.66, 95% confidence interval = 0.45-0.96, P = 0.03, under a dominant model for the C allele) and decreased as smoking exposure level increased (P~trend~ < 0.001).

Conclusion
These findings suggest that the rs2910164C>G in pre-miR-146a may contribute to genetic susceptibility to lung cancer, and that miR-146a might be involved in lung cancer development.

Background
ATM is a nuclear protein that plays a central role in the cellular response to DNA double stand breaks (DSBs), the DNA damage response (DDR). In mammalian cells, the DDR consists of co-ordinated multi-facet cellular processes, including DSB repair, cell cycle checkpoints and apoptosis which together act as barriers to tumor-genesis. Germ-line mutations in ATM result in the cancer-predisposing human disorder A-T (Ataxia telangiectasia). Inactivation of ATM due to gene mutations, epigenetic silencing and altered protein expression is found in many cancers including NSCLC. The role of ATM loss in NSCLC is incompletely defined. Early loss of ATM would be expected to cause an increased predisposition to mutation accumulation and the generation of neo-antigens that increase a tumour’s immunogenicity and lead to an increased sensitivity to PARP inhibition and radiation. We set out to determine the prevalence and significance of ATM loss in NSCLC cell lines and in resected NSCLC samples.

Methods
A panel of 7 NSCLC cell lines was screened for ATM protein expression levels using western blot. ATM signaling was investigated using specific antibodies to determine p-S1981 ATM, p-S824 KAP1 and p-S15 p53 following treatment of the cells with IR. ATM functionality was assessed by clonogenic survival assay to assess cellular viability after IR treatment. Clinical data was collected retrospectively through chart review of NSCLC patients diagnosed at the Tom Baker Cancer Centre from 2003 to 2006 and entered into the Glans-Look Lung Cancer Database. Archived formalin-fixed paraffin-embedded (FFPE) resected NSCLC tumour samples were retrieved and tissue microarrays constructed. Automated image acquisition was performed using a HistoRx PM-2000™. The ATM expression index was defined as the minimum ratio of the malignant cell specific AQUA score as compared with the non-malignant tumour-associated stromal cell specific AQUA score. Differences in the survival were compared using the log-rank test for low versus high ATM expression index groups and ATM expression groups as well as the stage subgroups. Cox proportional hazards regression was used to assess the prognostic effect of both ATM expression index.

Results
ATM deficiency was found in two NSCLC cell lines. The ATM deficient cell line, H23 demonstrates increased sensitivity to IR, disrupted ATM signaling and has the least surviving fraction of cells after treatment with single agent PARP inhibitor and topotecan. Our methodology identified an ATM-EI cutpoint of 0.716, defining 36/165 (21.8%) of patients as being ATM-deficient, many with stage I disease. After adjusting for histology, gender, age, and adjuvant treatment, the ATM-EI had no impact on stage I disease, but was a significant adverse prognostic factor for disease free survival (HR: 4.75, 95% CI: 2.02 to 11.17, p<0.001) and overall survival (HR: 5.09, 95% CI: 2.07 to 12.52, p<0.001) among those with stage II/III disease.

Conclusion
This study confirms ATM loss occurs in NSCLC cell lines and has therapeutic implications. It also demonstrates that ATM loss is seen in early stage NSCLC and is the first to show the prognostic consequences of this molecular deficiency. ATM status should be considered in designing new NSCLC clinical trials.

Background
The ataxia telangiectasia mutated (ATM) gene produces a protein essential in mechanisms responsible for regulating the controlled growth and proliferation of cells, as well as the DNA damage response. The lack of ATM expression can lead to a predisposition to cancer due to the inability for the protein to actively respond to DNA damage. Previous work in the Bebb lab has shown that the lack of ATM gene expression in non-small cell lung cancer (NSCLC) cells increases sensitivity to ionizing radiation, however little is known about whether ATM status can influence sensitivity to commonly used chemotherapeutic agents. In this study we will explore the chemosensitivity of NSCLC cells in relation to their ATM status, as well as the synergistic effects of combining multiple agents to help develop new treatment strategies for patients with low or absent ATM expression.

Methods
Several NSCLC cell lines were screened to identify the ATM and p53 status. Of the cell lines screened, four NSCLC cell lines: NCI-H460, NCI-H226, NCI-H23, and NCI-H1395 were chosen to treat with various chemotherapeutic agents. Optimization of cell proliferation for each cell line was conducted to determine the appropriate concentration of cells that allows for continued proliferation after a 72 hour period. Each of the agents used for cytotoxicity assays target a different mechanism of the cell, including topoisomerase I inhibitors, DNA damaging and alkylating agents, and mitotic and cell cycle inhibitors. The Chou-Talalay method for drug combinations was utilized as the combination index theorem provides a quantitative definition for examining additive, synergistic and antagonistic effects with our primary focus of observing synergy.

Results
Several NSCLC cell lines were characterized for their ATM status, and four were selected for cytotoxicity assays: H460 and H226 (ATM normal), and H23 and H1395 (ATM deficient). We observed a noticeable difference in growth rate between cell lines in addition to the differences in ATM and p53 status. Similarly, the cytotoxic response to the chemotherapeutic agents varied greatly, and the relationship of drug sensitivity to ATM status in the cells, as well as the synergistic response to combinational therapy will be presented and discussed.

Conclusion
The results of this study have several important implications. Our previous work has shown that resected, early stage NSCLC patients with low ATM expression have worse overall survival, however our demonstration that ATM deficient cell lines are more sensitive to cancer therapy could indicate that these same patients would respond better to certain lines of treatment. Similarly, increased sensitivity to combinational therapy will help mitigate detrimental side effects, leading to better quality of life during treatment periods.

Background
Lung cancers in smokers and never smokers (NS) are distinct clinical diseases. Specific molecular differences identified in these two groups include: EGFR and KRAS mutation, DNA methylation levels at specific loci, and most recently, global mutation spectra. However, much remains to be understood about the biology driving lung tumourigenesis in smokers and NS in order to improve treatment outcome. To date, no multi-dimensional integrative genomics (i.e. multi-omics) analysis designed to specifically compare current (CS) and NS lung tumours has been performed. We hypothesize that a multi-omics analysis which considers each tumour as its own unique perturbed system (as opposed to a grouped approach) will reveal molecular mechanisms of lung adenocarcinoma (AC) biology that are common or different in CS and NS.

Methods
Copy number, DNA methylation, and gene expression profiles were generated for lung AC and matched non-malignant lung tissues from 34 CS and 30 NS. PCR was performed to determine EGFR and KRAS mutation status. Copy number, methylation and expression alterations were integrated for 14,000 genes on an individual tumour basis. Disrupted genes were ranked according to the magnitude of alterations they exhibited using a novel algorithm we developed denoted MITRA. Of the genes scored by MITRA, those ranking in the 99th and 1st (top) percentiles for up- and downregulation, respectively, were subjected to Ingenuity Pathway Analysis (IPA). IPA was performed separately on all 64 lung tumours and pathway results for CS and NS were compared.

Results
We identified 361 genes that ranked in the top percentiles for up- or downregulation in at least 20% of the lung ACs we assessed. Identification of recurrent RASSF1A downregulation, and EGFR upregulation predominantly in NS demonstrates the ability of our ranking algorithm to prioritize genes known to be involved in lung tumour biology using multi-dimensional genomics data. To determine cellular pathways and functions likely deregulated as a consequence of gene disruption, we performed IPA on each tumour and determined the frequency of individual pathway disruption across tumours. This analysis revealed 88 annotated pathways with a minimum disruption frequency of 15% in either or both CS and NS. Commonly affected pathways involved: adhesion and extravasation implicating tumour invasion and migration; various catabolic and anabolic processes implicating cell metabolism; and several specific signaling pathways including atherosclerosis and Wnt/β-catenin signaling implicating inflammation and cell proliferation. Comparison of the pathways identified in CS and NS revealed 13 differentially disrupted pathways (Fisher's Exact test p < 0.05 and disruption frequency difference > 15%). Eleven pathways were preferentially disrupted in CS and affected metabolic, immune response, and inflammatory pathways. Anandamine degradation and ephrin receptor signaling were preferential to NS.

Conclusion
Our novel, multi-omics tumour system based approach revealed genes prominently disrupted in CS and NS lung AC which were associated with several cellular pathways commonly or differentially disrupted in these two groups. Pathways affected by genes disrupted at both the DNA and RNA level may contribute to the distinct clinical characteristics associated with CS and NS lung cancer and may serve as targets for intervention.

Background
In resected lung cancer, no reliable clinical or molecular predictors are currently available for identifying patients with high risk for developing recurrent disease. In a previous study, we compared gene expression profiling from adenocarcinoma specimens and normal lung tissue with non relapse (NR) and early relapse (ER), using Affimetrix human microarray HG-U133Plus 2.0. We selected 5 genes up-regulated and 4 genes down-regulated were predictive for clustering patients in ER and NR (Siggillino et al. ECCO 2011). Here, we validate our results using an independent cohort of patients with lung adenocarcinoma stage I to identify novel genes involved in the risk of ER compared to NR disease.

Methods
From tissue banking of 180 consecutive resected NSCLC stage I patients at two Italian institutions, we selected 58 frozen specimens of lung adenocarcinoma tissue with corresponding normal lung. Total RNA was isolated from tumor and normal lung specimens using RNA Universal Tissue Kit and automatically purified by Biorobot-EZ1 instrument (Qiagen). Quantification of mRNA expression levels of 9 genes (5 up-regulated: CLCA2, FABP3, H19, TFPI2, AKR1B10 and 4 down-regulated: CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1, were analyzed by real-time one-step RT-PCR using QuantiFast technology by RotorGeneQ instrument (Qiagen), and the results were compared considering β-actin as the internal reference gene and as calibrator the pool of normal tissues of analyzed patients. The gene expression of all evaluated genes and their association with relapse disease measures were assessed by t-test and logistic regression model was used for multivariate analysis.

Results
Fifteen-eight adenocarcinoma stage I patients were evaluable, 17% of which had an ER. Patients characteristics were as follows: median age was 65.8 years (38.7-81.5), 67.2% were male, 91.3% were PS 0, 70.7% were ever-smokers. The expression median values of 9 genes: CLCA2, FABP3, H19, TFPI2, AKR1B10, CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1 were 0.30, 0.71, 0.34, 1.10, 0.26, 0.24, 0.42, 0.53, 0.09, respectively. Among all genes evaluated, the NR vs ER mean expression levels of two genes down-regulated (CYP3A5, 1.09 vs 0.30; SCGB3A2, 2.28 vs 0.98) and two genes up-regulated (AKR1B10, 4.53 vs 34.20; FABP3 1.25 vs 1.55) were superimposable respect to the results of previous microarray analysis. The median disease free survival (DFS) and overall survival (OS) were 21 and 23 months, respectively. In the logistic multivariate analysis the mean expression levels of all genes showed a tendency to predict the ER in the overall population (p=0.07). Nevertheless considering only the expression levels of genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) identified as significant with t-test, the covariates in multivariate analysis increased their capacity of ER prediction (p= 0.028).

Conclusion
Our results indicate that it is possible to define, through gene expression, a characteristic gene profiling of early relapse tumor patients with an increased risk of relapse disease. The contemporary expression levels of 6 genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) predicted a worse DFS. Such features may have important implications for future targeted therapies. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

Background
Approximately 25% of lung cancers occur in lifelong never smokers. Although no dominant risk factor has been identified yet, the discover of molecular drivers potentially targetable with biological agents, makes lung cancer in never smokers a unique disease, candidate for a personalized therapy. Through the FISH test, we performed a screening for ALK, ROS1, and RET rearrangements, in a highly selected population of lung adenocarcinoma never smoker patients, previously demonstrated to be wild-type for EGFR and K-RAS mutations.

Methods
We collected archived histological material of 28 EGFR and K-RAS wild-type patients (pts), from a 200 never-smoker advanced lung adenocarcinomas database, to be analyzed for the presence of rearrangements in ALK, ROS1 and RET genes. All pts were treated at the Division of Medical Oncology of the S Maria della Misericordia Hospital in Perugia from October 2003 to February 2013. 20 specimens were included in a tissue microarray (TMA) analysis, whereas 8 were screened in separate subset, due to the scarce samples. FISH test was performed using a combination of commercial reagents and custom designed probes. Median overall survival (OS) of mutated pts compared to the pan-negative ones, was evaluated by Cox multivariate analysis.

Results
Clinicopathological characteristics: among the 28 patients, 27 were never smokers and 1 former light smoker, with a good performance status; 20 (72%) presented with a metastatic disease at diagnosis, 8 (28%) were locally advanced; median age was 56 years-old, with a predominance of female sex (18/28, 64%). All cases were invasive adenocarcinomas and classified into 18 (64%) solid predominant type, 1 (3.5%) mixed acinar/lepidic pattern, 1 (3.5%) papillary, no predominant subtype for 8 (28%) patients, because of unsufficient histological material available. Of the 28 never smoker cases, we identified 7 gene fusions (25%), including 2 pts ALK+ (7.1%), 3 pts ROS1+ (10.7%) and 2 RET+ cases (7.1%), one compatible with KIF5B:RET and other with CCDC6:RET fusion. Median OS for the entire cohort was 24.5 months (mo), 61.2 mo for mutated pts (any rearrangement) vs 24.1 mo for not-mutated, respectively (P = .292).

Conclusion
Molecularly selected never smoker lung adenorcinomas associates with a high incidence of driver genes mutations and further investigations to confirm our frequencies in larger cohorts are needed. In line with literature data, our findings suggest a different survival outcome among genotypes, and identification of specific subsets in this special population can lead to successful treatment with target therapies.

Background
Lung cancer is the leading cause of cancer-related deaths worldwide, where non-small cell lung cancer (NSCLC) accounts for 85% of cases. While cisplatin-based chemotherapy remains the gold standard treatment for lung cancer, response rates are low due to increasing development of resistance to cisplatin. Circumventing cisplatin resistance, therefore, remains a critical goal for anti-cancer therapy. The aim of this study is to examine the role of miRNA’s in the regulation of cisplatin resistance in NSCLC using a panel of isogenic cisplatin resistant NSCLC cell lines developed in our laboratory, and to examine the putative cancer stem cell markers within this chemoresistant phenotype.

Methods
MicroRNA profiling of a panel of isogenic cisplatin resistant (CisR) NSCLC cell lines, and age-matched parent cells (PT) was carried out using a 749 miRNA in-situ hybridisation array platform (Nanostring Technologies). The miRNA signature obtained was validated by qPCR using miRCURY LNA[TM ]Universal RT miRNA PCR technology (Exiqon). In order to determine the role of these miRNA’s in conferring cisplatin resistance, transfection of cell lines using miR-specific antagomirs and pre-miR’s will be carried out to examine this effect on sensitising lung cancer cells to cisplatin using clonogenic survival assays, apoptosis (APC), proliferation (BrdU) and DNA damage repair (γH2AX) assays. Furthermore, the characterisation and potential role of exosomes and exosomal-derived miRNA in modulating the cellular response to cisplatin chemotherapy will also be examined in this model of resistance. Putative stem cell markers (Oct-4, Sox-2, Nanog, SSEA4, Klf-4 and c-Myc) will be assessed in holoclones derived from resistant sublines. An in vivo model will be used to The tumourigenic potential of putative cancer stem-like cells within the cisplatin resistant population will be investigated in vivo using NOD/SCID mice. In parallel, asymmetric division assays will also be used.

Results
MicroRNA profiling of MOR, H460, A549, SKMES-1 and H1299 cisplatin resistant cell lines deduced a 3-miR signature across all five chemoresistant cell lines. Validation of this miR signature by qPCR showed differential expression of only one miRNA, miR30-c. miR-30c was significantly up-regulated (15-40 fold) in MOR, H460, SKMES-1 and H1299 CisR NSCLC cell lines and significantly down-regulated (5-fold) in A549 CisR cells relative to PT cells. The effects of miR-30c antagomirs and pre-miR’s in reversing the cisplatin resistant phenotype of NSCLC cells are currently being examined.

Conclusion
Differential expression of 3 specific miRNA’s was demonstrated in CisR lung cancer cells, relative to their parent counterparts, using an in-situ miRNA profiling hybridisation platform. While validation of this panel of miRNA’s identified only one differentially regulated miRNA species, miR30-c, further studies are warranted to explore the role of miR-30c in the cisplatin resistance phenotype of NSCLC. Exosome and cancer stem cell studies are currently under investigation.

Background
Lung cancer remains a leading cause for cancer mortality worldwide. A key feature of lung cancer development is genomic instability resulting from an accumulation of DNA lesions. In normal settings, these DNA lesions, such as double strand breaks and oxidised DNA, are rapidly repaired to prevent cytotoxicity and loss of genetic information. However, the molecular basis for the loss of genome integrity during cancer development remains to be determined. Herein, we have examined the involvement of hSSB1 in maintenance of genome stability and its potential role in lung cancer progression. hSSB1 is a critical component of the repair of DNA double strand breaks. As part of this study, we examined if hSSB1 is also involved in the repair of oxidative stress-induced DNA modifications. The most common nucleotide modification, 8-oxo-7,8-dihydro-guanine (8-oxoG), is repaired by the enzyme OGG1. Failure to repair these modifications results in mismatch mutations which are common in cancer. Therefore, understanding the molecular basis for genomic instability is key to the development of future therapeutics with clinical relevance.

Methods
To determine if hSSB1 expression is associated with lung cancer progression, a tissue microarray (TMA) with cores from 550 patients was stained with an anti-hSSB1 antibody. Kaplan-Meier survival curves were generated with the clinical data and patient prognosis was correlated with hSSB1 expression levels. To determine an in vitro association with lung cancer, A549 lung cancer cells were treated with hSSB1 specific siRNA. Cell survival was determined by microscopy and MTT assay. To examine the role of hSSB1 in repair of oxidised DNA, U2OS cells were treated with 500 µM H~2~O~2~. Cells treated with H~2~O~2~ were fixed, stained with antibodies for hSSB1 and 8-oxoG and examined by deconvolution microscopy. Lysates were collected from H~2~O~2~ treated U2OS cells and subjected to immunoprecipitation and Western blot analysis. Genomic DNA isolated from scrambled or hSSB1 siRNA U2OS cells treated with H~2~O~2~ were immobilised on nylon membrane and stained with antibodies for 8-oxoG.

Results
Significantly, TMA staining of lung cancer tissues indicated universal overexpression of hSSB1. Survival curves generated from the patient data indicated a poorer prognosis for patients with increased hSSB1 expression versus lower expressing tumours. Interestingly, in vitro inhibition of hSSB1 using siRNA significantly reduced cell survival. These data highlight the potential prognostic value of hSSB1 expression. As oxidative stress is prevalent in lung cancers, we also tested whether hSSB1 is capable of repairing 8-oxoG DNA lesions. Following oxidative DNA damage, hSSB1 localises rapidly to chromatin. hSSB1 also directly interacts with OGG1 to facilitate OGG1 recruitment to chromatin and repair of DNA damage. Importantly, cells lacking hSSB1 display ineffective repair of 8-oxoGs.

Conclusion
Our data highlight a potential role for hSSB1 in lung cancer progression and a novel role in maintenance of genome integrity. As tumours have increased genomic instability, elevated hSSB1 expression in lung cancer may enable tumours to cope with genomic instability. Taken together, these data present hSSB1 both as a prognostic marker and a novel therapeutic target.

Background
Lung cancer is the leading cause of cancer-related death worldwide. Nowdays, the histological subtypes of lung cancer and corresponding genetic polymorphisms play an important role in deciding the treatment options. KCTD11, a novel tumor suppressor, acts on antagonizing Hedgehog (Hh) signaling pathway . The deregulation of KCTD11 might activate glioma-associated oncogene homolog 1(Gli) transcription factors and lead to tumorigenesis. The present study was designed to analyze expression of the KCTD11 in different pathological lung cancer cell lines and lung cancer tissues, also the mechanism of KCTD11 inactivation and the possible affected downstream signal pathway.

Methods
Lung cancer cell lines and tissues were used to detect the expression of KCTD11. The clinical significance was analysed.Methylation detection was completed. KCTD11 vectors were transfected to squamous cell lung cancer cell lines to detect the Gli1 expression.

Results
The expression of KCTD11 was detected in Small airway epithelial cell (SAEC) and 14 lung cancer cell lines. RT-PCR showed reduced expression in lung cancer cell lines, especially in small cell lung cancer cell (SCLC) lines and squamous cell lung cancer cell (SCC) lines compared with SAEC. In all 14 tested cell lines (5 adenocarcinoma, 2 large cell, 3 SCC and 4 SCLC), we found these CpG islands were densely methylated in 3 of 4 SCLC cell lines, 2 of 3 SCC cell lines and 1 of 2 large cell lung cancer (LCC) cell linesbut none of the 5 adenomacarcinoma cell lines and SAEC. TMA staining showed significantly lower expression of KCTD11 in SCC lung cancer tissues than in normal tissues (24.2% vs 87.5%, p=0.000) .Also 5 of 10 SCC tissues compared with 1 of 12 adenocarcinoma showed methylation status. IHC analysis showed that there was 44.1% (55/118) positive Gli1 expression in group of negative KCTD11 expression. Correlation analysis showed they might exist marginally negative correlation (r=-0.165, p=0.052). H2170 cell lines with KCTD11 plasmid transfection showed significantly reduced Gli-1 expression compared with control plasmid transfection. (picture 1) Figure 1

Conclusion
In summary, we found the reduced KCTD11 expression in lung cancer. Hypermethylation was the important mechanism of KCTD11 epigenetic change in non-adenocarcinoma lung cancer cell lines and tissues. KCTD11 silencing resulted in the high expression of Gli1, a downstream mediator of Hh signaling pathway, and might affected the tumorigenesis and development in lung cancer. Targeting the KCTD11 treatment and thus controlling the Hh pathway might a new treatment target in non-adenocarcinoma lung cancer.

Background
Lung cancer is a highly lethal disease, and is believed to develop through a multistep carcinogenic process, which involves numerous genetic and epigenetic alterations. Among these alterations, mutations in “driver genes” such as KRAS and EGFR are found in non-small cell lung cancer (NSCLC) and they are demonstrated to contribute to a phenomenon, oncogene addiction. Recently, the EML4-ALK (echinoderm microtubule-associated protein–like 4 anaplastic lymphoma kinase) fusion gene has been discovered as a novel driver gene in a subset of NSCLC. We evaluated the oncogenic transformation ability of EML4-ALK by using an hTERT/CDK4-immortalized normal human bronchial epithelial cell (HBEC) model.

Methods
We used two HBEC lines, HBEC3 and HBEC4. Mutant KRAS[V12]-expressing HBEC was used as a positive control for oncogenic transformation. A lentiviral vector system was used to generate HBECs stably expressing EML4-ALK. EML4-ALK protein expression was confirmed by westernblotting, and downstream pathways were analyzed by westernblotting with phospho-specific antibodies. Malignant phenotypes of EML4-ALK-expressing HBECs were examined by WST-1 proliferation assay and liquid and soft agar colony formation assays.

Results
Westernblotting analysis showed that EML4-ALK was expressed in HBECs. Analysis of downstream pathways did not show significant differences between EML4-ALK-expressing and control HBECs. Introduction of EML4-ALK in HBECs increased the number of soft agar colonies but its effect was not as strong as KRAS[V12].Figure 1 A. Soft agar colony formation assay showing that EML4-ALK increased the number of colonies compared to control cells to a lesser extent than did KRAS[V12]. B. Cell proliferation assay (MTS-1) showing no significant difference between EML4-ALK-expressing and control HBECs.

Conclusion
EML4-ALK alone did not induce dramatic oncogenic changes in HBECs. To acquire more malignant phenotype, additional genomic alterations may be required and this is now under investigation.

Background
RANKL is an essential mediator of osteoclast differentiation, function, and survival. Tumor cells can induce RANKL expression in the bone stroma, causing activation of osteoclasts, leading to bone breakdown and subsequent tumor growth. In mouse models of lung cancer bone metastasis, RANKL inhibition by OPG-Fc can prevent tumor-induced osteolysis, decrease skeletal tumor burden, and increase survival. The effects on disease progression and survival may be explained by direct effects on the tumor in addition to indirect effects via osteoclast suppression. Recent clinical studies in patients with advanced cancer, including lung cancer, showed that RANKL inhibition with denosumab reduced the risk of skeletal-related events. Denosumab also improved overall survival compared with zoledronic acid in patients with lung cancer. In addition to the well-defined expression in the bone compartment, RANK and RANKL expression has also been demonstrated in human lung cancer cell lines and the functional expression of RANK has been confirmed through the demonstration of RANKL-dependent responses. The current study assessed the expression of human RANK and RANKL in human primary lung cancer samples in order to gain a potential mechanistic understanding of these clinical observations.

Methods
RANK and RANKL expression was analyzed in a panel of human primary lung cancer samples. Specific, monoclonal antibodies against human RANK (N-1H8, N-2B10; Amgen) and human RANKL (M366, AMG161; Amgen) were validated and optimized for immunohistochemistry (IHC) and used for expression analysis. mRNA was quantified using RT-PCR. Expression of RANK and RANKL was determined by IHC in the carcinoma element, tumor adjacent normal lung, and infiltrating cells. Incidence was scored as a positive IHC signal (any intensity). The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry, and Western blots of positive and negative control cells and xenograft samples.

Results
Analysis of primary human lung cancer using IHC demonstrated RANK staining in the tumor epithelium of 9/16 (56%) non-small cell lung cancer (NSCLC) adenocarcinomas, 9/26 (34%) squamous cell carcinomas (SCC), and 5/10 (50%) small cell carcinomas (SCLC). RANKL staining was observed in the tumor epithelium of 12/16 (75%) adenocarcinomas, 5/26 (19%) SCC, and 3/10 (30%) SCLC. RANK and RANKL were also observed in infiltrating macrophages and lymphocytes, respectively, within the majority of tumors examined. In addition, RANKL expression was frequently observed in type II pneumocytes of the alveoli, Clara cells of the terminal bronchioles, and in lymphocytes within the bronchus-associated lymphoid tissue (BALT); RANK expression was observed in alveolar macrophages proximal to the tumor and in M-cells of BALT. Analysis of mRNA levels indicated significantly greater levels of both RANK and RANKL in primary human NSCLC as compared with normal lung.

Conclusion
RANK and RANKL expression was observed in the epithelial carcinoma element in human primary lung cancer. Whether the expression of RANK and/or RANKL on lung cancer will directly contribute to tumor progression and/or metastatic activity and whether RANKL inhibition has a potential direct anti-tumor effect in lung cancer beyond the well established bone-targeted mechanism remains an objective of ongoing research.

Background
Rearrangement of the proto-oncogene RET is a newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) such as sunitinib, sorafenib, and vandetanib target RET kinase activity, suggesting that the patients with RET fusion genes may be treatable with a kinase inhibitor. However, the mechanisms of resistance to these agents remain largely unknown. Cancer cell microenvironments can critically affect cancer cell behaviors, including drug sensitivity. We determine whether microenvironmental factors trigger RET inhibitor resistance in LC-2/ad cell with CCDC6-RET fusion gene.

Methods
We investigated the effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on the susceptibility of CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, sorafenib, vandetanib and E7080)

Results
CCDC6-RET lung cancer cell was highly sensitive to RET inhibitors. EGF receptor (EGFR) ligand, EGF, activated EGFR and triggered resistance to sunitinib, E7080, sorafenib and vandetanib by transducing bypass survival signaling through Erk1/2 and Akt. The resistance to RET inhibitors was not induced by EGF in EGFR siRNA-treated cells. EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors even in the presence of EGF. Endothelial cells, which were known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cell to RET inhibitors, an effect inhibited by anti-EGFR antibody (cetuximab). HGF, MET receptor ligand, affected a drug response of sunitinib, not sorafenib, vandetanib and E7080.

Conclusion
Paracrine receptor activation by ligand from the microenvironment may trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, suggesting that receptor ligands from microenvironment may be additional targets during treatment with RET inhibitors. LC-2/ad cell line was sensitive to E7080, which inhibited RET and its downstream targets. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Background
Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. The Succinate Dehydrogenase Complex, Subunit B, IronSulfur Protein (SDHB), is a subunit of the Succinate dehydrogenase or succinate-coenzyme Q reductase (SQR) or respiratory Complex II, and has recently been identified by us as an important element in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of SDHB by RT-PCR. Levels were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR and western blot. Finally the expression of SDHB in a large cohort of MPM specimens with clinical data was asessed by IHC.

Results
Expression of SDHB occurs in all cell lines. Significantly higher expression of SDHB is observed in the malignant tumour material versus benign pleura. There was a trend towards better survival for patients expressing higher levels of SDHB, but this was not statistically significant.

Conclusion
SDHB, a key member of oxidative energy metabolism is significantly altered in MPM. This may have important future implications for the management of MPM.

Background
Environmental factors contributed toward lung carcinogenesis. Tobacco-smoking is the major etiological factor related with lung cancer in 90% in the world compared with Mexico with 66% of cases. In previous works we have demonstrated that other factors such as chronic exposure to wood smoke (WSE) are related to non-small-cell lung cancer (NSCLC) and it´s clinically and pathologically different from lung cancer arisen from tobacco-exposure, regarding on tumor histology, mutation profiles, brain metastasis incidence, response rate and overall survival. This work is aimed to estimate expression profiles related WSE on adenocarcinoma histology associated to pathological mechanisms that differ from other carcinogenic factors, such as tobacco exposure.

Methods
This study used clinical, longitudinal, prospective and observational. From January 2008 to June 2011, patients were admitted in Cancerology Institute with lung adenocarcinoma in stage IV, eligible for the inclusion. Clinical variables, WSE, gender and age. WSE was defined to being exposed to fumes by burning wood in fireplaces and wood stoves for five years for at least 4 hours per day. The WSE index was calculated multiplying the number of daily hours exposed by years of exposure. Collaboration agreements with the Institute of Medical Genomics and approved within the Health Research Sectorial Fund, CONACyT-México (SALUD-2009-01-115552). Primary biopsies were taken by guided tru-cut needle by tomography. Samples were analyzed by the Pathology for histological/quantification of neoplasic celularity, and stored at -80°C. RNA was extracted from tumor biopsies. RNA integrity (RIN>6) was analyzed using the Agilent 6000. We used an Affymetrix Human GeneChip® 1.0 ST. Two-Cycle Target Labeling was followed for microarrays. Analysis was done by Affymetrix console and “R” software language. Matrices employ 29 microarrays with experimental contrasts. Differential expressed genes were analyzed by linear model that analyze contrasts between experimental contrasts. The Partek Genomic Software 4 and SAM (Significant Analysis) was used for microarray comparisons and integration of genomic data.

Results
Using computational genomics using fold changes and confiability data we found differences in genomic expression. In brain metastasis (BM) patients the Fold Change values >1 shows 6 upregulated and 11 downregulated genes compared with or without WSE. Patients without BM the Fold Change values >1 shows 90 downregulated and 7 upregulated genes with or without WSE. Prior both analysis were confirmed with B statistics data (significant value ≥0). No difference were found using computational Genomics SAMS (False discovery rate), neither PARTEK (p-value, fold change), on gene expression evaluation.

Conclusion
Our results demonstrate that gene expression profiles are different in advanced lung adenocarcinoma patients with brain metastasis with or without WSE. These results could be used for predictive models related with BM in advanced lung cancer. The best genomic statistical value was obtained with Affymetrix console and “R” software language in this work. These results must be confirmed by increasing the experimental samples and validating in an independent cohort by PCR.

Background
Acquisition of ability to uncontrolled migration is one of the fundamental properties of cancer cells, enabling them to infiltrate tissues and metastasize. PAI-1 is the major physiological inhibitor of urokinase (uPA), which plays a key role in migration and invasion of tumor cells.

Methods
The aim of present study was to analyze the impact of increasing concentrations of PAI-1 mutated forms: VLHL PAI-1 with very long half-life time, Vn neg PAI-1 - devoid of affinity towards vitronectin and wPAI -1 on lung (A549, NCI-H1299) and prostate (LNCaP, DU145) cancer cells invasive activity. Selected cell lines are characterized by different (normal and high) urokinase production.

Results
No effect of PAI-1 proteins on invasiveness of lung cancer cells was observed, while dose-dependent significant inhibition was demonstrated in both prostate cancer lines (DU145 and LNCaP) cultured with VLHL PAI-1 (respectively p<0,05 and p<0,01) and Vn PAI-1 neg (p<0,05). Not surprisingly wPAI-1 significantly stimulated prostate cancer cells invasiveness in all concentrations.

Conclusion
PAI-1 inhibitory effect on prostate cancer invasive activity is associated with anti-proteinase activity. Lung cancer cells invasiveness regulation seems not to be PAI-1-urokinase regulated.

Background
Lung cancer is the first cause of death among all cancers in the world and every year more than 1.6 million patients die from lung cancer. In Polish population morbidity and mortality from lung cancer persist in the first location and amount respectively 21.4% and 31.2% of all cancers. Over the last 30 years the development of targeted therapy significantly influenced the routine practice in pathology. The treatment based on identification of the type of tumor is still crucial information however the importance of predictive or prognostic molecular markers has become significant step in the choice of treatment. Identification of mutations in the EGFR gene in non-small cell lung cancer (NSCLC) fully illustrates the impact of molecular biology in treatment decisions. The use of one of the small molecule tyrosine kinase inhibitors, gefitinib or erlotinib depends on confirmation presence of activating somatic EGFR mutation.

Methods
The aim of the study was to evaluate the EGFR mutations in two types of material cytological and histological. 189 histological, paraffin-embedded materials as well as 12 fresh and 72 fixed cytology specimens obtained by trans-thoracic, computed tomography supported biopsy or during bronchofiberoscopy were included. Material and methods. 273 patients with confirmed NSCLC were entered into the study. Each specimen contained at least 50% of tumor cells. The macrodissecion was performed on histological specimens to maximize tumor cells content. DNA was extracted from both types of material and the EGFR mutation was analyzed in exons 18, 19 20 and 21 using the direct sequencing method

Results
The mean age of patients was 61.5 years (range 25 – 84 years) with males predominance (151 males vs. 122 females). The histological types included: adenocarcinoma 186 (69%), squamous cell carcinoma 44 (16%), NSCLC, not-otherwise specified 30 (11%), large cell carcinoma 9 (3%) and adenosquamous carcinoma 4 (1%). Both types of material were equally sufficient to evaluate EGFR mutations. The ratio of molecularly non-diagnostic material due to DNA degradation or too scant DNA probe was respectively for cytological and histological material 1.2% vs. 4.75% (p<0.01). The percentage of EGFR somatic mutations were 10.62%. Females suffered more frequently from adenocarcinoma (females 70.49% vs. males 66.23%, p <0.01) and had significantly higher rate of EGFR mutations (females 17.21% vs. males 3.97%, p<0.01). Mutations in exons 21 and 19 together accounted for 87% of all types of mutations of the EGFR gene. Apart from these, two patients had co-existence of activating/inhibiting mutations: L858R (exon 21) with T790M (exon 20) and G719C (exon 18) with S768I (exon 20).

Conclusion
This study allows for the first time to estimate an EGFR mutation frequency in Polish population. Moreover the results have strengthened the importance of reusing cytological material in molecular evaluation. Cytological material recovered from fixed preparations and stained with hematoxylin and eosin showed comparable DNA quality to fresh tumor cells. The cytology especially fine needle aspirates as well as small biopsy material provide molecular biological information which is a key issue for the targeted treatment options.

Background
NLBP (novel-LZAP binding protein, also known as KIAA0776 or Maxer) was originally indentified as a reciprocal binding partner of LZAP. LZAP possesses dual functionality as a tumor suppressor and an oncogene in human cancers. Despite its strong association with LZAP, the biological roles and underlying molecular mechanisms of NLBP remain unknown. Recent studies indicating a possible link between p120 catenin (p120ctn) and NLBP in tumorigenesis.

Methods
We examined NLBP expression in 29 non-small cell lung cancers using immunohistochemical staining and immunoblotting, as well as an in vitro analysis of NLBP activity during cell proliferation in lung cancer cell lines. We next examined whether p120ctn plays a role in the molecular mechanism underlying NLBP activity

Results
In this study, we found that NLBP expression was increased in human lung adenocarcinomas, particularly early-stage adenocarcinomas, compared to squamous cell carcinomas. Furthermore, the overexpression of NLBP in H1299 cells resulted in an increase in cell proliferation. We next identified p120ctn as a novel NLBP-binding protein, and identified the reciprocal binding regions of these proteins in lung cancer cell lines. We also demonstrated that NLBP promotes cell proliferation by regulating the stability of p120ctn. This effect is mediated through the inhibition of ubiquitination, which occurs as a result of NLBP binding to p120ctn, leading to an increase in protein stability. Figure 1

Conclusion
In conclusion, the data presented here show that NLBP may act as a novel oncogene in the early stages of lung adenocarcinoma, and may promote cell proliferation in lung adenocarcinoma through interactions with p120ctn. The interaction between NLBP and p120ctn provides new implications for interplay between signaling pathways and protein networks in tumor development

Background
Among many molecular markers associated with tumor progression, the Wnt family has been shown to encode the multifunctional signaling glycoproteins that are involved in the regulation of a wide variety of normal and pathological processes in lung epithelium. Defects in Wnt signaling are associated with several tumor types, including lung cancer. Numerous reports have demonstrated aberrant Wnt (wnt-1, 2, 5a, and 7) activation in lung cancer. The Wnt signaling pathway has been extensively investigated in NSCLC, but not in lung carcinoid tumors.

Methods
Sixty formalin-fixed paraffin embedded typical (TC) and atypical (AC) human lung carcinoid tumor samples were analyzed by qRT-PCR for Wnt-1, 5a, 7a, 10b, 13 and Fz2 and Fz5 gene expression as potential tumor-associated markers, using SYBR Green-based qRT-PCR.

Results
The heatmap (Figure) shows low expression of Wnt genes (Wnt-1, 5a, 7a, 10b and 13) in almost all samples analyzed. Otherwise, Wnt-ligands are frequently positive, strongly positive for Fz2 and with lower levels for Fz5 in both carcinoid types (AC and TC). Wnt-1 and 13 expression was found negative in all TC and AC samples; Wnt-7a was expressed in 0% AC and 4.55% TC; Wnt -10b was expressed in 0% AC and 11.36% TC; and Wnt-5a was expressed in 18.18% AC and 20.45% TC. Regarding Wnt receptors, Fz2 was positive in 90.9% AC and 95.45% TC, and Fz5 was positive in 45.45% AC and 20.45% TC. Figure 1

Conclusion
In the current study, we assessed the clinical-pathological implications of changes in Wnt expression across a serie of lung carcinoids. Our data indicate that Wnt gene family is scarcely expressed both in TC and AT lung carcinoids. Conversely, ligands (Fz2, Fz5) are positively expressed in both types of lung carcinoids. Whereas the Wnt pathway has been shown to have a role in NSCLC, there are limits on the contribution of this signaling mechanism in lung carcinoids (TC and AC).

Background
EGFR mutation and EML4/ALK rearrangement have become standard genetic tests for patients with advanced adenocarcinoma of the lung. Other driven mutations and genetic abnormalities are still investigational including c-Met expression. c-Met overexpression has shown to confer resistance to EGFR TKI. By targeting c-Met, studies suggest that lung cancer cells may respond again to EGFR TKI therapy. The incidence of c-Met in NSCLC is not clear yet as well as how it does interact with other biomarkers.

Methods
A retrospective study of 32 consecutive biomarker profile tests from patients with either unresectable or stage IV chemonaive adenocarcinoma of the lung were analyzed. We adopted a comprehensive biomarker panel as part of a common decision made from a lung cancer consortium. All patients’ samples were sent to Response Genetics Inc with an order to perform a panel of 9 biomarkers. We report the incidence of c-Met expression and its co-existence with other biomarkers.

Results
The tumor sample of 32 patients with unresectable or stage IV chemonaive adenocarcinoma of the lung were analyzed. All patients were Caucasian; gender: 18 females/14 males; median age: 76 (range, 58-89); distribution of staging was: stage I: n=3, stage II: n=1, stage III: n=7 and stage IV: n= 21 patients. c-Met was ordered in 26 patients: 9 patients had c-Met overexpressed (34%), 5 had low-expression (19%); 12 had no enough tissue for the test. None of the c-Met high expression patients had EGFR mutation; in this cohort, 4/27 patients had EML4/ALK (15%) rearrangement; 2 of them had high c-Met expression; the other 2 did not have enough tissue. The presence of K-ras (25 tumor samples tested), PI3K (25 tumor sample tested), and ROS-1 (n=26 tumor samples tested) mutations were 12/25 (48%), 1/25 (4%), and 0/26, respectively.

Conclusion
Our cohort is small, and this may explain the high EML4/ALK and low EGFR incidence (sample size and patient selection). However, it is crucial to increase our knowledge on how these biomarkers interact themselves and what kind of role do they play in lung carcinogenesis. Some novel trials propose a double target approach in patients who have two pathways overexpressed. Hence, it is imperative that we develop a molecular phenotype data to understand these co-existence genetic phenomenon. Our cohort confirmed the high incidence of K-ras mutations in adenocarcinoma of the lung and also showed that c-Met high expression may be common in chemonaive patients without prior exposure to EGFR TKI. Interestingly, our 2 EML4/ALK patients also had c-Met highly expressed. PI3K and ROS-1 mutations seem to be rare genetic abnormalities.

Background
Squamous cell carcinoma is the most commonly diagnosed lung cancer type in Turkey; however lung adenocarcinoma diagnosis has risen among women and nonsmokers. MPM is common in Turkey because of the high asbestos exposition rates. Mostly, lung adenocarcinoma cannot be clearly differentiated from malignant pleural mesothelioma (MPM). Because of The limitations in the immunohistochemical methods there has been a growing interest in the use of gene expression profiling for diagnosis in many cancers. So we aimed to evaluate the gene expression profiles in tumor cells by using RT-PCR array, between two separate groups of lung adenocarcinoma and MPM patients.

Methods
Ten newly diagnosed patients with adenocarcinoma and paraffin-embedded tissues of 12 patients with MPM were included in this study without considering gender differences. Eight healthy individual were recruited as a control group. After processing the fresh samples of lung adenocarcinoma stored at -80°C for RNA isolation, cDNA synthesis and the expression of 84 genes were associated with DNA repair were analyzed with RT-PCR Array. Paraffin tissues of patients with MPM were deparaffinized and the same procedure was applied. Fold change values of gene expressions in each group are calculated in “SA Bioscience” data analysis expression page.

Results
ACBT, B2N, GADPH AND RPLPO genes were identified as housekeeping genes. Table 1 and 2 shows the comparisons of fold change values ​​of the gene expression differences between lung adenocarcinoma, MPM and control groups.

Table 1: Fold changes ​​of the gene expressions of lung adenocarcinoma and MPM tumor cells comparing to control group
Gene	Adenocarcinoma/control fold change	MPM/control fold change	p value (comparing to control group) (adenocarcinoma/MPM)
APEX2	1,6763	6,1243	>0.05 / >0.05
BRCA1	9,5919	20,2646	>0.05 / 0,01558
BRCA2	4,3804	10,8169	>0.05 / 0,012071
CCNH	2,3922	4,4773	>0.05 / 0,032102
CDK7	3,909	15,4192	>0.05 / 0,019161
LIG4	2,2608	13,7822	0,044834 / >0.05
MLH1	2,5581	7,5515	>0.05 / 0,013792
MLH3	5,9579	15,4275	>0.05 / >0.05
MSH3	2,2494	10,2785	>0.05 / >0.05
MSH4	6,5356	12,0767	>0.05 / >0.05
NEIL3	14,3334	80,5092	0,015299 / >0.05
PARP1	0,9019	2,0543	>0.05 / >0.05
PARP2	4,7127	7,4604	0,043874 /0,009579
PARP3	2,3613	8,2119	>0.05 / 0,049911
PMS1	1,8236	7,3582	>0.05 / 0,039034
RAD50	2,6223	10,2765	>0.05 / 0,03758
RAD51	2,6223	10,2765	>0.05 / >0.05
RAD51B	4,3683	16,7622	>0.05 / >0.05
RAD51D	3,2769	7,0688	>0.05 / >0.05
RAD52	2,0126	5,1099	>0.05 / >0.05
RPA3	2,8581	7,9353	>0.05 / >0.05
PRKDC	0,5309	2,9778	>0.05 / >0.05
SMUG1	6,7053	18,0914	>0.05 / >0.05
TREX1	0,5115	4,1669	>0.05 / >0.05
UNG	7,422	26,6752	0,027669 / 0,009662
XPA	2,0485	8,8342	>0.05 / >0.05
XRCC2	5,6758	17,6367	>0.05 / >0.05
XRCC4	5,9765	17,0836	>0.05 / >0.05

Table 2: The genes that fold change values ​​were statistically significant (p <0.05) in MPM and adenocarcinoma tumor cells relative to each other
Gene	MPM / Adenocarcinoma fold change	P
CDK7	4.39	<0.05
MLH1	5.32	<0.05
TREX1	9.29	<0.05
PRKDC	7.64	<0.05
XPA	5.54	<0.05
PMS1	5.19	<0.05
UNG	4.93	<0.05
RPA3	2.97	<0.05

Conclusion
We showed that adenocarcinoma and MPM tumor cells have different expression profiles of DNA repair genes. Our study suggests that TREX1, PRKDC, PMS1 genes can be significant in support of differential diagnosis between MPM and lung adenocarcinoma.

Background
αB-Crystallin has been shown to correlate with the invasion of several tumors. In this study we investigate the role and mechanism of αB-Crystallin in non-small cell lung cancer (NSCLC).

Methods
Twelve cases of NSCLC and matched nontumorous samples were used to analyze αB-Crystallin expression at the level of protein. Then, we up- and down-regulated the expression of αB-Crystallin in NSCLC cells with specific vshRNA and αB-Crystallin cDNA , and assessed the role of αB-Crystallin in the proliferation, invasion of NSCLC cell line, Furthermore, The molecular mechanism of αB-Crystallin in NSCLC cells were determined by genetic and functional screens. Finally, expression of αB-Crystallin was further examined by Immunohistochemistry (IHC) in tissue microarray (TMA) consisting of 208 cases of NSCLC, and the prognostic role of αB-Crystallin in NSCLC was evaluated by Kaplan-Meier and Cox regression analysis.

Results
The expression of αB-Crystallin in NSCLC tissues was much higher than those in nontumorous samples, and forced loss of αB-Crystallin expression suppressed the invasive potential of lung cancer cell lines, whereas up-regulated αB-Crystallin expression enhanced the cells invasion both in vitro and in vivo. Furthermore, we demonstrated overexpression of αB-Crystallin enhanced the invasion ability of lung cancer cells by resulting in lung cancer cells epithelial-mesenchymal transition (EMT) through ERK1/2/slug pathway. Clinically, we showed that αB-Crystallin overexpression is associated with lymph node metastasis and poor prognosis of NSCLC. Together, our findings indicate that αB-Crystallin may represent a potential therapeutic target and a novel prognostic marker of NSCLC.

Conclusion
Our findings indicate that αB-Crystallin may represent a potential therapeutic target and a novel prognostic marker of NSCLC.

Background
The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer.

Methods
In this study, the function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The PAX6 mRNA level in 53 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR.

Results
Suppression of PAX6 expression inhibited cell growth and colony formation by A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was suppressed. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines.

Conclusion
Our data support the hypothesis that PAX6 accelerates cell cycle progression by potentiating G1-S progression.

Background
NSCLC is a heterogeneous neoplasm comprising several histologic types, etiology, genetics, survival and response to therapy. Accurate analysis of these subtypes has increased sample requirements, which is challenging in the era of minimally invasive procedures. A recent survey of 90 US pathologists presented at ASCO 2013 meeting, concluded insufficient sample availability in 6% of all NSCLC samples recently handled by these pathologists. Moreover, subcellular localization of marker expression linked to tumor pathobiology necessitates methodological advancement. With the development of a new platform that allows in situ, multiplexed sub-cellular analysis of over 60 proteins, this project aims to demonstrate the feasibility of detailed in situ molecular profiling and perform comparative analysis of known cancer pathways and prognostic markers on the same serial section.

Methods
Multiplex immunofluorescence staining and imaging of over 30 biomarkers, including several RTKs, cell adhesion molecules, select members of PI3K, MAPK/ERK, JAK/STAT pathways, angiogenesis, hypoxia, proliferation and chemotherapy resistance markers were performed on replicate FFPE tissue microarrays (TMA) from 382 samples. Cell-level and subcellular-level marker expressions were quantified using image analysis algorithms and compared between serial sections. Associations between marker expressions and histological subtypes and survival were investigated in European male smokers. Multivariate analysis was performed using logistic regression and Cox proportional hazard models on over 300 quantitated features of marker expression. All models controlled for age. Serial sections were modeled separately and combined to improve confidence in associations. EGFR and cMET positivity was evaluated using whole cohort median expression values to define positive cells and the summary statistics are reported using 10% positive cells as cutoff for characterizing positive samples.

Results
In concordance with previous reports, differential expression of RRM1, CK5 and CK7 was observed in SCC vs AD in the high grade, early stage male smokers (N=86). With a 10% cell positivity threshold, 72.7% (76.3%, serial section (SS)) of all male smokers (N=183 (190, SS)) were positive for EGFR. EGFR positivity was higher in SCC, 83.9% (86.0%, SS) compared to AD 54.9% (61.8%, SS). Opposite was observed for cMET with 81.7% (78.9%, SS) of AD characterized as positive compared to only 58.0% (57.0%, SS) SCC. Among the several previously reported prognostic markers evaluated in this study, only CA9 expression was associated with overall patient survival with a hazard ratio of 1.47, p-value 0.0005 (N=278). Again, analysis of serial section produced a similar result confirming the robustness of the platform.

Conclusion
The study demonstrates the capabilities of multiplexing technology (MultiOmyx[TM]) for assessment of limited lung samples, encompassing topographic expression features and the ability to observe relationships between markers through in situ pathway profiling. Additionally, by evaluating markers on exactly the same sample set (same section), a direct comparison of their relative significance in predicting course of disease is now feasible.

Background
Lung cancer is a leading cause of cancer related deaths, 80% of which are classified as non-small cell carcinomas (NSCLC). Differentiating the two main sub-types of NSCLC, adenocarcinoma (AD) from squamous cell carcinoma (SCC) is crucial for therapeutic decision-making. Current methods for characterizing subtypes may involve DAB stains on up to 7 tissue sections, depending on complexity of diagnosis. This process may deplete precious tissue required for molecular studies sequencing and other predictive markers. The goal of the current study was to measure 11 proteins on a single section using a novel multiplexed immunofluorescence (IF) technology (MultiOmyx[TM]) and evaluate performance of analytical workflows in automatic biomarker scoring and in AD, SCC discrimination, with reference to the Pulmotype® test of 5markers

Methods
The protein markers included in the study were comprised of the five antibodies from the Pulmotype® test - Muc1, CK5/6, TRIM29, CEACAM5 and SLC7A5. Six additional markers TTF1, p40, CK7, CK20, p63, NapsinA were selected based on literature reports. These markers were applied to two separate cohorts of NSCLC cases. The entire set of 11 markers was multiplexed on a 378 core tissue microarray (TMA) containing 213 cases of AD or SCC diagnosis (cohort 1). A second 74 core TMA with 50 cases of AD or SCC was stained with the Pulmotype® markers. Manual scores were generated for the immunofluorescence protein images and DAB stained serial tissue sections were used to generate manual ground truth protein expression scores. The first cohort was used to model diagnosis of AD or SCC using an implementation of Breiman and Cutler’s Random Forest and compared to the performance of a previously published lung classifier using manual DAB scores. Image and data analysis algorithms were developed to aid automated biomarker scoring. These algorithms segment the immunofluorescence images into tumor and stromal areas and compute a large number of biomarker-related metrics. Linear regression modeling was used on a down-selected set of metrics to generate automated protein expression scores per biomarker for the second cohort.

Results
Manual scoring of all 11 targets demonstrated excellent concordance between fluorescence and DAB. Concordance was also demonstrated between manual DAB scores and automated IF metrics for the five Pulmotype® markers with an overall sensitivity and specificity of 95% and 87%, respectively. Statistical modeling indicated that 9 (of the 11) multiplexed markers provided 97% specificity and 90% sensitivity in classifying AD versus SCC. The observed 7% indeterminate rate measures well against the existing published indeterminate rates for the Pulmotype® test (11%) and the classic IHC marker combination TTF-1/p63 (29%).

Conclusion
Multiplexed analysis of a single tissue section allows maximum use of limited sample and enhanced protein profiling in context of tissue histology. We have shown that differential diagnosis of AD and SCC may be achieved using a multiplexed panel of markers in a single tissue section, when compared to the Pulmotype® test panel. Concordance between fluorescence and DAB shows transferability of the two detection methods. Furthermore, we demonstrated that image and data analysis tools can be applied for consistent automatic biomarker scoring.

Background
Figure 1Epidermal growth factor receptor (EGFR) mutation status is the primary issue on the appropriate therapy of EGFR-tyrosine kinase inhibitor (EGFR-TKI) in non-small lung cancer. The point mutation of EGFR T790M (C→T) is known to be an acquired and the most common resistance against EGFR-TKI. Highly sensitive mutation detection system has been desired considering the difficulty in　obtaining tissue specimens during disease progression. Eprobe is new fluorescence labeled probe with high affinity toward target ssDNA and works as competitive probe (Figure 1). Here we describe a novel method to identify T790M mutation using Eprobe on real-time monitoring of PCR and melting curve analysis.

Methods
Figure 1Eprobe was designed to bind wild type allele including T790M region with competing primer, which enriches mutant allele amplification (Figure 2). The T790M mutation was detected by melting curve analysis following real-time monitoring of PCR. We verified detection ability by genomic DNA containing wild type and mutated EGFR T790M (cell lines H1975) gene. For clinical evaluation, 338 tumor tissues from the patients with lung adenocarcinoma, of which EGFR gene mutation status had been revealed by the nucleic acid-locked nucleic acid PCR clamp (PNA-LNA PCR clamp), were assayed by Eprobe method. We compared the two methods on T790M mutation assay.

Results
The T790M mutant genome could be detected when it accounted for as little as 0.5% of a mixture of wild type genome by enrichment of mutation amplicon. The activating EGFR mutation (exon19 deletion, L858R, and L861Q) had been detected in 143 out of 338 samples (42.3%) but no T790M mutation was identified by PNA-LNA PCR clamp. Among 143 samples harboring activating EGFR mutation, T790M mutation was identified in 2 samples (1.4%) by Eprobe method.

Conclusion
The Eprobe method is sensitive for detecting EGFR T790M. Since Eprobe works in simple manner, current method is expected to apply to other gene detections.

Background
MicroRNAs (miRNA) are endogenous, small regulatory nucleotides that negatively regulate gene expression post-transcriptionally. They are involved in a wide range of cellular functions, including growth, development, and apoptosis. Given their widespread roles in biological processes, changes in their expression are likely to be associated with the development and progression of diseases. Understanding their patterns of expression could provide new insights into complex biological processes and the possible clinical implications of miRNA dysfunction. As such, global miRNA expression profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used microarrays and quantitative real-time PCR (qRT-PCR). With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options.

Methods
To compare the attributes of different profiling methodologies, five pairs of non-small cell lung cancer cell lines and their corresponding xenograft models were analysed using a microarray platform (Illumina Human microRNA Expression Profiling v2), NGS (Applied Biosystems SOLiD™ 3 Plus and 4 Systems and Illumina HiSeq2500), and the NanoString nCounter System (Human miRNA Expression Assay v1). The platforms were evaluated according to the following criteria: (i) inter-platform concordance, (ii) concordance with an independent validation method, qRT-PCR, and (iii) detection of differentially expressed miRNAs in a biologically relevant setting.

Results
Inter-platform correlations ranged from 0.62 – 0.80, while correlations with qRT-PCR, the current gold standard for validating expression profiling studies, was highly statistically significant for all platforms, with Spearman’s ρ ranging from 0.79 – 0.86. The accuracy in detecting differential expression was the highest for NGS (88%). Overall, sequencing technologies had the greatest detection sensitivity, along with the largest dynamic range of detection, and highest concordance with qRT-PCR. To assess the technical reproducibility of NGS, the same set of samples was profiled in duplicates. Using unsupervised hierarchical clustering, technical replicates for each biological sample clustered together, with Spearman’s ρ > 0.93 in all cases. miRNA analysis of formalin-fixed paraffin-embedded tissue (FFPE) was also evaluated. FFPE samples represent a rich source of archived specimen for retrospective studies of human disease. The feasibility of miRNA analysis with FFPE tissues would offer many opportunities to evaluate such large banks of archival materials. Three pairs of matched frozen and FFPE xenografts tumors were profiled using the Illumina HiSeq2000 platform. Hierarchical clustering showed similarity between expression profiles of paired frozen and FFPE samples (Spearman’s ρ > 0.88); whereas, samples of different biological origin were less correlated (Spearman’s ρ < 0.81).

Conclusion
These results show the superior sensitivity, accuracy and robustness of NGS for global miRNA profiling in both frozen and FFPE tissue. Although microarrays and qRT-PCR have been used more extensively for expression profiling and are highly reproducible, they are limited to the detection of only known targets identified at the time of assay development and manufacturing. With the rapid increase in miRNAs being discovered and deposited in public databases, sequencing will offer a more comprehensive view of the miRNA transcriptome.

Background
The personalized therapies against specific genetic targets in tumoral cells like EGFR mutations or EML-ALK translocation, or the development of minimized invasive procedures for diagnosis and cancer staging have dramatically improved the outcome of patients with lung cancer. The combination of the wide use of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and genetic testing to individually tailor chemotherapy should become the standard of care. However, the rapid evolution of personalized medicine is toward the routine testing of high number of biomarkers per patient. Therefore, we have tested whether the recently developed DNA arrays and multiplex technologies could increase the suitability of very small size biopsies performed by EBUS-TBNA for the high-throughput molecular diagnosis

Methods
Fourty consecutive EBUS TBNAs with histologically diagnosed lung adenocarcinoma were tested using the high throughput array and sensitive mass spectrometry technology (Massarray, Sequenom) for the detection of 216 mutations in 26 cancer genes (Lungcarta).Each of the 40 DNAs extracted from only one 19- to 22-gauge needle aspiration spread on a glass slide per patient has been successfully amplified and analyzed within one-day experiment for all of the 238 mutations

Results
Five patients (12.5 %) exhibited EGFR mutations and received either gefitinib or erlotinib, 1 of them exhibiting the T790M resistance mutation. One patient had the BRAFV600E mutation confirmed 1-y later on a skin metastasis and thus entered a phase II trial to receive anti-BRAF therapy. There were EBUS-TBNA exhibiting mutations in KRAS (n=7), PI3K (n=1) and cMET (n=1), and insertion in HER2 (n=2).

Conclusion
In conclusion, only one 19-22 gauge needle aspiration is suitable in routine care for the detection of at least 238 mutations, to guide treatment decisions and offer patients to benefit from current and future individually targeted drugs

Background
Circulating tumour cells (CTCs) are present in the blood of a proportion of patients with lung cancer. However, it is currently unclear how suitable CTCs are for use in the detection of predictive genetic mutations. We sought to determine the utility of DNA extracted from CTCs to screen for the underlying primary tumour mutation.

Methods
Using ScreenCell™ MB devices, from 20/01/12 to 25/01/2013, CTCs were captured in peripheral blood of 100 patients who underwent surgery for lung cancer at The Royal Brompton Hospital. DNA was extracted using QIAamp DNA Micro kit (QIAGEN) followed by whole-genome amplification using GenomePlex® SingleCell WGA kit (Sigma). DNA from matched primary tumours was used as reference. Mutation detection in EGFR and KRAS genes was undertaken using cobas®4800 (Roche) and single-strand conformation analysis for BRAF gene. Sensitivity and specificity analyses were undertaken to measure predictive performance of mutation testing in CTCs.

Results
The DNA extracted from CTCs, were of sufficient quality to allow mutation analyses to be successfully performed in 100%, 99%, and 98% of samples for EGFR, KRAS, and BRAF genes, respectively. In CTC DNA, the KRAS mutation rate (codons 12/13 and 61) was 9.1% and concordance with the primary tumour was 78.8%. Six mutations were detected in CTCs, but not in primary tumours, and 13 mutations in primary tumours were not detected in corresponding CTC samples. Three mutations were detected in matched CTC and primary tumour specimens. One mutation in EGFR was detected in CTC DNA and 3 mutations were detected in primary tumours. In all cases, the mutations were detected in discordant specimens. The concordance between mutations detection in CTCs and primary tumours was 95.8%. BRAF V600E mutation was not detected in any sample. In general, the results suggested low sensitivity but high specificity (Table). Due to low number of EGFR mutations detected, test performance results require further validation.

The performance of mutation testing in circulating tumour cells

Statistic	KRAS	EGFR
Sensitivity (95% CI), %	18.8 (4.05-45.6)	0.0 (0.0-70.8)
Specificity (95% CI), %	91.8 (83.0-96.9)	98.9 (94.1-100)
Positive predictive value (95% CI), %	33.3 (7.49-70.1)	0.0 (0.0-97.5)
Negative predictive value (95% CI), %	83.8 (73.8-91.1)	96.8 (91.0-99.3)

Conclusion
The result of our study indicates that the DNA extracted from CTCs can be used to screen for primary tumour mutations with reasonable concordance. Differences in the mutation results from the CTC and primary tumours needs to be explored in more detail and may be due to issues related to processing and / or tumour versus CTC heterogeneity.

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.