Virtual Library

Background:
Stereotactic body radiotherapy (SBRT) has demonstrated high rates of local control with low morbidity and has now emerged as the new standard of care for medically inoperable, early-stage non-small cell lung cancer (NSCLC). However, the impact of lung SBRT on survival in the elderly population is less clear given competing co-morbid conditions. An analysis of the National Cancer Data Base (NCDB) was undertaken to determine whether definitive SBRT in patients 70 and older improves survival relative to observation alone.

Methods:
The NCDB, a retrospective national database capturing up to 80% of all patients treated for cancer, was queried for patients ages 70 or higher with early stage (T1-T3N0M0) NSCLC from years 2003-2006. Overall survival was compared between patients treated with stereotactic body radiotherapy alone and patients receiving no treatment. Extended Cox proportional hazards model was applied to estimate the treatment effect of SBRT.

Results:
A total of 3,147 patients met the selection criteria for this analysis. SBRT was delivered to 258 patients (8.2%) and 2889 patients (91.8%) received no treatment. There was no significant difference in the distribution of Charlson/Deyo comorbidity index scores between the two groups (p=0.076). Multivariable analysis revealed improved overall survival with SBRT compared with observation for the entire cohort (HR 0.64, p<0.001), as well as for each age group as follows: 70-74, HR=0.72; 75-79, HR=0.66; 80-84, HR=0.59; 85 and above, HR=0.56.

Conclusion:
SBRT is associated with improved survival in elderly patients with early stage NSCLC with concurrent comorbid conditions compared to observation alone . The data support the use of SBRT for treatment of elderly patients with early stage NSCLC that have limiting co-morbid conditions.

Background:
To determine the safety and efficacy of lung SBRT in older patients and to compare their outcomes to those of younger patients.

Methods:
Patients with primary lung cancer treated with SBRT were identified from a multi-institutional (5) database of 1192 cases. Details of patient factors, treatment specifics, toxicity and clinical outcomes were extracted from the database. All events were calculated from the end of radiotherapy. Estimates of local (LR), regional (RR), and distant metastases (DM) were calculated using the competing risk method. Cause specific (CSS) and overall survival (OS) were calculated using the Kaplain-Meier method. Outcomes were compared for those <70, 70-79, >=80. Toxicity was graded per CTCAE V3.0. The 90 day mortality was reported for those <70, 70-79, >=80. Univariable analysis was performed to determine associations with CSS in patients aged >70.

Results:
The median follow-up was 1.7years (1-10y) and median age 75 (41-94). There were 364 patients age <70 (28%), 546 age 70-79 (42%) and 387 age ³80 (48%). 621(48%) were female, 1125(87%) were peripheral and 852(66%) were biopsied. There was no difference in baseline SUV (p=0.6), histology (p=0.4), radiation dose (p=0.1), gender (p=0.3) or biopsy rate (p=0.2) among the three age groups. Patients aged >=80 had significantly more T2 tumors 21% vs 23% vs 32 % (p<0.01). There was no difference in 5 year LR (10% vs 11.5% vs 10%, p=0.7), RR (22% vs 10% vs 9%, p=0.1), DM (17% vs 16% vs 21%, p=0.07) or CSS (80% vs 80% vs 75%, p=0.6). Those age ³80 had significantly lower 5 year OS (75% vs 44% vs 23%, p<0.01). The grade 3+ pneumonitis rate was 1.3% vs 1.6% vs 1.5% (p=0.9) in patients ages <70,70-79, >=80 respectively. The 90 day mortality rates for patients aged <70,70-79, >=80 were 1.4%, 2.7%, and 2.6% respectively. In patients aged >70 CSS was associated with tumor size (p<0.01; HR1.4) and baseline SUV max (p=0.03; HR1.04).

Conclusion:
SBRT is a safe treatment modality in elderly patients (aged >80). Despite larger tumor volumes, the tumor control outcome were identical to the younger patients treated with SBRT. All patients, regardless of age, should be considered for treatment of early stage lung cancer (T1-T2) with SBRT.

Background:
An anatomical surgical resection is considered to be the standard of care in fit patients, but non-randomized comparative effectives studies suggest that survival outcomes may be similar following SABR. An antitumor immune microenvironment was found to be a prognostic factor in surgically resected early stage NSCLC. SABR has been reported to activate the immunesystem in malignant diseases via a number of mechanisms. We investigated the impact of both surgery and SABR in early stage NSCLC on the immunesystem, studied in peripheral blood over time.

Methods:
This is a non-randomised trial. Treatment by either surgery or SABR treatment for early stage (cT1-T2aN0M0) were determined by an institutional multi-disciplinary tumorboard, and in accordance with the patient’s preference . SABR was typically delivered in 3-8 fractions in 1-2 weeks, based on risk-adapted radiotherapy schemes that delivered a biologically effective dose of >100 Gy. Surgery generally involved a VATS lobectomy. Blood was collected prior to treatment, and at weeks 1, 2, 3 and 6 after start of treatment. The peripheral blood mononuclear cell (PBMC) fraction was isolated and was stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin, to activate the T cells. Subsequently, the T-cells cells were harvested and analyzed by flow cytometry on the expression of CD4 and/or CD8, granzyme B and interferon (IFN) γ. As PD-1 expression is induced in T-cells after antigen exposure the expression of PD-1 was determined. Changes of population proportions between the different time points were analyzed with the related-samples Wilcoxon signed rank test.

Results:
23 early stage non-small cell lung cancer (NSCLC) patients were included in the study. Of these, 13 patients underwent surgical resection at a mean age (±standard deviation) of 62,9± 8,4 years, and 10 patients who underwent SABR at a median age of 70,0 ±10,4 years. SABR patients had more comorbidities, and a poorer WHO performance score, but clinical tumor stage was comparable. A significant increase in the proportion of IFNγ[+]Granzyme B[+] CD8 T cells (p<.05) was observed at week 2 in the SABR treated group, whereas no difference was found after surgical resection. The PD1[+] fraction of CD4[+] T cells was significantly increased at week 2 in the SABR treated group (p<.05), whereas no differences were seen at two weeks after surgical resection. Proportions of PD1[+ ]CD4 T cells remained elevated in the SABR group at week 3 and 6. A similar trend was observed in the CD8[+] T cell population, although this did not reach statistical significance (p<.1).

Conclusion:
SABR but not surgery, enhances T-cell activation and PD-1 upregulation. The results of our study warrant further investigation as to whether SABR induces an anti-tumor response in patients with early stage NSCLC . The upregulation of PD-1 inherently accompanied with this activation of the immune system potentially warrants combination treatment with PD-(L)1 blockade.

Background:
This study aimed to evaluate the importance of interfraction variations in gross tumor volume (GTV) during stereotactic body radiotherapy (SBRT) for early lung cancer patients and assess its impact on dosimetric GTV coverage

Methods:
Forty-seven consecutive patients undergoing SBRT were treated with 48 Gy in 4 fractions (group 1: n=35) or 60 Gy in 8 fractions (group 2: n=12). For each patient, Cone-Beam Computed Tomography (CBCT) imaging obtained at each fraction and initial planning 4DC (CT) were analyzed. GTVs were delineated on all CBCTs, and individual treatment planning was recalculated on each CBCT. Statistical analyses were performed to compare differences between independent samples: the Mann-Whitney U test was used for non-normal continuous variables analyses between groups and the χ2 test for proportions within each SBRT group. Wilcoxon signed rank test was also used to assess changes in volume, dosimetric parameters, and tumor localization. All significance tests were two-tailed and p<0.05 was considered significant

Results:
A total of 236 CBCTs were processed and analyzed. Median total treatment times were 8 days for group 1 and 19.5 days for group 2. There was a significant tumor volume change between the initial CT and the 1st CBCT (p=0.003) in group 1. This was not found in group 2 (p=0.67). GTV was significantly larger at the 2nd CBCT (p=0.003 for group 1 and p=0.049 for group 2) compared to the 1st CBCT. Volume changes were not significantly different at the 3rd fraction compared to 1st CBCT. In group 1, GTV volume significantly decreases at the 4th fraction compared to the 2nd (p=0.047). In group 2, the significant decrease in volume occurs at the 6th fraction (p=0.026). There was no association between the overall treatment time and tumor volume variations. Taken individually (n=47) 83% of tumors have at least one occurrence of a greater than 15% volume change during SBRT compared to the 1[st] CBCT. Variations of more than 20%, 30% and even 40% were observed in ~60%, 40%, and 17% of treatments, respectively. No factor that would predict a significant volume change during SBRT for the patients analyzed could be identified. In group 1, tumor coverage factor (>95%) for any given fraction deviated no more than 5% from optimised coverage obtained in the initial treatment plan. Although sample size is smaller, there was a trend towards lower tumor coverage factors in group 2 compared to group 1. Conformity index for all tumors still ranged from 3.41 to 13.35 in group 1, and 0.95 to 10.48 in group2, without any association with tumor volume variations or treatment time

Conclusion:
There was considerable variation in tumor volumes and more frequent than initially expected for patients undergoing lung SBRT. However, these volume changes did not significantly impact dosimetric parameters. Whether these results affect treatment and/or patient outcome remains to be investigated in prospective clinical trials

Abstract not provided

Background:
Fibrotic changes after SBRT for stage I NSCLC are difficult to distinguish from local recurrences (LR), hampering proper selection for salvage therapy. Huang et al. (1) defined CT high risk features (HRF) for detection of LR. This study attempts to validate these HRFs in an independent patient cohort.

Methods:
From a multicenter combined database of patients treated with SBRT for stage I NSCLC between 2006 and 2012, 53 LR were detected of which 14 were biopsy proven. The biopsy proven LR (N=14) were matched 1:2 to patients without LR (n=28) based on: 1) dose 2) PTV 3) follow up time 4) central/peripheral location 5) lung lobe. Of the resulting 42 patients 18 were male and 24 female with a median age of 73 years (range 56-89years). Median tumor size, PTV and dose were 2.3 cm (range 1.0-4.9cm), 49cc (range 9-166cc), 48 Gy (range 48-60Gy) in 4 fractions (range 3-8) respectively. Most tumors were peripheral (76%) and located in the upper lobes (55%). Median follow up (FU) was 36 months (range 14-78months) and median time to LR was 18 months (range 12-45months). For all patients, planning CT scans and at least two follow up scans were available. Two blinded observers scored eight HRFs for each scan. Sensitivity and specificity in predicting LR were assessed and compared using Fisher’s exact test. Analysis for best fit was done using AUC.

Results:
Results of sensitivity and specificity are shown in Table 1. The best performing HRF was cranio-caudal growth: sensitivity 86%, specificity 82%. The odds of LR increased on average by 2.6 (95%CI1.5-4.3) for each additional HRF detected, while the AUC was 0.86. The presence of ≥ 3 HRFs resulted in the best cut-off with sensitivity 79% and specificity 86%. Loss of linear margin and bulging margin were scored identical and therefore only the latter was included in the model. The two best combinations of HRFs were: 1) bulging margin & cranio-caudal growth, with a sensitivity of 93% and specificity of 82% or 2) bulging margin & enlarging opacity after 12 months, with a sensitivity of 86% and specificity of 89%. Table 1

CT high risk factor for local recurrence	Sensitivity (%)	Specificity (%)	p-value
Any HRF	93	64	.001
enlarging opacity (≥5mm and ≥20%)	86	68	.003
sequential enlarging opacity	57	89	.002
enlarging opacity after 12 months	71	89	<.001
bulging margin	64	100	<.001
loss of linear margin	64	100	<.001
loss of air bronchograms	7	100	0.33
cranio-caudal growth (≥5mm and ≥20%)	86	82	<.001
new pleural effusion	14	93	0.59

Conclusion:
In this matched group of biopsy proven LR and controls, cranio-caudal growth was the best individual predictor of LR after SBRT. Combining HRF bulging margin with either cranio-caudal growth or enlarging opacity after 12 months resulted in higher sensitivities and specificities than number of HRFs. 1)Huang et al. Radiotherapy&Oncology 2013

Background:
A large Canadian multicentre randomized trial (LUSTRE) has recently opened to determine if stereotactic body radiotherapy (SBRT) to 48 Gy in 4 fractions (peripheral) or 60 Gy in 8 fractions (central) improves outcomes compared to conventionally hypofractionated radiotherapy (CRT) to 60 Gy in 15 fractions in early stage non-small cell lung cancer. Given the rapid diffusion of lung SBRT technology across Canada, a unique radiotherapy quality assurance (RTQA) program was devised to minimize variations in practice. This study describes the RTQA experience to date.

Methods:
Centres participating in LUSTRE are required to satisfy three RTQA requirements prior to being accredited: (a) Respond to a survey describing treatment equipment, planning system details and image guidance parameters in order to confirm that their centre is compliant with protocol guidelines; (b) Assess SBRT delivery accuracy using a thoracic phantom produced by IROC (Imaging and Radiation Oncology Core); and (c) Successfully complete four treatment plans from the developed trial planning guide using SBRT and CRT for one centrally and one peripherally located cancer.

Results:
Currently 13 centres are undergoing RTQA: (a) Surveys have been completed in 8 centres, 2 require revision, and 3 are incomplete. (b) Phantom testing has been completed in 9 centres, 2 are incomplete, 1 has results pending, and 1 is being resubmitted. Although 6/13 centres were identified as having active SBRT programs (>3 patients/month), only 2/6 had completed the IROC phantom prior to study accreditation (most having in-house end-to-end tests). (c) 8/13 centres have successfully submitted their test cases. All 8 submitting centres passed on SBRT/CRT distributions and conformality indices. However, 5/8 centres required resubmission for contouring revisions. In one case, the GTV/ITV was incorrectly contoured. In another case, it was contoured on the incorrect dataset. In the remainder, normal organs (lungs, bronchi, esophagus) had contouring errors, particularly the bronchial tree; contours excluded the major bronchi, and in 2 cases, normal lung parenchyma was included. Some centres did not follow standardized nomenclature for targets and normal organs, as they were likely new to this naming convention. Some issues were related to misinterpretation of the planning guide, prompting the trial group to work with centres to ensure a seamless future workflow.

Conclusion:
Preliminary results show that most well-established lung SBRT centres rely on their own in-house standards, while others are using LUSTRE RTQA to implement new SBRT programs. Our experience shows that when centres participate in an independent review, alterations are recommended that can improve their own existing QA processes, and contribute to standardized practice nationwide. Such an RTQA process can be a model worth considering in future radiotherapy randomized trials, and also when instituting new radiotherapy technologies into existing clinical programs.

Background:
Three prospective randomized control trials (RCTs) comparing stereotactic ablative radiotherapy (SABR) and surgery in early stage non-small cell lung cancer (ES-NSCLC) failed to complete accrual. Numerous other comparative effectiveness studies have been published, but such studies may be more prone to bias, and conclusions may vary based on study quality. The goal of this study was to perform a systematic review of comparative effectiveness studies that compare both treatment modalities in this patient population, to assess study quality and conclusions.

Methods:
In accordance with PRISMA guidelines, a systematic review was conducted on studies reporting on comparative outcomes of surgery versus SABR for ES-NSCLC. Studies published in the English language over a 10-year period (April 2006-March 2015) were identified using PUBMED with an inclusive search strategy, using the National Library of Medicine’s medical subject headings. Eligible study designs included RCTs, population analyses, match pair comparisons, propensity-match score comparisons, retrospective case-control series, decision analyses, and cost-effectiveness analyses. Letters, editorial and systematic reviews were excluded. Abstracts identified were independently reviewed by two investigators to determine eligibility, with discrepancies settled by a third investigator. Using a standardized data abstraction form, study, patient, tumor, and treatment characteristics were abstracted. As patients undergoing surgery and SABR often differ in their baseline characteristics, we determined the proportion of studies reporting statistical adjustment for baseline characteristic imbalances (e.g. matching in patient studies, sensitivity analyses in modeling studies). The Fisher’s exact test was used to determine if there was an association between the use of statistical adjustment and differences in overall survival (OS) findings.

Results:
Of the 568 studies identified by our search strategy, 22 were eligible for analysis. Primary study design was retrospective (n=11), population-based (n=7), or model-based (n=4). Most patient studies (n=17) reported on a statistical adjustment for differences in baseline characteristics, with propensity score matching (n=12) being the most common technique employed. All studies, except for 1, reported details of the type of surgery performed. SABR doses employed ranged from 30 Gy in 1 fraction, to 60 Gy in 3 fractions. The weighted average pathologic confirmation of malignancy rate for SABR patients was 72% (range 22-100%). Of the 20 studies reporting on overall survival, 12 found that SABR and surgery were equal, or sensitive to variability in baseline patient, treatment, or tumor factors. The remaining 8 studies reported an overall survival benefit of surgery over SABR, however, 4 of these studies did not employ statistical adjustments for baseline characteristics. In the other 4 studies reporting overall survival superiority of surgery when controlling for various co-variates, at least one other recurrence endpoint (local, regional, or distant) was found to be equal between surgery and SABR. All but 2 studies stated in their conclusion that future clinical trials are warranted to investigate the role of SABR in the potentially operable ES-NSCLC patient.

Conclusion:
A systematic review of the comparative effectiveness literature indicates that the results of well-controlled studies comparing surgery and SABR argue for clinical equipoise. Results of a pooled analysis of two international RCTs that closed prematurely are expected shortly.

Background:
As Stereotactic Ablative Body Radiotherapy (SABR) is increasingly used to treat early non-small cell lung cancer, a larger proportion of elderly patients are now receiving radical therapy. This review aims to assess whether age significantly influences overall survival (OS) in patients with early stage lung cancer treated by SABR according to a standard provincial protocol, and to determine if a maximum age guideline should be introduced.

Methods:
Using a population database all lung-SABR patients were divided into age categories <70 yo (n=45), 70-74 (n=28). 75-79 (n=39),80-84 (n=33) and ≥85 (n=22). Patient and tumor characteristics were collected including: sex, Charlson comorbidity index (CCI), ECOG performance status, tumor diameter, maximum tumor SUV (SUVmax), forced expiratory volume in 1 second (FEV1), and whether a pathologic diagnosis was obtained. For each cohort, OS from date of SABR was calculated. Variability among tumor characteristics between cohorts was evaluated by Chi-squared test and OS was calculated by Kaplan-Meier.

Results:
185 patients were treated from 2009 to 2013. Median age was 76 (range 49-94). The percentage of patients with pathologic diagnoses and the percentage of males was similar among age categories. FEV1 values, ECOG status, SUVmax values and tumor length were not significantly different among the age categories. Older patients had significantly greater CCI scores (see table p=0.001). Median OS for all patients was 36 months and was not reduced in the oldest cohorts (<70: 34 months, 70-74: 24 months: 75-80: 39 months, 80-84:36 months, ≥85: 36 months).

	Age Categories (years old)
	<70	70-74	75-79	80-84	85+
CCI
0-1	5 (%)	0	0	0	0
2-4	59 (%)	39	35	15	10
5-7	34 (%)	54	57	76	60
8 or more	2 (%)	7	8	9	30

Conclusion:
Based on 5 years of population based data, an age cutoff for lung-SABR is not endorsed. In our treated population, patients ≥85 yo have similar OS as younger patients despite greater CCI scores.

Abstract not provided

Background:
Abdominal compression (COMP) for motion management began with our lung stereotactic body radiotherapy (SBRT) practice. From 11/2009, breath hold technique by automatic breath control (ABC) device is selectively employed typically for fit oligometastatic patients (pts). We now compare local failure (LF) results for COMP versus ABC.

Methods:
Our IRB-approved SBRT registry was queried for pts. treated for either a primary lung cancer (PRIME) or an oligometastatic (OLIGO) diagnosis with a minimum 6 months follow up. SBRT was delivered by a stereotactic-specific LINAC platform with vacuum-bag based immobilization, and infrared-based X-ray positioning system+/- CBCT for image-guidance. With COMP, tumor excursion was limited to <1cm and the ITV was created one of two way dependent on treatment era: 1. Fused GTV excursion CTs from free breathing, fixed inhale and exhale travel or 2. by 4DCT, with PTV created from the MIP ITV after adding a 5mm margin. With ABC, 3 serial CT image sets confirmed target immobilization, with the PTV generated after 5 mm was added to the static GTV. SBRT was delivered either with dynamic arcs or step-and-shoot intensity–modulated beams. SBRT dose/fractionation schedules evolved over time and reflected treatment era, tumor location, clinician preference, and trial-based experience. LF was defined as progressive and increasing CT scan abnormalities confirmed by progressive and incremental increases in a lesion’s SUVs on serial PET imaging, with or without biopsy.

Results:
For the interval 10/2003 to 7/2014, 873 pts with 931 lesions were treated. Overall pt. characteristics were: 455 (52.1%) female; 83.9% Caucasian; median age 73 years (range 37-97); median KPS 80 (range 40-100); median BMI 26.2 (range 12.1-56.3). Overall tumor characteristics were: median tumor size 2.2 cm (range 0.7-10.0); median PET SUVmax 7.5 (range 0.8-59), per RTOG 0813 definitions 234 (25.4%) were central lesions, with no significant tumor differences between COMP and ABC cohorts. 830 (89.2%) lesions were PRIME, 101 (10.8%) were OLIGO. ABC was used significantly more for OLIGO vs. COMP (34.4% vs.8.3%, p<0.0001). Median follow-up and SBRT dose were 16.4 months (0-109.5) and 50 G/5 fractions respectively. Overall crude rate of LF was 9.9%. Use of ABC was not associated with increased LF compared to COMP: hazard ratio (HR)=1.043 [95% CI 0.48-2.29; p=0.92] Three-year actuarial rates of LF for ABC vs. COMP were 13.8% and 16.5%, respectively. After stratifying by OLIGO/PRIME, neither ABC nor COMP was significantly associated with LF. There is a suggestion that centrality may be associated with LF with ABC (HR =2.087, p=0.066)On univariate analysis, BMI, tumor size, PET SUVmax and central location were associated with failure, with size the most significant.

Conclusion:
Although form of motion control overall did not predict for LF in lung SBRT, LF for central tumors may be associated with ABC use.

Background:
Radiation therapy (RT)-induced lung injury is one of the major causes of morbidity in patients with thoracic cancer. Extensive work has been done to understand the predictors of lung injury in patients receiving conventionally fractionated RT. However, less work has been done in the setting of hypo-fractionation. Further, conventional methods to consider lung injury typically assess global lung function (e.g. symptoms, pulmonary function tests), are affected by many other (non-radiation) factors, and are thus non-specific. Single photon emission computed tomography (SPECT) perfusion imaging affords an objective quantitative manner to assess the effects of RT on regional lung function. We herein report the preliminary results of a prospective study to assess the magnitude of RT-induced reductions in regional lung perfusion following hypo-fractionated stereotactic RT.

Methods:
Four patients undergoing hypo-fractionated stereotactic lung RT (SBRT: 12 Gy x 4 fractions or 10 Gy x 5 fractions) had a pre-treatment SPECT (single-photon emission computed tomography) perfusion scan providing a 3D map of regional lung perfusion. Scans were repeated 3-6 months post-treatment. Pre- and post SPECT scans were registered to the planning CT scan (and hence the 3D dose data). Changes in regional perfusion (counts per cc on the pre-post scans) were computed in regions of the lung exposed to different doses of radiation (in 5 Gy intervals), thus defining a dose-response function. SPECT scans were internally normalized such that total counts in the regions receiving <5 Gy were equal between pre- and post-treatment scans.

Results:
3 months post-RT, changes in perfusion are highly variable. At 6 months, there is a consistent dose-dependent reduction in regional perfusion. Average percent decline in regional perfusion was 10% at 15-20 Gy, 20% and 20-25 Gy, and 30% at 25-30 Gy representing a relatively linear dose response with an approximate 2% reduction per Gray for doses in excess of 10 Gy. Subtle increases in perfusion were seen in lung receiving <10 Gy. Figure 1



Conclusion:
Hypo-fractionated stereotactic RT appears to cause a dose-dependent reduction in regional lung perfusion. There appears to be a threshold effect with no apparent perfusion loss at doses <10 Gy, in both normalized and unnormalized dose-response curves. Additional data is needed from a larger number of patients to better assess this issue. This sort of data can be used to assist optimizing RT treatment plans that minimize the risk of lung injury.

Background:
Comparative effectiveness research has demonstrated similar rates of disease control and overall survival (OS) for patients with stage I non-small cell lung carcinoma (NSCLC) who are treated with either surgery or SABR. It was therefore hypothesized that the introduction of SABR might improve patient selection for surgery, lead to the referral of high operable risk patients for SABR, and consequently reduce the lung cancer surgery perioperative mortality rate.

Methods:
Cancer registry data identified all patients with stage I NSCLC who underwent surgery or SABR between 1993-2014 at a Veterans Affairs medical center. Mortality rates from the pre-SABR and post-SABR (after 2007) eras were compared. Clinical records in the Computerized Patient Record System were queried to analyze rates of disease control and overall survival (OS).

Results:
A total of 284 patients underwent surgery for stage I NSCLC in the pre-SABR (n=171) and post-SABR (n=113) eras. The majority of patients were male (96.6%) and the median follow-up was 4.1 years. Operative procedures included a pneumonectomy (n=10), lobectomy (n=206), or wedge resection (n=68). The 90-day mortality rate was 3.2%, whereas the 6-month mortality rate was 7.0%. Comparing mortality rates in the pre-SABR to post-SABR eras, there were no declines at 90-days (3.5% vs. 2.7%, p=0.47), or 6-months (7.0% vs. 7.1%, p=0.36). Patients referred for SABR have included 27 medically inoperable patients and 0 operable patients. The mortality rate after SABR was 0% at both 90-days and 6 months. Comparing SABR and surgery, the rate of disease progression was similar (p=0.47); found in 18.5% after SABR (1 distant, 4 regional), 23.4% after lobectomy (9 regional, 2 regional and distant, 11 distant), 33.3% after wedge (3 local, 3 distant), and 0% after pneumonectomy. Two-year OS was numerically superior with SABR (69.4% vs. 63.1%), although this was not statistically significant (p=0.52).

Conclusion:
The introduction of SABR neither influenced patient selection for surgery, nor reduced the perioperative mortality rate for patients with stage I NSCLC. These data suggest comparative effectiveness research alone may be insufficient to improve outcomes for this disease. Efforts to complete a prospective randomized trial of surgery vs. SABR should not be abandoned.

Background:
Radiation pneumonitis is a potentially life-threatening complication of curative radiotherapy in individuals with lung cancer. Predicting which patients are at higher risk of pneumonitis is constrained by limited understanding of its causes. The aim of this study was to examine patient characteristics and radiological factors associated with increased risk of radiation pneumonitis in individuals with lung cancer receiving curative radiotherapy.

Methods:
Individuals with lung cancer treated with curative radiotherapy between January and June 2009 were identified from our departmental database. Data were extracted on patient sex, age and smoking status, lobe affected by cancer, pathology, T and N stage, radiation dose delivered, the use of concurrent chemotherapy, and the grade of fibrosis present on the diagnostic CT scan. The CT scans were reviewed and the fibrosis scored by two pulmonary radiologists. CTCAEv3.0 toxicity scores were used to grade the pneumonitis. Mann-Whitney, chi-squared and Fisher exact tests were used to determine the impact of the various factors on the risk of developing pneumonitis.

Results:
84 patients were identified who underwent curative radiotherapy for lung cancer between January and June 2009. The minimum follow-up for the cohort was 5 years. One year and 3 year survival were 61.9% and 29.8% respectively. 8/84 patients (9.5%) developed significant pneumonitis (CTCAEv3 grade 3 - 5). 6/22 (27.3%) patients with fibrosis on their diagnostic CT developed grade 3 - 5 pneumonitis compared with 2/58 (3.4%) of patients with no fibrosis on the diagnostic CT (Fisher exact test, p=0.0042). Low grade pneumonitis had no impact on survival (grade 0, median survival 80 weeks, grade 1 - 2, median survival 78 weeks) whereas median survival was reduced to 16 weeks in those with grade 3 - 5 pneumonitis. One out of 8 patients in this group survived one year. Only the presence of fibrosis on the diagnostic CT scan and continued tobacco use affected the risk of developing pneumonitis with fibrosis increasing the risk of developing pneumonitis (relative risk 7.9, p < 0.04) and continued tobacco use reducing the risk (relative risk 0.3, p < 0.02). There appeared to be a trend between the fibrosis score on the baseline scan and the risk of developing pneumonitis which did not achieve statistical significance

Conclusion:
The data from this small study suggest radiation pneumonitis affects approximately 1 in 10 individuals receiving curative radiotherapy for lung cancer. The presence of pulmonary fibrosis on the diagnostic CT scan increased the risk of developing pneumonitis. Consideration should be given to alternative treatment options for these patients.

Abstract not provided

Background:
Stage III NSCLC has large variations in primary and nodal metastatic tumor burden and its treatment is controversial.We determined the benefit to overall survival (OS) of these patients from surgery, and potentially complementary role of FDG-PET/CT-based metabolic tumor volume of primary tumor (MTV~T~), nodal metastasis (MTV~N~), and whole-body (MTV~WB~) in selecting patients for surgery.

Methods:
With IRB approval, we retrospectively reviewed 239 stage III NSCLC cases with pre-therapy FDG-PET/CT scans treated 2004 – 2013 (141 IIIA and 98 IIIB, 115 men and 124 women, median age 67.2 years), and measured MTV~T~,MTV~N~, and MTV~WB~. Kaplan-Meier curves and log-rank test were used for determining survival differences between surgically and non-surgically treated patients. Multivariate Cox regression analyses were conducted. Logistic regression analysis was used to evaluate whether each covariate was associated with receiving surgery(including surgery alone and surgery in combination with chemo or radiation). Wilcoxon rank-sum tests were performed for determining differences of primary, nodal, and whole-body MTV between the groups.

Results:
30% (42/141) of IIIA patients and 10% (10/98) of IIIB patients had surgical treatment (p<0.001, Chi-square test). OS was different between surgically and non-surgically treated patients (p<0.001) at 1 year(86% vs. 54%), 2 years(64% vs. 32%), 3 years(52% vs. 21%), and 5 years(39% vs. 14%), with median survival of 37.3 months vs.13.6 months, respectively. Covariates associated with OS were: surgery (0.43 ≤ HR ≤ 0.46, p≤0.001), log~10~MTV~T~ (HR=1.54, p<0.001), log~10~MTV~N~ (HR=1.63, p<0.001), and log~10~MTV~WB~ (HR=2.06, p<0.001) (Figure 1). Log~10~MTV~T~, Log~10~MTV~N~, and Log~10~MTV~WB ~were inversely associated with receiving surgery, with odds ratio of 0.53(p=0.01), 0.55(p=0.036), and 0.38 (p=0.002), respectively. MTV~T~, MTV~N~, and MTV~WB~ were smaller in surgically treated patients, with median of surgically vs. non-surgically treated patients of 17.8 vs. 55.0, 5.3 vs. 15.1, and 27.8 vs. 92.0 cc, respectively (p≤0.004). Additionally, those with stage IIIB disease were significantly less likely to receive surgery after controlling for age, gender, and MTV. No statistically significant interactions were found between surgery and stage or between surgery and log~10~MTV~T~, log~10~ MTV~N~, or log~10~MTV~WB~.Figure 1



Conclusion:
Surgery and smaller MTV are associated with better OS of stage-III NSCLC patients. Smaller MTV and stage IIIA (vs. IIIB) are associated with receiving surgery. FDG PET/CT-based metabolic tumor volume can potentially inform surgical treatment decisions to further improve survival outcome.

Background:
Recent developments of radiological examinations have been able to bring more accurate information about the biological malignancy of primary tumors in non-small cell lung cancer (NSCLC). The aim of this study is to elucidate the optimal candidate of lobe-specific selective lymph node dissection (LND) that reduces the extent of mediastinal LND according to clinical information including radiological evaluation of primary tumor on thin-section computed tomography (TSCT) and tumor location in clinical(c)-stage I NSCLC patients.

Methods:
Eight hundred and seventy-six patients with c-stage I NSCLC (adenocarcinoma and squamous cell carcinoma), who underwent complete surgical resection between January 2003 and December 2009 were included in this study. For all tumors, we obtained the maximum dimension of the tumor (tumor) and solid component (consolidation) using a lung window level setting from the TSCT scan images, and estimated the consolidation-to-tumor ratio (C/T ratio) for each tumor. We elucidated the lymph node metastatic incidence and distribution according to the primary tumor lobe location and extracted the associated clinicopathological factors with mediastinal lymph node involvement.

Results:
The patients included 490 men and 386 women, with a median age of 66 years old. The radiological findings were ground glass opacity (GGO)-predominant (C/T ratio ≤ 0.5) in 134 patients and solid-predominant (C/T ratio > 0.5) in 742 patients. There were 744 adenocarcinoma cases and 132 squamous cell carcinoma cases, and the incidences of mediastinal lymph node metastasis were 9.9% in adenocarcinoma cases and 4.5% in squamous cell carcinoma cases, respectively. There were no cases with hilar and mediastinal lymph node metastasis in GGO-predominant tumors. There was no significant association of clinical factors with subcarinal lymph node metastasis in right upper-lobe and left upper-division lung adenocarcinoma. In 257 bilateral lower-lobe lung adenocarcinomas, a total of 32 cases (12.5%) were positive for mediastinal lymph node metastasis, and seven cases (2.7%) were negative for subcarinal lymph node metastasis but positive for upper mediastinal lymph node metastasis (mediastinal skip metastasis). An elevated preoperative serum carcinoembryonic antigen (CEA) level (p < 0.001) showed only a significant association with upper mediastinal lymph node metastasis in the patients with bilateral lower-lobe primary lung adenocarcinoma.

Conclusion:
It would be acceptable to perform selective LND in patients with c-stage I NSCLC with GGO-predominant tumor. Elevated serum CEA was associated with upper mediastinal lymph node involvement in lower-lobe primary lung adenocarcinoma with radiologically solid-predominant tumor. We should be careful when applying selective LND to patients with solid-predominant tumor, especially located in the lower lobe.

Background:
Mediastinal lymph node evaluation in Non Small-Cell Lung Cancers (NSCLC) is of paramount importance for optimum planning of treatment. Recently, it was claimed that subcarinal lymph node dissection could be spared in upper lobe NSCLC resections because of the low incidence of metastatic disease. These data, however, were single institution reports. We used complete national data to investigate patterns of unsuspected mediastinal lymph node involvement in patients operated for NSCLC.

Methods:
A national registry was used to identify every single patient operated for NSCLC during an 11-year period (2003-2013). Unsuspected mediastinal lymph node involvement was investigated by comparison of clinical and final pathological nodal stage, and patients with clinical mediastinal lymph node metastases were excluded. For every patient we extracted information about tumor location, histopathology, clinical and pathological TNM-stage. All preoperative imaging and staging investigations were recorded.

Results:
An unsuspected mediastinal lymph node metastasis was found in 426 patients (9.8%) of 3953 patients and 167 (4.4%) had unsuspected subcarinal metastases, which were significantly more frequent in patients with lower lobe or middle lobe cancers compared with upper lobe cancers 7 % (101/1440) versus 1.8% (42/2258), (p<0.01). Preoperative invasive mediastinal staging was used in 57% (n=2253) of all patients and significantly more frequent in upper lobe cancers (62% (n=1400), p<0.01), in patients who had unsuspected mediastinal lymph node metastasis (75% (n=320), p< 0.01) and in patients with subcarinal metastases (74% (n=124), p< 0.01).

Location of the tumor	All patients	Patients with N2 disease	Metastasis in station 7
RUL	1254	121	28 (2.2 %)
LUL	1004	116	14 (1.4 %)
RLL	672	78	52 (7.7 %)
LLL	585	62	36 (6.2 %)
Middle lobe	183	16	13 (7.1 %)
Bilobectomy	255	33	24 (9.4 %)
Total	3953	426	167

Conclusion:
A substantial number of patients undergoing surgery for NSCLC have unsuspected subcarinal mediastinal lymph node involvement despite 74% had preoperative invasive mediastinal staging. Unsuspected subcarinal metastases were most common in lower and middle lobe cancers but were also frequent in upper lobe NSCLC. Subcarinal lymph node dissection should therefore be a routine part of surgery for NSCLC - regardless of tumor location to avoid undiagnosed subcarinal lymph node metastasis.

Background:
Intra-operative lymph node sampling during lung cancer resection is a key surgical performance measure. It informs prognosis, treatment selection for adjuvant chemotherapy and surveillance programs following treatment. This study aimed to analyse the relationship between adequacy of intra-operative lymph node sampling and survival at a large thoracic oncology centre in the United Kingdom.

Methods:
A retrospective review of pathological reports for all patients undergoing lung cancer resections at the University Hospital South Manchester from 01/01/2011 to 31/12/2013 was undertaken. Intra-operative lymph node sampling was assessed for adequacy against standards set out by the IASLC Staging Manual in Thoracic Oncology. Survival data was obtained through national death registry data and provided a minimum of twelve months follow-up for all patients at the time of analysis in January 2015.

Results:
A total of 987 patients underwent surgical resection for NSCLC in the study period. Overall, there was no significant difference in survival between patients with adequate intra-operative lymph nodal sampling and those with inadequate sampling (log rank p=0.66). Median survival times were not estimable for pN0 and pN1 patients as only a small proportion died. However there was a significant difference in the median survival time of pN2 patients according to whether the intra-operative lymph node sampling was adequate or inadequate (Figure 1). Figure 1Figure 2





Conclusion:
Few patients have died in the pN0 and pN1 categories limiting interpretation. As the data matures we expect to see a survival difference according to adequacy of intra-operative nodal sampling that is supported by the differential median survival data of pN2 patients according to nodal adequacy. The data supports that the survival difference is due to inaccurate staging, inadequate resection (R1) and inappropriate omission of adjuvant.

Abstract not provided

Background:
Currently adjuvant chemotherapy is not recommended for patients with completely resected stage I lung cancer. The ability to sub-stratify survival within stage I is an important consideration as it is assumed that survival is heterogeneous within this sub-group. Liang et al recently published a Chinese multi-institutional logistic regression derived model to predict post-operative survival in over 5000 patients undergoing lung cancer surgery for all stages. The aim of our study is external validation of their published nomogram in a British cohort focusing on stages IA and IB to determine applicability in selection of adjuvant chemotherapy within stage I.

Methods:
We retrospectively analysed data from a prospectively collected database from our institutions. Patient variables were extracted and the score individually calculated. Receiver operative characteristics curve (ROC) was calculated and compared with the original derivation cohort and the discriminatory ability was further quantified using survival plots by splitting our (external) validation cohort into three tertiles and Kaplan Meier plots were constructed and individual curves tested using Cox regression analysis on Stata 13 and R 3.1.2 respectively.

Results:
From April 2007 to February 2015 a total of 1442 patients underwent surgery for primary lung cancer at our institution. We excluded 118 patients with carcinoid tumours (not in the original Chinese development set) and 86 patients without complete lymph node assessment leaving 1238 patients for validation. For all patients from stage IA to IIB the mean (SD) score was 9.95 (4.2). The ROC score comparing patients who died versus those that remained alive was 0.62 (95% CI 0.58 to 0.67). This was lower than the 0.71 reported by the Chinese group when split into 1,3 and 5 year survival. When divided into prognostic score tertiles, survival discrimination remained evident for the entire cohort, as well as those for stage IA and IB alone. The P value comparing survival between the middle and highest score with baseline (low score) was P=0.031 and P=0.034 respectively. Figure 1. Survival discrimination within Stage I Figure 1



Conclusion:
Our results of external validation suggested lower survival discrimination than reported by the original group, however discrimination between survival remained evident for stage I.

Background:
Video-assisted thoracoscopic wedge resection of multiple small, non-visible, and nonpalpable pulmonary nodules is a clinical challenge. We propose an indocyanine green (ICG) injection and intraoperative fluorescence detection with a near-infrared (NIR) fluorescence for localization of small sized pulmonary nodules.

Methods:
Fluorescence properties of ICG topically injected into the lung parenchyma were determined using a resected porcine lung and previously reported by the authors. In clinical study, 15 cases of VATS pulmonary resection for small sized pulmonary nodules were enrolled in the study. The ICG mixed with iopamidol was injected into the pulmonary nodules by CT-guided percutaneous injection. ICG fluorescence was visualized by a near-infrared (NIR) thoracoscope, then the target nodule was excised by VATS procedure.

Results:
Topically injected ICG / iopamidol mixture spot remained at the injected point of the lung parenchyma for more than 6 hours in each case, and each ICG fluorescence was identified at the pulmonary nodule with the NIR thoracoscope. Each target nodule was successfully removed with negative surgical margin.

Conclusion:
CT guided ICG injection and intraoperative NIR thoracoscopic detection is a feasible method to localize small sized pulmonary nodules.

Background:
Current British Thoracic Society (BTS) guidelines advocate the use of a global risk prediction score such as Thoracoscore to estimate the risk of death prior to radical surgical management in those with non-small cell lung cancer (NSCLC). A recent publication by Powell et al(1) used the National Lung Cancer Audit (NLCA) linked to Hospital Episode Statistics (HES) to produce a score to predict 90 day mortality. The aim of this study is to validate this score, henceforth called the NLCA score, and compare its performance with Thoracoscore.

Methods:
We identified data on all patients in the NLCA who received curative surgery for NSCLC between 2010 and 2012. We calculated the proportion that died in hospital and within 90 days of surgery. Each person was given a score based on the coefficients and constants in the NLCA score and Thoracoscore. The discriminatory power of both scores was assessed by a receiver operating characteristic (ROC) and an area under the curve (AUC) calculation.

Results:
We identified 2858 patients for whom we had complete data to form our validation cohort. The 90 day mortality was 5%. We generated ROC curves to assess the discrimination of the NLCA score in predicting 90 day mortality and to test the ability of Thoracoscore to predict in-hospital mortality. Area under the ROC curve was 0.68 and 0.60 respectively. We performed a post hoc analysis using data from the NLCA on all 15554 patients who underwent curative surgery for NSCLC between 2004 and 2012 to derive summary tables for 90 day mortality, stratified by procedure type, age and performance status (table 1).

Conclusion:
These results suggest that although the NLCA score performs slightly better than Thoracoscore neither performs well enough to be advocated for routine use to risk stratify patients prior to lung cancer surgery. It may be that the addition of physiological parameters to demographic and procedural data or use of physiological measurements alone would better predict mortality; however this would form the basis of a further project. In the interim we advocate the use of our summary tables that serve to provide clinicians and patients the real-life range of mortality according to performance status and age for both lobectomy and pneumonectomy. 1. Powell HA, Tata LJ, Baldwin DR, Stanley RA, Khakwani A, Hubbard RB. Early mortality after surgical resection for lung cancer: an analysis of the English National Lung cancer audit. Thorax. 2013;68(9):826-34. Figure 1

Background:
ENB is limited by diagnostic accuracy of 60-80%[ [1]]. We hypothesize that using intraoperative CBCT with real time 3D overlay onto fluoroscopic images to confirm placement of biopsy tools in the lesion will increase the diagnostic accuracy of ENB biopsies. [1] Wang Memoli JS, Nietert PJ, Silvestri GA. Meta-Analysis of Guided Bronchoscopy for the Evaluation of the Pulmonary Nodule. Chest. 2012;142(2):385-393. doi:10.1378/chest.11-1764

Methods:
Patients with undiagnosed small pulmonary nodules (<20 mm) underwent biopsy where an initial CBCT of the chest under breath hold was performed, followed by a 3D model reconstruction of the lesions while the surgeon started the ENB. At the end of the bronchoscope navigation, the 3D model of the lesion was fused and automatically registered in real time over the 2D fluoroscopy, allowing an evaluation of the biopsy tool positioning in 3-dimensions. Multiple samples were collected after confirmation of the tool position using various oblique views. Figure 1



Results:
In our initial experience with 10 cases, CBCT acquisition, reconstruction and 3D-overlay was successful in all cases. This procedure enabled confirmation of biopsy tool position within the target lesion in all cases. In one case, the new information obtained successfully discriminated a diaphragm implant from what previously had been interpreted as a basilar parenchymal nodule. In a second case, CBCT reconstruction enabled biopsy of a 15mm lesion thought to be a solitary metastasis. The biopsy was interpreted as normal, albeit in clinical circumstances which were suspicious for malignancy. The patient elected non-surgical treatment of an esophageal primary, precluding definitive pathologic confirmation. A third case provided a biopsy interpreted as normal in a patient who ultimately proceeded to resection for growth of the nodule. While frozen section suggested a benign entity, final pathology demonstrated scattered elements of malignancy. In the remaining cases, CBCT and 3D overlay assisted in successful and accurate biopsy of nodules <20mm.

Conclusion:
Intraoperative CBCT and real time 3D overlay onto fluoroscopic images to confirm appropriate positioning of the biopsy tools in the lesion during ENB is technically feasible. It effectively combines the advantage of real time CT imaging with the advantages of ENB biopsy. This has the potential to increase the diagnostic accuracy of ENB aided tissue diagnosis of small pulmonary nodules. This novel technique will facilitate early accurate diagnosis of lung cancer in small nodules with a minimally invasive approach.

Abstract not provided

Background:
Sublobar resection of small pulmonary nodules by minimally invasive techniques can be a challenge, as this approach reduces the haptic feedback often required to reliably localize small lesions. Use of Electromagnetic Navigational Bronchoscopy (ENB) is a relatively new technique that has potential to assist in real time operative localization of such lesions, as ENB can deliver visual cues for their location in the form of either a dye marking or a radio-opaque clip, or both. There is limited data available on the feasibility of this approach. We want to describe our experience with this technique.

Methods:
A retrospective review of cases in which ENB was used to localize small pulmonary nodules was done from August 1, 2013 to February 1, 2015. We start by using ENB to navigate to the target lesion. In our initial experience, methylene blue was injected into the parenchyma around the mass, and dye migration to the pleural edge was used as a visual cue for location. We then amended our protocol to include placement of both methylene blue dye and a radio-opaque clip in the parenchyma immediately adjacent to the target lesion. Fluoroscopy was then used to triangulate the location of the clip, and by extension the mass, via markings on the chest wall with the lung deflated prior to incision. The visual cue of the dye marking as well as the fluoroscopic localization of the clip served to confirm each other. This was followed by minimally invasive resection of the lesion using these cues to assist in port placement. Figure 1



Results:
A total of 28 cases were identified. ENB was successful in navigating to the lesion in all cases. ENB dye localization alone was successful in 5 of 6 cases. After the first unsuccessful dye localization, our amended protocol of dye marking and clip placement led to successful localization in 22 consecutive cases.

Conclusion:
Use of electromagnetic navigational bronchoscopy to localize small pulmonary nodules is a feasible approach and is technically straightforward. As we see broader implementation of lung cancer screening protocols, thoracic surgeons can expect to encounter many more small pulmonary nodules requiring resection. There is accumulating data that sublobar resection is equivalent to lobar resection for small, peripherally located lung cancer. Use of the algorithm – ‘Navigate, Triangulate and Resect’ will enable thoracic surgeons to more successfully perform sublobar resections of small pulmonary nodules by minimally invasive techniques.

Background:
The rate of mediastinal lymph node metastasis is controversial for patients with clinical N0 non-small cell lung cancer. The primary advantage of video-assisted mediastinoscopic lymphadenectomy(VAMLA) over conventional mediastinoscopy or videomediastinoscopy is to reduce the false-negative rate. We aimed to analyze to evaluate the value of routine VAMLA for patients with clinical T1a-T2aN0 patients prospectively.

Methods:
From March 2010-January 2015, 41 patients with non-small cell lung cancer with clinical stage T1-T2aN0 by postireon emission tomography/computed tomography underwent routine VAMLA before planned resectional surgery.Routinely, stations #2L, 2R, #4R, #4L, 7 were nearly completely resected. In some patients, #10R and #8 lymph nodes were biopsied. The prevalence of mediastinal lymph node metastases at VAMLA and lung resection was recorded.

Results:
There were 5 females (12.2%) and 36 (87.8%) males. The mean age was 62.5 . years. A total of 5 patients were had cT1a-bN0, whereas 36 patients had T2aN0. Eleven patients (26.8%) had occult mediastinal lymph node metastasis. A total of 26 patients underwent lung resectional surgery; only one patient (3.8%) were upstaged to pN2, whereas 3 patients (11.5%) were upstaged to pN1.

Conclusion:
VAMLA seems to disclose considerable number of mediastinal lymph node metastasis in these patients with T1 and T2 clinically staged N0 by positron emission tomography/computed tomography. Routine use of VAMLA is recommended with limited use of mediastinal lymph node evaluation in patients during resectional surgery.

Background:
One measure of the quality of thoracic surgery for non-small cell lung cancer (NSCLC) is the adequacy of nodal evaluation; the rate of pathological nodal upstaging can introduce bias in patient selection for surgical therapy. Robotic-assisted thoracic surgery (RATS) offers the ability to sample nodal stations not easily assessed with conventional open surgical methods. We sought to determine the rate of nodal upstaging as a function of the frequency of various lymph node stations sampled in clinical stage I NSCLC patients undergoing RATS.

Methods:
We retrospectively reviewed the charts of patients with right-sided clinical stage I NSCLC who underwent robotic-assisted pulmonary resection with mediastinal lymph node dissection at our institution from 2013 to 2015. CT or PET scan was used to determine clinical stage. The DiPasquale Quality Index (DQI) defines a complete lymph node dissection (LND) as sampling LN 4R, 7, and 9 for right-sided tumors. Our institutional policy for the initial two years of our RATS program was to limit such to right-sided tumors.

Results:
Robotic anatomic lung resection was performed in 70 patients with right-sided clinical stage I NSCLC. The majority were of the upper lobe (41; 58.6%). The most frequent lymph node stations sampled robotically were LN 4R, 7, 9, 10, and 11 (60.6%, 90.1%, 66.2%, 49.3%, and 64.8%, respectively). According to the DQI, 31 (44.3%) tumors underwent complete LND. Pathologic nodal upstaging occurred in 5 patients (7.1% [pN1 4, 5.7%; pN2 1, 1.4%]). Hilar (pN1) upstaging occurred in 2.8%, 0%, and 20.0%, respectively, for cT1a, cT1b, and cT2a tumors. Comparatively, historic hilar upstage rates of video-assisted thoracoscopic surgery (VATS) versus thoracotomy versus recent robotic data for cT1a, cT1b, and cT2a were 5.2%, 7.1%, and 5.7%, versus 7.4%, 8.8%, and 11.5%, versus 3.5%, 8.6%, and 10.8%, respectively. The 1-year overall survival was 97% and the disease-free survival was 98% at 1 year.

Conclusion:
When patients are appropriately selected and proper lymph node sampling is performed, the rate of upstaging with RATS is comparable to VATS and lower than thoracotomy. The rate of hilar upstaging with robotic resection, however, increases with increasing clinical T stage and appears superior to both VATS and thoracotomy for cT2a tumors. This also has implications for patients who may be considered for therapies like stereotactic radiation therapy. Larger studies comparing matched open, VATS, and robotic approaches are necessary to quantify long term survival and local failure rates.

Background:
Surgery is the treatment of choice for early stage lung cancer (LC). While lobectomy (L) is the historic standard, whether long term outcomes of sub-lobar resection (SL) are comparable is still under debate. The only randomized trial was conducted 20 years ago; 5 subsequent meta-analyses showed inconclusive or conflicting results. We present a comprehensive review of the literature on 5 year-survival after SL compared to L for early stage LC.

Methods:
A priori inclusion criteria were: 1) observational studies, 2) L compared to SL for early stage LC, 3) at least CT staging, 4) 5-year survival reported. A Medline search through January 2015 resulted in 32 studies, representing 24 distinct datasets. The absolute difference in 5-year survival was calculated and plotted for each study.

Results:
There were 4,702 cases treated with L, 2,323 treated with SL. Of 20 studies reporting the reason for SL, 11 indicated that SL was performed because of comorbidities, or impaired cardiopulmonary function. Among all 24 studies, 4 showed no difference in 5-year survival, 13 favored L, and 7 favored SL (Figure 1). Of the two studies using propensity scores, one favored L and the other SL. No meta-estimate could be calculated due to high statistical heterogeneity. Of 21 studies reporting recurrence rate (Figure 2), 11 favored L and 10 favored SL. Figure 1



Conclusion:
Studies comparing 5-year survival rates of SL to L are heterogeneous, and traditional meta-analytic summary estimates of survival and recurrence could not be calculated. SL survival is often similar to L survival, despite the fact that SL is performed in patients with comorbidities or impaired cardiopulmonary function. New approaches to comparing L to SL survival are needed.

Abstract not provided

Background:
The risk of recurrent lung cancer decreases markedly after 4 years. It is unknown whether frequent surveillance after this time period would be beneficial in order to diagnose and treat secondary lung cancers. The purpose of investigation is to assess whether there is an increasing frequency of second lung cancers and whether the first primary reduces Overall Survival(OS)/Lung Cancer Specific Survival(LCSS) as compared to similar patients presenting with their first lung cancer (new primary, NP).

Methods:
The SEER databases were used to investigate incidence (1973-2010) and OS/LCSS (1998-2010) of secondary lung cancer. Incidence was examined by frequency and trend analyses. A SP population was chosen who was originally treated for Stage I-III NSCLC and developed a new primary at least four years after diagnosis of their original primary lung cancer (N=1,699). The OS/LCSS of their SP NSCLC were compared to patients presenting with a new primary (NP) NSCLC. OS/LCSS in NP and SP were analyzed by Kaplan-Meier estimation, multivariate proportional hazards modeling and log-rank tests in the overall group and in a favorable sub-group (stage I, < 4cm).

Results:
The annual incidence rates per 100,000 persons for SP NSCLC has increased almost 5 fold in last three decades (2.5 in 1973; 12 in 2010; p<0.001), more so in male patients. OS and LCSS in SP were higher than NP in the log rank tests (p<0.001). In the subgroup of NP and SP who had favorable tumor characteristics, OS/LCSS was significantly different between NP and SP (P=0.0032; P=0.0015), but did not remain so after accounting for treatment, tumor factors, and patient characteristics (HR=0.983, p=0.8493; HR=1.154, p=0.1770). Rates of OS and LCSS improved significantly with increasingly aggressive treatment in the SP group. Patient and tumor characteristics of the first primary NSCLC were not signantly linked to mortality.

Conclusion:
Patients presenting with a second primary lung cancer had a similar or better OS/LCSS as compared to patients presenting with a new primary lung cancer. The SP population also benefitted from increasingly aggressive treatment. Continued surveillance for new primary lung cancers after 4 years may be beneficial to lung cancer survivors.

Background:
Surgical resection is the most important curative treatment modality for early-stage non-small-cell lung cancer (NSCLC). However, incomplete (margin-positive) resection is associated with inferior survival. We sought to develop a valid facility-based quality metric to measure surgical quality, adjusting related patient demographic and clinical characteristics.

Methods:
We identified facilities that performed cancer-directed surgery for patients diagnosed with AJCC stage I-IIIA NSCLC in the NCDB between 2004 and 2011. We used a multivariate logistic regression model, adjusting for patient risk-mix in each facility, to predict the expected number of risk-adjusted margin positivity (RAMP) cases for each facility. We divided the number of observed margin positivity (OMP) cases by the expected number of RAMP cases to obtain an observed: expected (O/E) ratio for each facility. We categorized facility performance as low outlier (O/E ratio<1 and p<.05), high outlier (O/E ratio>1 and p<.05), or non-outlier. Facility characteristics across performance categories were compared by chi-square test. Five-year unadjusted overall survival (OS) rates were estimated by Kaplan-Meier analyses and compared across categories with the log-rank test.

Results:
A total of 96,596 NSCLC stage I-IIIA patients underwent surgery in 941 facilities. The overall OMP rate was 4.6%. We identified 73 facilities as low outliers (mean O/E ratio=0.41), 755 as non-outliers (mean O/E ratio=1.28) and 113 as high outliers (mean O/E ratio=2.78). Compared to patients treated at high-outlier facilities, patients treated at low-outliers were more likely to be privately insured (34.7%[Low] vs. 32.9%[High]), reside in high-income neighborhoods, have no comorbidity (51.7% [Low] vs. 41.9 [High], p<.001), have adenocarcinoma (62.4%[Low] vs. 58.1%[High], p<.001), stage IA disease (41.6%[Low] vs. 39.6%[High], p<.001) and receive sub-lobectomy (11.7%[Low] vs. 9.9%[High], p<.001). Low-outlier facilities were more likely to be teaching/research or NCI-designated programs (54.8% [Low] vs. 18.5% [High], p<.001) and in the highest quartile of total cancer surgical volume (90.4% [Low] vs. 34.5% [High], p<.001) and lung cancer surgery volume (42.5% [Low] vs. 29.2% [High], p<.001) (Table 1). They also had smaller proportions of uninsured/Medicaid patients (45.2% [Low] vs. 36.2% [High], p=.006). The 5-year unadjusted OS estimates were: 0.62 (low-outliers), 0.58 (non-outliers), 0.57 (high-outliers); log-rank p<.001. Table 1. Facility characteristics across performance categories

	High-Outlier(N=113)	Non-Outlier(N=755)	Low-Outlier(N=73)	p-value
	N(%)
Census_region
Northeast	18(15.9)	154(20.4)	19(26.0)	0.03
Midwest	39(34.5)	223(29.5)	15(20.6)
South	37(32.7)	257(34.0)	35(48.0)
West	19(16.8)	121(16.0)	4(5.5)
Facility_type
Community_Cancer_Program	23(20.4)	164(21.7)	0(0.0)	<0.001
Comprehensive_Community_Cancer_Program	62(54.9)	419(55.5)	28(38.4)
Teaching/Research	17(15.0)	128(17.0)	28(38.4)
NCI_program	4(3.5)	17(2.3)	12(16.4)
Other	7(6.2)	27(3.6)	5(6.9)
Proportion_of_Medicaid/uninsure_patients
Q1(low)	25(22.1)	206(27.3)	13(17.8)	0.006
Q2	16(14.2)	204(27.0)	20(27.4)
Q3	41(36.3)	174(23.1)	21(28.8)
Q4(high)	31(27.4)	171(22.7)	19(26.0)
Lung_cancer_surgery_as_a_proportion of_all_surgery
Q1(low)	8(7.1)	73(9.7)	0(0.0)	<0.001
Q2	37(32.7)	224(29.7)	9(12.3)
Q3	35(31.0)	226(29.9)	33(45.2)
Q4(high)	33(29.2)	232(30.7)	31(42.5)
Total_cancer_surgery_volume
Q1(low)	12(10.6)	98(13.0)	0(0.0)	<0.001
Q2	32(28.3)	193(25.6)	0(0.0)
Q3	30(26.6)	253(33.5)	7(9.6)
Q4(high)	39(34.5)	211(28.0)	66(90.4)

Conclusion:
Facility performance in lung cancer surgery can be captured by using the RAMP rate. Low-outlier facilities delivered superior OS than high-outliers. RAMP metrics could allow facilities to understand their performance and serve as a quality improvement benchmark.

Background:
Lymph node (LN) metastasis is an important prognostic factor for patients with surgically resected NSCLC. We have previously described the extent of missed N1 LN metastasis in a cohort of patients treated at metropolitan institutions. With long-term follow up, we now quantify the survival impact of missed LN metastasis.

Methods:
We conducted a prospective cohort study to retrieve intrapulmonary LNs from discarded NSCLC resection specimens after completion of routine pathology examination. Retrieved materials were histologically examined and classified as LNs with and without metastasis. Survival information was retrieved from institutional tumor registries. Survival distributions were plotted using the Kaplan-Meier method and evaluated with proportional hazards models controlling for gender, race, pathologic N-category, tumor size, margin status, and Charlson score.

Results:
We evaluated 111 patients who were 47% male with a median age of 66 years. Clinical characteristics are summarized in Table 1. Discarded LNs with metastasis were found after re-dissection in 25 (23%) patients. Patients with discarded LN metastasis had an increased risk of death (Figure 1) with an unadjusted hazard ratio (HR) of 2.0 (p-value=0.06) and an adjusted HR of 1.8 (p-value=0.23) compared to those with no discarded LNs with metastasis. When >2 discarded LNs with metastasis were found, patients had 4.8 (p-value=0.0002) times the hazard of death compared to those with no discarded LNs with metastasis (adjusted HR=4.4, p-value=0.0032).

N(%)	No LN Metastasis	LN Metastasis	Total
Bi-lobectomy	8	2	10
	9%	8%
Lobectomy	75	16	91
	87%	64%
Pneumonectomy	3	7	10
	3%	28%
N0	71	6	77
	83%	24%
N1	6	12	18
	7%	48%
N2	9	7	16
	10%	28%
T1	45	3	48
	52%	12%
T2	29	11	40
	34%	44%
T3	10	8	18
	12%	32%
T4	2	2	4
	2%	8%
Margin Negative	83	22	105
	97%	88%
Margin Positive	3	3	6
	3%	12%
Mean(SD)
Charlson Score	1.8	1.8	1.8
	1.6	1.7	1.6
Tumor Size(cm)	3.2	5.0	3.6
	1.8	2.1	2.0

Figure 1



Conclusion:
The presence of metastasis in inadvertently discarded LNs in NSCLC resection specimens has significant implications for patients’ post-operative clinical course. Additional LN metastasis found on re-dissection was associated with reduced survival. A more rigorous protocol for gross dissection of lung resection specimens is needed, and should prove beneficial to patients’ long-term survival.

Background:
A right-sided pneumonectomy after induction therapy for non-small cell lung cancer (NSCLC) has been shown to be associated with significant perioperative risk. We examined the impact of laterality on long-term survival using the National Cancer Data Base (NCDB).

Methods:
Perioperative and long-term outcomes of patients who underwent pneumonectomy following induction chemotherapy ± radiation from 2003-2011 in the NCDB were evaluated using Kaplan-Meier method, multivariable logistic regression analysis and multivariable Cox proportional hazards modeling.

Results:
During the study period, 1,652 patients met inclusion criteria, of whom 740 (45%) underwent right-sided pneumonectomies. Right-sided patients were more likely to have adenocarcinomas, cN2 disease and lower co-morbidity scores (Table). The 30-day mortality rate was higher for right-sided procedures in univariable (11% [84/740] vs 4% [39/912], p<0.001) and multivariable (OR 9.1 [1.8-50.0], p<0.01) analysis. However, 5-year overall survival between right and left pneumonectomy were not significantly different (figure) after a median follow up of 30.2 months. Right-sided procedure also did not impact overall survival in multivariable analysis (hazard ratio (HR), 1.41 [95% CI: 0.87-2.27], p=0.16), while increasing age (HR, 1.02 [95% CI: 1.01-1.03]), Charlson co-morbidity Score of 2 (HR, 1.42 [95% CI: 1.04-1.93]), adenosquamous histology (HR, 1.72 [95% CI: 1.18-2.51]), cN1 status (HR, 1.27 [95% CI: 1.02-1.58]), cN2 status (HR, 1.38 [95% CI: 1.14-1.66]), cN3 status (HR, 1.84 [95% CI: 1.19-2.83]), cM1 status (HR, 2.04 [95% CI: 1.42-2.92]) and incomplete resection (HR, 1.45 [95% CI: 1.14-1.84]) all predicted worse survival. Figure 1

Table: Baseline characteristics.

Variable	Right-sided (n=740)	Left-sided (n=912)	p
Induction chemoradiation	461 (62%)	584 (64%)	0.47
Age (median, IQR)	59 (52-66)	60 (52-67)	0.07
Charlson/Deyo Comorbidity Score			0.02
0	518 (70%)	610 (66%)
1	190 (26%)	243 (27%)
2	32 (4%)	68 (7%)
Histology			0.02
Adenocarcinoma	227 (37%)	243 (32%)
Squamous	310 (50%)	450 (59%)
Large cell	28 (5%)	19 (2%)
Adenosquamous	20 (3%)	21 (3%)
Neuroendocrine/carcinoid	4 (1%)	7 (1%)
BAC	28 (5%)	23 (3%)
Clinical N			< 0.01
0	190 (27%)	269 (31%)
1	134 (19%)	187 (21%)
2	368 (52%)	381 (44%)
3	16 (2%)	34 (4%)

There were no significant differences between the groups with regards to sex, race, facility type, and clinical T and M status.



Conclusion:
In this population analysis, right-sided pneumonectomy after induction therapy was associated with a significantly higher perioperative but not worse long-term mortality compared to a left-sided procedure. These findings can be used in the risk/benefit analysis when considering patients for pneumonectomy following induction therapy.

Abstract not provided

Background:
Outcomes of pneumonectomy after neoadjuvant chemoradiastion therapy (CCRT) for patients with stage IIIA-N2 non-small cell lung cancer (NSCLC) have been well-known as grave. Whenever possible, we have tried sleeve resection in patients to avoid pneumonectomy(PN). We evaluated whether the sleeve resection (SL) could have avoided the postoperative mortality/morbidity and achieved comparable long-term outcomes with pneumonectomy.

Methods:
We retrospectively reviewed medical records of 574 consecutive patients with clinical stage IIIA-N2 non-small cell lung cancer who underwent surgery after neoadjuvant CCRT from 1997 to 2013. Clinical outcomes were analyzed and compared in 98 consecutive patients who had either SL (n = 25) or PN (n = 73) after neoadjuvant CCRT in a single institution.

Results:
Thirty-day postoperative mortality were 0% (0/25) in SL group, and 5.5% (4/73) in PN group (p=0.120). Ninety-day postoperative mortality were 12.0% (3/25) in SL group, and 17.8% (13/73) in PN group (p=0.498). The most common cause of ninety-day mortality was acute respiratory distress syndrome (n=11). Morbidity rate was 48.0 % (12/25) in SL, and 49.3% (36/73) in PN. The 5-year survival was lower in the PN group (PN, 24.7 % versus SL, 45.1%, p=0.086). The recurrence pattern (locoregional versus distant) did not differ between two groups (p=0.726). When recurrences occurred (n = 50), the site of first recurrence was local (stump site) in 0 % (0/25) of patients with SL and in 4.1% (3/73) of patients with PN.

Conclusion:
Following neoadjuvant CCRT for patients with stage IIIA-N2 NSCLC, SL showed a comparable or even better early and long term clinical outcomes with PN. Therefore, SL should be considered, whenever possible.

Background:
Biopsy of APW (levels 5/6) lymph nodes can be important for lung cancer staging, but the APW is not accessible by routine mediastinoscopy or EBUS. Although some consider ECM potentially dangerous, we reviewed our ECM experience to determine safety and accuracy and to define/validate parameters for patient selection.

Methods:
With IRB approval we reviewed two institutions' databases for patients undergoing ECM between 3/1/97 and 12/31/11. Physical parameters (PP) that were thought to impact on the difficulty and safety of ECM, ie., clavicular head (CH), thoracic inlet (TI), and anterior mediastinal (AM) dimensions, were measured using 55 CT scans available from the first 100 pts.

Results:
Of 190 patients, 128 (67.3%) were male and ages ranged 28-91 yrs. Indication for surgery was either cancer (>95% with lung cancer >80%) or adenopathy (<5%). All procedures were performed by a single surgeon during routine mediastinoscopy. There were no intraoperative complications and blood loss was <25 cc in all cases. Morbidity occurred in 15 (7.9%) with 1 (0.55%) major complication and no mortality. A pathologic diagnosis was obtained in 189 (99.5%). Postop pain was easily controlled with bupivicaine. PP were compared to those in an additional 12 control patients with failed procedures (Table). Although each PP alone was not useful, the APWI (TI X AM product) did predict degree of difficulty (p=0.015) and divided patients into 3 groups predictive of the degree of difficulty: Straightforward (APWI>17), Intermediate (APWI=6-17), and Prohibitive (APWI<6) (Figure). The APWI was then prospectively validated with excellent accuracy in the next 90 patients. The APWI can be helpful in the selection of patients for thoracic surgeons, particularly those learning ECM. A short video demonstrating the technique of ECM will be presented.

Table: Physical Parameter Measurements (values were obtained from CT scans available on 55/100 initial patients comparing with a separate group of 12 patients with unsuccessful ECM

Parameter	Successful ECM (cms)	UnSuccessful ECM (cms)
Clavicular Head (CH)	2.3+0.36	2.28+0.36
Thoracic Inlet (TI)	6.32+1.07	5.99+0.62
Anterior Mediastinum (AM)	2.53+0.82	1.89+0.82
APWI (TI X AM)	16.2+6.77	11.1+4.4*
*p=0.015

Figure 1



Conclusion:
ECM is straightforward, safe, and accurate in mediastinal staging. Our novel APWI helps to safely select patients for any thoracic surgeon's skill and comfort level.

Background:
In our cancer network, single station N2 NSCLC may be managed with surgical resection followed by adjuvant chemotherapy as meaningful survival has been shown in such circumstances. Multi-station N2 disease diagnosed pre-operatively is not considered a surgical entity. However, sometimes occult multi-station pN2 may only be identified during intra-operative nodal sampling. This study aimed to analyse the survival of such patients at a large thoracic surgical centre in the United Kingdom.

Methods:
A retrospective review of all pathological reports from NSCLC resections at the University Hospital South Manchester from 01/01/2011 to 31/12/2013. Based on the histological results from intra-operative nodal sampling, patients were stratified into nodal categories pN0, pN1, pN2 single station and pN2 multi-station. Survival data was obtained through national death registry data allowing a minimum of twelve months follow-up for all patients at the time of analysis in January 2015.

Results:
987 surgical resections for NSCLC were performed during the study period 2011 to 2013 at UHSM. A total of 132 patients had pN2 disease; 85 with single station pN2 and 47 with multi-station N2. The median survival time for those patients with multi-station pN2 was 798 days (95% CI 405-1191 days) and 762 days (95% CI 616-908 days) for those with single station pN2. Median survival times were not estimable for patients with pN0 and pN1 as only a small proportion of patients died. For pairwise comparisons between N categories, a Bonferroni adjustment for multiple comparisons used a critical value of 0.008 for significance. Patients with single station pN2 and multi station pN2 had significantly lower survival times than patients with N0, but there was no statistically significant difference in survival between patients with pN1, single station pN2 and multi-station pN2 (Figure 1).Figure 1



Conclusion:
Interestingly, patients with multi-station pN2 had a similar survival to those with single station pN2. This cohort of multistation pN2 patients is likely to represent those with microscopic nodal metastases and does not represent all multistation N2 disease. Although the numbers are small it does raise interesting questions about the exclusion of patients with radiologically-occult multi-station N2 disease, detected during the pre-operative systematic sampling of small mediastinal nodes through endoscopic or surgical techniques, being excluded from surgery as part of their multi-modality treatment in our network.

Abstract not provided

Background:
Current nodal staging for non-small cell lung cancer (NSCLC) only take into account the anatomic location of lymph node (LN). Although among patients with same pathologic N1 NSCLC, they are known to have heterogeneous prognosis and prognostic significance of extent of LN involvement is still uncertain. The objective of current study was to evaluate whether LN ratio (LNR) is a marker of prognostic factor for survival in patients with pathologic stage II/ N1 NSCLC after complete resection

Methods:
A total of 4,089 consecutive patients underwent curative surgical resection for NSCLC between 2004 and 2012. Of these, 413 patients who found to have pathologic stage II/N1 NSCLC after complete resection were retrospectively analyzed. For LNR, the optimal cutoff value was determined using chi square score, which were calculated using the Cox proportional hazards regression model. The prognostic value of the LNR was calculated by Cox regression hazard model analysis.

Results:
The study included 337 males and 76 females with a mean age of 62 years. The mean numbers of metastatic and dissected LN were 1.84 and 26 respectively and the mean LNR was 0.082. The number of the metastatic LN was significantly correlated to the LNR (r=721; p<0.0001). Based on the maximum chi square score and minimum p value approach, the optimal cutoff value of LNR was 0.1 and patients were classified into two groups according to LNR. Both 5-year overall survival rate and the lung cancer-specific survival rate in the high LNR group (LNR ≥0.1) were significantly lower than those in the low-LNR group (overall survival: 55.4.% vs 69.8%, p=0.003; lung cancer specific survival rate: 58.4% vs. 77.0%, p<0.0001) Also, disease free survival (DFS) rates according to LNR were 56.8% in low-LNR group (LNR<0.1) and 35.0% in high-LNR group (LNR≥0.1). DFS rate in the low-LNR group was significantly higher than that in the high-LNR group (p<0.001). LNR is an independently related prognostic factor with overall survival (OR=2.288; 95% CI=1.513~3.459; p<0.0001), lung cancer-specific survival (OR=2.740; 95% CI=1.709~4.395; p<0.0001) and DFS (OR=2.191; 95% CI=1.543~3.110; p<0.0001) after adjustments of clinical variables including sex, age, stage, surgical extent, histology and adjuvant treatment.

Conclusion:
LNR is an independent prognostic factor of survival in patients with pathologic N1 NSLC after complete surgical resection.

Background:
Conversion to open thoracotomy occurs when thoracoscopic manipulation becomes difficult as a result of particular situations during complete thoracoscopic lobectomy after the surgeon starts to dissect blood vessels Based on special intra-operative situations, conversion to open thoracotomy can be divided into active and passive conversion. Active conversion to open thoracotomy implies that the surgeon gives up the thoracoscopic manipulation voluntarily and performs open surgery under direct vision as a result of the difficulty of thoracoscopic manipulation when encountering problems, such as adhesions of lymph nodes and difficulty of exposing huge tumors, which may result in massive bleeding, tumor rupture, and undue extension of the operative time. Passive conversion to open thoracotomy implies that the surgeon has to discontinue thoracoscopic manipulation and perform open surgery under direct vision because of urgent or serious intra-operative complications, including blood vessel breakage and bronchial membrane rupture, which are difficult to treat thoracoscopically. Lymph nodes are an important etiology affecting the conversion of complete thoracoscopic lobectomy to open thoracotomy.Five hundred consecutive patients with non-small cell lung cancer underwent complete thoracoscopic lobectomy at the Department of Thoracic Surgery of Peking University People’s Hospital, and the conversion to open thoracotomy was performed in 47 cases (9.4%). Lymph node interference means that a lymph node cannot be separated easily, and was the reason for conversion to open surgery in 31 cases (65.9% of 47 cases).The effect of lymph node interference on surgery has not been thoroughly addressed to date. We studied the data of patients who underwent complete thoracoscopic lobectomy in our hospital, and analyzed the effect of lymph nodes on the conversion to open thoracotomy and corresponding factors.

Methods:
Between September 2006 to April 2013, 1006 patients （545 men, 461women, median age 60 years, range from 13 to 86 years)received completly thoracoscopic lobectomy, including segmectomy(n=13), simple lobectomy(n=846), compound lobectomy(n=131), pneumonectomy (n=8), sleeve lobectomy(n=8). The main procedure was completely video-assisted anatomical lobectomy with mediastinal lymphadenectomy as we have reported.

Results:
All procedures were carried out smoothly without serious complication. 83 cases converted to thoracotomy(8.2%), including 70 cases of initiative conversion and 13 cases of passive conversion in which 59 cases was interference by doornail lymph nodes. Pathological result show 821 cases of malignant disease and 185 cases of benign disease. All patients recovered well.the average operative time in the conversion thoracotomy group was significantly longer (272.7 ± 67.2min versus186.9 ± 58.1min, P = 0.001)compared with completely endoscopic surgery group, the average blood loss was significantly increased(564.2 ± 507.7ml versus 158.0 ± 121.0ml, P = 0.001), the drainage time was significantly longer(8.9 ± 5.0d versus 6.6 ± 3.5d, P = 0.001) and the postoperative hospital stay was significantly longer(12.5 ± 7.7d versus 9.2 ± 5.8d, P = 0.001).

Conclusion:
Interference of lymph doeds was the main reason for conversion to thoracotomy on VATs lobectomy. It may prolonged the operative time, increase the blood loss in operation and delay the postoperative recovery of the patients. Select the proper indication of conversion thoracotomy may reduce the negative effects of conversion thoracotomy.

Background:
Bronchoplasty has become widely accepted as a reliable and safe lung-saving procedure for lung cancer. The purpose of this study was to evaluate the factors contributing to the outcomes of bronchoplasty for lung cancer by analyzing a single institution’s data for a 30-year period.

Methods:
In the 2416 patients who underwent lung resections for lung cancer at Nagasaki University Hospital from 1980 to 2010, there were 222 bronchoplastic procedures. After excluding patients who underwent carinoplasty, 213 patients (161 bronchoplasty and 52 broncho-angioplasty) were included. The patients were divided into two groups by the date of surgery: the 1[st] period was 1980 to 1995, and the 2[nd] period was 1996 to 2010.

Results:
Bronchoplasty and broncho-angioplasty were performed in 100 (75.8%) and 32 (24.2%) patients, respectively, in the first period and 61 (75.3%) and 20 (24.7%) patients, respectively, in the second period. Overall 90-day operative morbidity and mortality rates were 25.8 and 9.8%, respectively, in the first period and 45.7 and 2.5%, respectively, in the second period. Thirty-day mortality rates were 6.8% in the first period and 0% in the second period. Five-year survival was 41.1% (n = 132) in the first period and 61.5% (n = 81) in the second period (P = 0.0003). Comparing bronchoplasty and broncho-angioplasty, the 5-year survival was 45.6 and 26.5%, respectively, in the first period (P = 0.0048) and 60.9 and 62.1%, respectively, in the second period (P = 0. 8131). Using multivariate analysis to identify potential prognostic factors, the type of operation (broncho-angioplasty), postoperative complications and histology (non-squamous cell carcinoma) were significant factors affecting survival in the first period, but none of the factors significantly affected survival in the second period. When the rates of pN2 or N3 histological type disease were compared in each period, the rate of pN2 or N3 disease in non-squamous cell carcinoma was 51.4% in the first period and 45.5% in the second period; both were significantly higher than in squamous cell carcinoma (31.6 and 16.9%, respectively; P = 0. 0365 and 0.0073). Figure 1



Conclusion:
The present study suggests that progress in the preoperative staging system and perioperative medical management, as well as surgery, has contributed to current improvements in patients undergoing bronchoplasty and broncho-angioplasty. However, since nodal status in non-squamous cell carcinoma is not precisely evaluated before the operation, the indication for bronchoplasty should be considered carefully.

Background:
Non-small cell lung cancer (NSCLC) may be treated with curative intent using radiotherapy, either as single modality or in combination with systemic chemotherapy. Most commonly, radiation treatment is planned based on findings at 18-Fluorodeoxyglucose Positron Emission Tomography (PET), following pathologic confirmation of involvement at a single mediastinal site. We hypothesized that systematic mediastinal evaluation with EBUS-TBNA in NSCLC patients considered for radical radiation therapy may identify disease extent discrepant with that indicated by PET-CT.

Methods:
This prospective ethics board-approved multi-centre cohort study in three Austrailan tertiary centres consented patients prior to mediastinal evaluation with Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) for NSCLC,where non-invasive imaging indicated the likely treatment modality would include radical radiotherapy. EBUS evaluation was performed systematically with sampling of any lymph node (LN) exceeding 6mm diameter.

Results:
Thirty eligible patients with NSCLC form the basis of this report. No procedural complications occurred during performance of EBUS-TBNA. LN sampling was performed from a mean 2.5 lymph node stations per patient (median 3,range 1–5). Adequate samples were obtained from all sites examined by EBUS-TBNA. Mean long-axis size of sampled LN was 16+7.8mm (median 13mm,range 5–36mm). 24% of sampled LN were 10mm or less. Discordant findings were observed in 10 of 30 patients (33%) (Figure 1) EBUS-TBNA identified a greater extent of mediastinal involvement than PET in four patients, with invasive sampling resulting in upstaging in three patients. In one further patient, extent of disease was greater than noted on PET due to more proximal involvement of LN disease not resulting in stage advancement. Median size of LN upstaged by EBUS was 7.5mm (range 7–9). In eleven mediastinal LN in six patients, EBUS identified a lesser extent of mediastinal disease than PET, including two patients down-staged from N3 à N2. Median size of LN down-staged by EBUS was 12mm (range 6–21). FIGURE 1. Flowchart of patients Figure 1



Conclusion:
Our findings demonstrate clinically significant discrepancy between two modalities frequently used to stage mediastinal disease extent in NSCLC patients being considered for radiotherapy. PET-based radiotherapy planning alone may not be appropriate given the risk of excessively large, or insufficiently large, radiation fields where planning is not based on invasive LN sampling. These results suggest minimally invasive comprehensive/systematic mediastinal staging should be considered for all patients prior to radiotherapy to accurately assess pathologic stage and extent of disease, and to ensure treatment fields most accurately encompass all sites of disease.

Abstract not provided

Background:
Purines are well known as intracellular sources for energy but also act as extracellular signaling molecules. In the last decade there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. The effects carried out depend on the concentration, expressed pattern of purinergic receptors and general dynamics of synthesis and degradation. In this study we analyze different purines and purinergic receptors in bronchoalveolar lavage (BAL) of patients with non-small-cell lung cancer (NSCLC) to provide further insight on their relevance in the tumor microenvironment.

Methods:
In this prospective clinical trial we enrolled 27 patients with NSCLC and 16 patients with chronic obstructive pulmonary disease (COPD) without signs of malignancy. The study was approved by our local ethics committee and registered as a clinical trial in the German Registry for Clinical Trials (DRKS-ID: DRKS00005415). BAL was performed using flexible bronchoscopes. The bronchoscope was wedged into a subsegment were the tumor was present and a total of 300 ml sterile saline was instilled. The BAL-fluid (BALF) was recovered by gentle aspiration. Purines (ATP, ADP, AMP, Adenosine and Inosine) were analyzed using fluorescence/luminescence based assays. Expression of purinergic receptors and Ectonucleotidases in NSCLC (P2X1, P2X4, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, P2Y14, CD39, CD73) were analyzed using qPCR.

Results:
Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD without signs of malignancy (p=0.006 and p=0.009). Inosine concentrations however are higher in patients with malignant disease (p=0.01). In the subgroup-analysis of metastasized versus non-metastasized tumors receptor-analysis revealed a higher expression of P2X4 (p=0.07), P2X7 (p=0.0008) and P2Y1 (p=0.009) as well as of the ectonucleotidase CD39 (p=0.007). Analysis of the purine metabolites in the respective groups showed no statistically significant differences. Furthermore there is a positive correlation of the proportion of macrophages in differential cell count in BAL with the expression of P2X7 (r=0.53, p=0.02).

Conclusion:
Previous data suggests pro-inflammatory, zytotoxic and thus anti-neoplastic effects of Adenosine-Triphosphate (ATP) and ADP. Also it has been shown that low ATP concentrations in the tumor microenvironment can lead to enhanced proliferation of tumor cells. In this first in vivo study on purinergic signaling in lung cancer we find lower concentrations of ATP and ADP in samples from NSCLC patients compared to COPD without signs of malignancy in accordance with these findings. Furthermore in aggressive, metastasized NSCLC we find a higher expression of the ectonucleotidase CD39. This enzyme degrades ATP and ADP to Adenosin and has previously been shown to hence induce immune escape in malignant disease. Furthermore we demonstrate elevated expression of P2X4, P2X7 and P2Y1 in the tumor microenvironment of metastasized NSCLC compared to non-metastasized tumors. This suggests a role of these receptors in tumor metastasis, however the exact mechanisms remain unclear. To further illustrate these interactions we are currently initiating a study to identify purinergic receptors in NSCLC tumor cells from pathologic specimen. With this knowledge future translational studies can be conducted to potentially provide new therapeutic targets.

Background:
Lung cancer growth was significantly repressed by the first generation CDK2/9/7 inhibitor seliciclib (R-roscovitine, CYC202, Cyclacel Ltd). This induced anaphase catastrophe and apoptosis to occur. Anaphase catastrophe happens when supernumerary centrosomes attempt mitosis by clustering extra centrosomes. If this clustering is inhibited, cells segregate chromosomes inappropriately and anaphase catastrophe occurs and leads to death of daughter cells. This study explored antineoplastic effects of a next generation CDK2/9 inhibitor: CCT68127 (Cyclacel) against lung cancer cells. CCT68127 inhibits CDK2/9 more potently and selectively than seliciclib (IC50s for CDK2 and CDK9 are 30nM and 110nM, respectively).

Methods:
Antineoplastic CCT68127 effects in murine (transgenic mouse-derived) and human lung cancer cells were compared to seliciclib using luminescent cell viability assays. Cell cycle arrest and apoptosis induction by CCT68127 were detected using fluorescence-based cell imaging after staining with propidium iodide (PI) and double-staining with Annexin V and PI. Multipolar anaphase cells were scored after a tubulin and DNA staining. RPPA (Reverse Phase Protein Assay) analyses were performed in CCT68127 and vehicle-treated lung cancer cells to uncover mechanisms engaged by CDK2/9 antagonism. Expression levels of nearly 200 key growth-regulatory proteins were examined before and after 6, 24, and 48 hours of CCT68127 versus vehicle treatments of murine: ED1 (wild-type KRAS) and LKR13 (mutant KRAS) and human lung cancer cells: H522 (wild-type KRAS) and Hop62 (mutant KRAS).

Results:
IC50s of CCT68127 in murine lung cancer cells (ED1, LKR13, and 393P) were <1µM while IC50 of seliciclib was >25µM. KRAS mutant murine lung cancer cells (LKR13 and 393P) were more sensitive to CCT68127 than the KRAS wild-type line (ED1). In contrast, growth inhibition in C10 immortalized murine pulmonary epithelial cells was negligible. IC50s in human lung cancer cell lines (Hop62, A549, H2122, H522, and H1703) were comparable to murine lung cancer cell lines. KRAS mutant lung cancer cells (Hop62, A549, and H2122) were more sensitive than KRAS wild-type lung cancer cell lines (H522 and H1703). Immortalized human bronchial epithelial cells (BEAS-2B) were resistant to CCT68127 treatment. CCT68127 triggered apoptosis in a dose-dependent manner in murine lung cancer cell lines and at much lower concentrations than seliciclib. CCT68127 caused G1 arrest. Its growth inhibition was partially reversed in washout experiments. CCT68127 also induced apoptosis in human lung cancer cells (Hop62, A549, H522, and H1703). A mechanism responsible for these effects was found. Anaphase catastrophe was triggered by CCT68127 treatment of murine and human lung cancer cell lines and was independent of KRAS mutation status. RPPA analyses uncovered distinct protein profiles after CCT68127 treatment. These included DNA repair, Hippo and Rab GTPase pathway members that were each markedly down-regulated.

Conclusion:
CCT68127 is a next generation CDK2/9 inhibitor that has more potent antineoplastic activity against KRAS mutant and wild-type lung cancer cells than the prior inhibitor, seliciclib. This occurred via induced anaphase catastrophe and was linked to changes in expressed growth regulatory proteins. Taken together, these findings implicate use of a next generation CDK2/9 inhibitor for human lung cancer cases.

Background:
Tumor initiating cells (TICs) are a small subpopulation within cancer that is thought to be resistant to conventional therapies and capable of reinitiating tumors. However, only a few biomarkers of TICs have been well elucidated.

Methods:
By the methods of QPCR, FACS, western blot, colony formation and statistic analysis, we have investigated whether CACNA2D1 (α2δ1) to enrich TICs of non-small cell lung cancer (NSCLC) and and tharget therapy of its antibody.

Results:
In comparison to α2δ1[-], α2δ1[+] cells demonstrated greater TICs properties with higher potential of self-renewal, differentiation and reconstituting tumors. Following treatment, these cells were enriched in clinical samples. We verified a monoclonal antibody of α2δ1 mAb which targets to α2δ1 had therapeutic treatment to TICs of NSCLC and further α2δ1 mAb combined with the common anti-cancer drug of carboplatin was obtained to suppress the established xenograft tumors. Importantly, the disease free survival and overall survival of NSCLC patients with increased α2δ1 expression was significantly shorter than that of patients with decreased expression. Mechanically, our results showed that a role of α2δ1 in up-regulating stemness of NSCLC cells was associated with NOTCH signaling.

Conclusion:
Collectively, our data indicate that α2δ1 could be used as a marker for identifying TICs of NSCLC and targeting these cells might provide a way to treat this disease.

Background:
Heparan sulfate D-glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1) participates in the biosynthetic steps of heparan sulfate (HS) and found to target VEGF in acute myeloid leukemia(AML) cells thus contributing the angiogenesis and proliferation of AML cells . However, the role of HS3ST3B1 in NSCLC has never been reported. In this study, we aim to investigate the role of HS3ST3B1 in NSCLC epithelial-to-mesenchymal transition.

Methods:
Expression of HS3ST3B1 was investigated by qRT-PCR in specimens of tumor and matched normal tissues of NSCLC patients and also in epithelial and mesenchymal NSCLC cell lines. A549 and HCC827 cell lines was induced to mesenchymal phenotype by TGF-β, and expression of HS3ST3B1, CDH1, and VIM were studied by PCR. HS3ST3B1 was knockdown by siRNA to analyze the effect of HS3ST3B1 on EMT. Computational predicting software was used to predict potential regulators of HS3ST3B1 and dual luciferase report system demonstrated that miR-218 may target HS3ST3B1 in cells. MiR-218 was tranfected into cells to analyze the association of miR-218 and HS3ST3B1 in cells.

Results:
HS3ST3B1 was significantly up-regulated in tumors compared with matched normal tissues(P=0.002). Its expression was also up-regulated in mesenchymal phenotype NSCLC cells lines compared with epithelial phenotype(P＜0.05). When epithelial phenotype NSCLC cells transformed to mesenchymal phenotype induced by TGF-β, HS3ST3B1 was also significantly up-regulated. Moreover, when HS3ST3B1 was knockdown by siRNA in mesenchymal phenotype NSCLC cell lines, cells were reversed to epithelial phenotype morphologically. With Targetscan, we found that HS3ST3B1 was one potential targets of miR-218 and dual luciferase report system demonstrated that HS3ST3B1 was direct target of miR-218 in cells. When miR-218 was transfected into cells, we found that HS3ST3B1 was down-regulated. Figure 1 Figure 2





Conclusion:
HS3ST3B1 may regulate EMT and it can be regulated by miR-218 in NSCLC.

Abstract not provided

Background:
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer including lung cancer, which is attributed to a greater number of deaths than prostate, breast and colorectal cancers combined. Paxillin is a 68 kDa protein that is an integral part of the focal adhesion and acts as an adaptor molecule. We initially cloned the gene for paxillin, and localized it to chromosome 12q24. We have previously reported that paxillin can be mutated (approximately 8%), amplified (5-7%), and/or overexpressed in almost 80% of lung cancer patient samples. Paxillin protein is upregulated in more advanced stages of lung cancer compared with earlier stages and is a prognostic factor for non-small cell lung cancer (NSCLC). Paxillin gene is amplified in some pre-neoplastic lung lesions as well as neoplastic lesions. We identified 22 different variants of paxillin mutation in our initial investigation especially between the LD and the LIM domains (Jagadeeswaran et al. 2008). There are mutations that have been validated in the TCGA set. We selected six mutants to perform further studies ((P52L, A127T, P233L, T255I, D399N, and P487L as well as wild-type as control). Our investigations focused on an effort to understand the contribution of molecular abnormalities found in paxillin and their relationship to mitochondrial functionality.

Methods:
HEK293 cells as well as a paxillin null NSCLC cell line H522 was used to overexpress the above paxillin mutants and wild-type paxillin. Live cell confocal microscopy was performed to evaluate cell motility, immunoprecipitation to determine interaction with other proteins, and gene expression analysis was performed to evaluate effects on gene expression.

Results:
Among the mutations we investigated, we found that the most common paxillin mutant A127T in lung cancer cells enhanced cell proliferation, focal adhesion formation and co-localized with the anti-apoptotic protein B cell CLL/Lymphoma 2 (BCL-2), which among other sites also localizes to the mitochondria. We further found that when these variant clones of activating mutations were expressed in HEK293 cells, they conferred phenotypic changes resembling neoplastic cells. In gene chip microarrays assay investigating gene expression modulation conferred by these mutations in these same HEK293 cells, we found that P52L, A127T, T255I, P233L and D399N mutations, compared to wild-type paxillin, indeed modulated the expression of a significant number of genes. In particular, there were a number of mitochondrial signature proteins that were altered in the various mutants. Analyzing mitochondrial functions by measuring the interaction of these mutants with mitochondrial proteins MFN2, and DRP1, we identified that they alter mitochondrial dynamics, with significant fission rather than fusion. Paxillin also translocated from the focal adhesion to the mitochondrial membrane. In relationship to cisplatin responsiveness, PXN and mutant overexpression lead to cisplatin resistance.

Conclusion:
These data suggest that wild-type and mutant paxillin variants play a prominent role in neoplastic changes with direct implications in lung cancer progression and hence, its potential as a therapeutic target needs to be explored further.

Background:
The methyltransferase enhancer of zeste homolog 2 (EZH2) belongs to the polycomb repressive 2 complex (PRC2). EZH2 is upregulated in several malignancies including prostate, breast and lung cancer. The EZH2 protein forms one of the critical protein complexes of PRC2 by partnering with EED (embryonic ectoderm development) protein. This EED/EZH2 complex has been shown to interact with histone deacytelase (HDAC). This interaction is highly specific and HDAC does not interact with any other PRC2 protein complexes. In the present study, we investigated the link between EZH2 and HDAC in lung cancer cell lines and in human tumor tissue microarrays (TMAs). We also further investigated EZH2 as a marker for response to HDAC inhibitors.

Methods:
We analyzed EZH2 and HDAC1 mRNA expression in two lung adenocarcinoma datasets (MDACC n=152, and TCGA n=308), and correlated the gene expression with tumors’ clinico-pathological characteristics and patients’ outcome. To study the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor suberanilohydroxamic acid (SAHA), we examined mRNA and protein expression by RT-PCR and Western blot, respectively, in twelve lung adenocarcinoma (LUAD) cell lines at baseline and after overexpression or knock-down of EZH2 or HDAC1 gene expression using siRNA. Response to (SAHA) in cell lines was measured by MTT assay and correlated with protein and mRNA expression levels of EZH2 and HDAC1.

Results:
Direct and positive correlation was found between EZH2 and HDAC1 expression NSCLC cell lines (P <0.0001). This correlation was confirmed in NSCLC specimens from MDACC (Spearman’s correlation r=0.416; p < 0.0001) and TCGA datasets (r=0.221; p <0.0001).Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of MDACC and TCGA datasets had lowest survival compared to the patients who had either or both low expressions. Overall survival in the univariate analysis (MDACC dataset; Hazard Ratio (HR)=2.97; p=0.031 and TCGA dataset; HR=2.6 and p=0.041) and multivariate analysis (MDACC; HR=2.92 and p=0.034 and TCGA; HR=3.17 p=0.016). When EZH2 expression was knock down, there was a significant reduction in HDAC1 expression; conversely, when HDAC1 was knocked down EZH2 expression was also decreased. These concordant change in expression was seen both at the protein and mRNA level. Importantly, while all 8 cell lines with high EZH2 protein expression responded to SAHA treatment with average inhibition rate reaching 73.1%, three out of four cell lines with low EZH2 expression had a significantly lower response rate to SAHA inhibition with average inhibition rate 43.2% (P<0.0001). Additionally, altering the expression of EZH2 concordantly altered the sensitivity to SAHA i. e. forced increased expression of EZH2 increased the response to SAHA and vice versa.

Conclusion:
Our data suggest that EZH2 and HDAC expression are correlated in LUAD cell lines in human tissue microarrays and overexpression of both is a negative prognostic indicator. Additionally we show that increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting LUAD patients with HDAC inhibitors.

Background:
Human bombesin receptors (BnR) are one of the most frequently over-expressed receptor families by human lung-cancers. It is known the activation of the classical members of the BnR family (GRPR, NMBR), causes a marked effect on cell-signaling and growth, often autocrine in nature, on lung-cancer cells. In addition, it has been discovered that the orphan receptor related to the BnR family, BRS-3, is widely distributed in central/peripheral normal tissues, and numerous tumors include lung-cancer cells. However, in contrast to the classical BnRs (GRPR, NMBR), BRS-3 has received little attention in lung-cancer in large part due to the fact its natural ligand is still unknown, and also because, until recently, the lack of specific pharmacological tools to study it. To address this, in this study, we examined the frequency of hBRS-3 expression in lung-cancer cells, and the effects of specific hBRS-3 activation on cell-signaling and cell-function (growth) in different lung-cancer cell lines.

Methods:
17 human lung-cancer (LC) cell lines were studied (9 NSCLC, 8 SCLC), as well as hBRS3 transfected H1299 and Balb 3T3 cells. The BRS-3 selective agonist, MK-5046 and selective BRS-3 antagonist, Bantag-1 were used. BnR expression was assessed by PCR using specific primers for hBRS3, hGRPR or hNMBR. Receptor activation was determined by assessing PLC and MAPK cascade activation using Western Blotting, and cytosolic Ca[2+] release. Proliferation was studied by clonogenic and [[3]H]-Thymidine assays, and EGFR transactivation was assessed using Western blotting.

Results:
Of the 17 LC cell lines, 9 (53%) express hBRS3 [H358, H460, H520, H720, H727, H69, H82, N417, H510], 14 (82%) express hGRPR [H28, H157, H358, H520, A549, H838, H1299, SK-LU-1, H720, H727, H69, H82, H345, H510] and 13 (77%) express hNMBR [H28, H157, H358, A549, H838, H1299, SK-LU-1, H720, H727, H82, H345, N417, H510]. MK-5046 stimulated PLC activation in 6/9 cells which express hBRS3 (H358, H720, H727, H69, H82, N417), and MAPK activation in all 9 hBRS3 cell lines. Cytosolic Ca[2+] increased with MK-5046 addition in all hBRS-3-containing cells, included Balb and H1299 transfected cells, except in H358 cells. Similarly, MK-5046 increased [3]H-Thymidine uptake in 5/9 cells (H460, H520, H720, H727, H82, H510), as well as in Balb and H1299 hBRS-3 transfected cells, and this increase was in a dose-response manner in H727, H69 and N417 cells. MK-5046 stimulated the clonal growth of N417 and H727 cells. MK-5046 addition to H358, H460, H727 (NSCLC) and H69, N417, H510 (SCLC) caused Tyr[1068] phosphorylation of the EGFR.

Conclusion:
These results show the orphan human BnR, hBRS-3 is present in more than one-half of human lung cancer cells. Furthermore, these receptors are functional with their activation effecting cell signaling (MAPK, PLC, Ca[2+]) and cell growth. Transactivation of EGFR is likely an important transduction cascade. These results suggest this orphan BnR, similar to classical BNRs, will be important to assess for growth effects and expression in human lung tumors, and its pharmacological inhibition may be a useful therapeutic approach.

Abstract not provided

Background:
In non-small cell lung cancer (NSCLC), activation of the phosphoinositide-3-kinase (PI3K)/mTOR pathway is common in tumors resistant to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). CC-115 (Celgene Corporation), an mTOR kinase inhibitor that targets both mTORC1 and mTORC2 as well as DNA-dependent protein kinase (DNA-PK), is currently under early clinical development. We evaluated CC-115 in combination with Erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.

Methods:
In the present study we investigated whether CC-115 is able to increase the therapeutic effect of the EGFR TKI Erlotinib in several different NSCLC cell lines which exhibit intermediate or high resistance to EGFR TKIs: A549, H1975, H1650, HCC95, H2122 and H23. Mechanisms of inhibition were analyzed with assays for proliferation, apoptosis, and cell cycle progression. Cell signaling activity was analyzed using phospho-specific antibodies in Western blotting. Xenograft mice studies were performed to confirm the results in vivo.

Results:
CC-115 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.07 up to 6.9 mM. The anti-proliferative efficacy of Erlotinib was increased in the NSCLC cells synergistically by combination treatment with CC-115 with combination indices down to 0.04-0.2, indicating strong synergy. The synergistic, anti-proliferative effect of the combination treatment could be explained by increased cell cycle arrest and inhibition of signaling components in the mTOR pathway, especially 4E-BP1. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of Erlotinib and CC-115.

Conclusion:
We demonstrate that the therapeutic effect of the EGFR tyrosine kinase inhibitor Erlotinib can be increased by simultaneous treatment with the mTOR kinase/DNA-PK inhibitor CC-115, justifying further clinical studies in lung cancer patients with primary or acquired resistance to EGFR TKIs.

Background:
In-frame insertions in exon 20 of EGFR are infrequent activating mutations in the tyrosine kinase domain that have decreased sensitivity to EGFR inhibitors and currently have no available targeted therapies. In vitro studies ectopically expressing some of the common insertions (3 to 21 bp between codons 762 and 770) show reduced sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients whose tumors harbor these mutations do not respond to EGFR kinase inhibitors. To date, there are no known patient-derived cell lines that harbor the EGFR exon 20 insertions that recapitulate patient insensitivity to EGFR TKIs. Here we report the isolation and characterization of a patient derived cell line with an EGFR exon20 insertion.

Methods:
The CUTO-14 cell line was derived from a malignant pleural effusion of a lung adenocarcinoma patient harboring the EGFR exon 20 insertion p.A767_V767dupASV after obtaining IRB-approved informed consent. PCR amplification of EGFR exon 20 and subsequent Sanger sequencing was performed on genomic DNA isolated from CUTO-14. H3255 (L858R) and HCC827 (exon 19 del) cell lines were used as controls because they harbor sensitizing EGFR mutations. Cell viability was evaluated by MTS proliferation assay. Phosphorylation status and signaling was analyzed by western blot and an EGFR phosphorylation array. For tumor xenograft studies, nude mice were injected with 1.5 x 10[6] cells in matrigel and evaluated weekly for tumor growth.

Results:
Genomic sequencing of CUTO-14 demonstrated that the cell line maintains the pA767_V767dupASV EGFR exon 20 insertion. CUTO-14 showed relative resistance to gefitinib inhibition compared to HCC827 and H3255 in ERK1/2 phosphorylation assays. CUTO-14 also demonstrated reduced sensitivity to gefitinib compared to HCC827 and H3255 in cell proliferation assays. Tumor formation was observed in mice after injection in nude mice.

Conclusion:
CUTO-14 cells represent a novel model for the investigation of therapeutic strategies for EGRF exon 20 insertions mutations. The cell line has the ability to develop tumors in vivo and importantly shows reduced sensitivity to EGFR TKIs mimicking the lack of response in patients with these mutations.

Background:
Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that the basic helix-loop-helix transcription factor, Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models, and that inhibition of Twist1 in murine models and KRAS mutant NSCLC cell lines led to oncogene-induced senescence (OIS) and in some cases, apoptosis. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.

Methods:
We used gene expression profiles from KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis to identify pharmacologic inhibitors of TWIST1. Growth inhibition was determined through the colony formation and MTS assays. Apoptosis (cl-PARP, active anti-C3) and OIS (SA-β-Gal) was assessed. Genetic (shRNA) and pharmacologic inhibition of the TWIST1-E2A pathway was performed. Lung tumor burden as well as levels of TWIST1 protein, apoptosis and proliferation were measured after treatment with harmine in the CCSP-rtTA/tetO-KrasG12D/Twist1-tetO7-luc(CRT) mice.

Results:
We found that several of our CMAP compounds had significant growth inhibitory effects in NSCLC cell lines. Interestingly, a family of related harmala alkaloids including harmine ranked highly in our CMAP analysis. We observed that harmine could inhibit growth in KRAS mutant NSCLC cell lines through the induction of OIS or apoptosis and phenocopied genetic inhibition of TWIST1. Remarkably, harmine treatment led to TWIST1 protein degradation as well as degradation of its binding partners, the E2A proteins, E12/E47. Furthermore, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1 and were independent of its ability to inhibit the DYRK kinases. In addition, we demonstrated that heterodimer formation of TWIST1/E12/E47 resulted in a reciprocal stabilization of each binding partner and that E12/E47 are required for TWIST1 mediated suppression of OIS and apoptosis. Importantly, we found that harmine preferential targets the TWIST1-E12 heterodimer for degradation and the growth inhibitory effects of harmine are in due in at least part to the ability to inhibit the TWIST1/E12/E47 heterodimer as overexpression of the E2A proteins can suppress harmine induced cytotoxicity. Finally, we have demonstrated that harmine treatment lead to Twist1 protein degradation and tumor growth inhibition in our Kras[G12D]/Twist1 murine model of lung adenocarcinoma. We are currently testing and designing structure analogs of the initial candidate agents to develop more specific and potent inhibitors of TWIST1.

Conclusion:
We have identified a novel TWIST1 inhibitor harmine that induces degradation of TWIST1 and its binding partners, E12/E47 and inhibits the growth of KRAS mutant NSCLC both in vitro and in vivo. Therefore, we believe that targeting the TWIST1-E2A pathway would be an effective therapeutic strategy. Since TWIST1 is essential not only for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, the development of this novel class of TWIST1 inhibitors could have a significant clinical impact.

Abstract not provided

Background:
Gene fusions of anaplastic lymphoma kinase (ALK), ROS1, and RET are targetable oncogenes present in approximately 9% of advanced NSCLC. Current assays for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHQ) and qRT-PCR. These tests, however, are complex and have disadvantages in terms of turnaround, sensitivity, cost and throughput. The nCounter platform allows joint detection, in a single tube, without any enzymatic reaction and in 72 hours, of multiple fusion genes by transcript-based method from formalin-fixed paraffin-embedded (FFPE) samples.

Methods:
A custom set consisting of 5´and 3´ probes and/or fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was evaluated. A panel of ALK-ROS-RET positive cell lines (H2228, H3122 [EML4-ALK], SU-DHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS], BaF3 pBABE [CD74-ROS], LC2/ad [RET]) and control fusion negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) were used for nCounter validation. To determine the minimum of tumor surface area for detection, ALK translocated cell line H2228 was tested in FFPE at increasing cell numbers (2500, 5000, 10.000, 25000, 50000) corresponding to a surface area of 0.27, 0.55, 1.1, 2.75 and 5.5 mm2, respectively, in the FFPE block. A total of 38 FFPE samples positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS (n=7) and RET (n=1) were also analyzed. Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and 4 house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative ALK fusion translocation was defined by a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively. Response to crizotinib by RECIST criteria was retrospectively collected in patients with ALK-positive NSCLC.

Results:
nCounter sensitivity to predict fusion transcripts ALK, ROS and RET in cell lines by using both methods (3’/5’ and direct reporter probes) was 100%. Results indicate that samples containing as few as 10% positive tumor cells and a 2.75 mm2 tumor surface area were sufficient for adequate gene fusion detection. The accuracy of prediction (AUC) of ALK 3’-5’ ratio score in 45 independent samples was 82.6% (95% CI 69.3-95.6) with a kappa coefficient score of 0.637. Among 28 samples ALK-FISH-positive, ALK 3’-5’ scoring was positive in 27 samples (96%). One sample was non-evaluable by ALK 3’-5’ scoring. Among the 17 samples ALK-FISH-negative, ALK 3’-5’ score was negative and positive in 10 (59%) and 7 (41%) samples, respectively. All patients with ALK-FISH-negative samples but ALK 3’-5’ score positive (n=7) were positive for ALK IHC and 5 of them were treated with crizotinib. Response assessment was available in 3 of these patients and response rate was 100%. One patient non-evaluable by FISH but positive 3’-5’ scoring also responded to crizotinib.

Conclusion:
The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening modality that might identify FISH-negative/non-evaluable NSCLC patients who could benefit from ALK inhibitors.

Background:
Mutation analysis plays a central role in the management of lung adenocarcinomas (LUAD). The use of multiple single gene or mutation specific assays, broadly adopted in many laboratories to detect clinically relevant genomic alterations, often leads to delays if sequentially performed, tissue exhaustion, incomplete assessment and additional biopsy procedures. Comprehensive assays using massively parallel “next-generation” sequencing (NGS) offer a distinct advantage when addressing the increased testing needs of genotype-based therapeutic approaches. Here we describe our experience with a 410 gene, clinically validated, hybrid-capture-based NGS assay applied to testing of LUAD.

Methods:
Consecutive LUAD cases submitted for routine mutation analysis within a 1 year period were reviewed. Unstained slides of formalin fixed, paraffin embedded tissue were received for each case (range 15-20 slides/case). Corresponding H&E stained slides were reviewed and cell counts were performed in a subset of cases with limited material to establish minimal tissue requirements. Testing was performed by a laboratory-developed custom hybridization-capture based assay (MSK-IMPACT) targeting all exons and selected introns of 410 key cancer genes (J Mol Diagn 17:251-264, 2015). Barcoded libraries from tumor / normal pairs were captured and sequenced on an Illumina HiSeq 2500 and analyzed with a custom analysis pipeline.

Results:
A total of 469 specimens were received for comprehensive testing (98 cytology samples, 239 needle biopsies, 132 large biopsies/resections) of which 93% (436/469) were successfully tested. Thirty four cases (7%, 34/469) failed due to very low tumor content or low DNA yield. Cell counts for failed samples averaged 239 cells / slide (range 10-270) while all successfully tested had over 1,000 cells / slide each. Failure rate was similar for cytologies and biopsies. An average of 10 genomic alterations were detected per patient (range 1-96). The most frequently mutated genes were TP53, EGFR, KRAS, KEAP1 and STK11. Copy number gains of NKX2-1 and EGFR genes and CDKN2A loss were most common. EGFR mutations and ALK fusions were detected in 28% and 4% of cases, respectively. Among the 299 EGFR / ALK WT cases, MSK-IMPACT uncovered targetable genomic alterations that would have remained undetected through focused EGFR/ALK testing alone. These included fusions in RET (10) and ROS1 (13), mutations in ERBB2 (11) and BRAF (19) and amplifications in MET (12, unrelated to EGFR), MDM2 (26) and CDK4 (20) among others. The higher than expected rates of RET and ROS1 fusions are related to enrichment of previously tested cases known to be negative for other driver alterations.

Conclusion:
Comprehensive hybrid-capture based NGS assays such as MSK-IMPACT are an efficient testing strategy for LUAD across sample types. This upfront broad approach enables more optimal patient stratification for treatment by targeted therapeutics.

Background:
Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung cancers, the vast majority of which are non-small cell lung carcinomas (NSCC). It is critical to further distinguish adenocarcinomas from squamous carcinomas in order to optimize the efficiency of the precision medicine analysis for the detection of active molecular targets for therapies. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin-A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin-A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung.

Methods:
Developmental reagents from Ventana Medical Systems, Inc. were leveraged for this study. Detection of CK 5/6 and p40 [BC28] was used to identify squamous cell carcinoma cells. Detection of Napsin A and TTF-1 was used to identify adenocarcinoma cells. Detection was accomplished using secondary antibody:enzyme conjugates and orthogonal chromogenic detection chemistries to simultaneously detect all 4 biomarkers. Fully automated multiplexed detection was performed on a Benchmark XT with 4 microns thick sections from formalin fixed paraffin embedded, non-small cell lung cancer specimens obtained from both the Ventana Medical Systems, Inc. tissue bank and from the Thomas Jefferson University’s Department of Pathology, Anatomy and Cell Biology laboratories. Detection of each marker in multiplex was compared to individual detections using diaminobenzidine deposition according to established Ventana Medical Systems, Inc. protocols. All detections were reviewed by a board certified pathologist.

Results:
Adenocarcinomas (7 of 7) and the adenocarcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for TTF-1 (yellow nuclear stain) and Napsin-A (pink cytoplasmic granular stain). Squamous cell carcinomas (5 of 5) and the squamous cell carcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for p40 (blue nuclear stain) and CK5/6 (brown cytoplasmic stain). The colors were clear, distinct, easily differentiated and recognizable. There was no discrepancy between the expression of the individual antibodies and the expression of the same antibodies in the multiplex setting.

Conclusion:
Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or IHC, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. This new quadriplex IHC offers the capability with a single 4 micron section to accurately diagnose primary lung adenocarcinoma, squamous cell carcinoma or adenosquamous carcinoma and while conserving tissue for additional molecular testing.

Abstract not provided

Background:
Next generation sequencing (NGS) has exceptional sensitivity, but at the expense of false positives. This can result in a less than optimal positive predictive value and eventually the futile treatment of patients. We have developed a unique set of quality control filters for both Ion Torrent and Illumina that minimize false positives, but have little negative impact on sensitivity. To address this paradoxical association of sensitivity and false positives, we developed a dual platform methodology of NGS using both the Ion Torrent and Illumina to solve this classical dilemma.

Methods:
A series of filters were developed to determine quality cutoffs for variant calls to minimize false positives that included the minimum quality score threshold (QUALT), minimum percent variant reads (MPVR), minimum variant reads (MVR), minimum variant reads threshold (MVRT), minimum variant allelic frequency threshold (MVAF), minimum variant reads positive predictive value (MVR-PPV), and systematic errors (SE). A parallel system of using the MiSeq and PGM to sequence all specimens within an IT systems control and a Classify Callsmatrix solution for mutational analysis was designed. Unique cohorts of patients with prior exome sequencing as part of TCGA were used as gold standard controls with matching fresh frozen and FFPE samples.

Results:
Table 1 provides the results of filters developed to maximize sensitivity versus PPV. Using our targeted sequencing panel the PGM consistently outperformed the MiSeq for the standard performance characteristics of sensitivity and PPV for both frozen and FFPE samples. Both platforms have systematic false positives that are unique and gene specific.

Table 1 Platform	Tissue	VAF setting	QUAL Cutoff	MVRT Cutoff	MVAF Cutoff	Mean Sensitivity	Range Sensitivity	Mean PPV	Range PPV
PGM	FF	0.2%	None	None	None	100%	93-100%	88%	70-96%
PGM	FF	0.2%	>99	>=20	>.035	99%	93-100%	95%	78-100%
PGM	FFPE	0.2%	None	None	None	99%	93-100%	58%	2-94%
PGM	FFPE	0.2%	>99	>=21	>.018	97%	63-100%	92%	40-100%
MiSeq	FF	1%	None	None	None	97%	79-100%	49%	31-66%
MiSeq	FF	1%	>99	>=5	>.017	95%	66-100%	82%	66-95%
MiSeq	FFPE	1%	None	None	None	94%	43-100%	10%	2-37%
MiSeq	FFPE	1%	>99	>=10	>.028	92%	39-100%	62%	6-100%

Table 2 provides the results for dual platform sequencing which show a marked reduction in false positives while maintaining sensitivity.

Table 2	FF	FF	FF	FF	FFPE	FFPE	FFPE	FFPE
	SNV(s)	SNV(s)	Indels	Indels	SNV(s)	SNV(s)	Indels	Indels
	Percent	VAF	Percent	VAF	Percent	VAF	Percent	VAF
Assay Sensitivity	99.8%	2.87%	100.0%	2.90%	98.3%	3.56%	100.0%	3.60%
Assay PPV	97.5%	2.87%	91.0%	2.90%	96.7%	3.56%	91.0%	3.60%

Conclusion:
Single platform NGS is plagued by false positives. Dual platform sequencing is a reliable method of diminishing false positives with minimal to no impact on sensitivity.

Background:
Molecular testing has become important in managing advanced non-small cell lung cancer (NSCLC), both in clinical practice, as well as in clinical trials. For the latter, tissue samples often have to be analysed in a central laboratory. We evaluated the turnaround time and possible delay in start of therapy in this process.

Methods:
We reviewed our prospective database on all molecular testing cases for clinical trial suitability in patients with advanced NSCLC between March 1, 2011 (start) and October 31, 2014. The following time points were considered: T1 (request for tissue sections from the pathology lab); T2 (receipt of sections and shipment); T3 (arrival of sections in central lab (CL)); T4 (receipt of biomarker result from CL).

Results:
251 patients were considered for biomarker-driven trials. Twenty-three cases did not have further analysis, as the request for central molecular testing was cancelled: insufficient tissue (n=11); exclusion criterion (n=10); patient refusal (n=2). Results for the remaining 228 patients were: failure of central biomarker analysis due to insufficient quantity of tissue (n=18), or quality of tissue (n=3, i.e. decalcification or poor fixation). Valuable results were obtained for 207 patients. In 91 of 228 (39.9%) samples sent, a biomarker of interest was documented. This led to 34 clinical trial inclusions. Other patients were no longer eligible due to loss of performance status (n=20), loss of contact (n=14), no trial slot available at the appropriate time (n=18), or exclusion criteria (n=5). The mean waiting time between signing informed consent (T1) and receiving results of the biomarker analysis (T4) was 25.1 calendar (SD 17.3) days (Table). The preparation of the unstained slides by the pathology lab took about 9.1 (SD 6.8) days, the time of the biomarker testing itself accounted for 12.8 (SD 7.3) days. For 18 of 228 (7.9%) patients, repeated sample shipments were needed because of insufficient tumor cells, their mean waiting time between informed consent and receiving the biomarker result was 62.2 (SD 38.4) days. Table: Waiting times (t) in molecular testing for 228 patients.

Time interval	Mean	StDev	Median	Range
Pathology lab (T2-T1)	9.1	6.8	7.0	1 - 70
Shipment (T3-T2)	1.8	1.6	1.0	0 - 17
Analysis (T4-T3)	12.8	7.3	12.0	2 - 58
Request to result (T4-T1)	25.1	17.3	22.0	7 - 184

Conclusion:
While molecular testing is important in many NSCLC trials, our results show that waiting times for central laboratory analysis can cause an important delay in treatment initiation, and even ineligibility for the trial(s) under consideration. Start of therapy based on properly validated local testing, with a posteriori central biomarker testing to guarantee the integrity of the trial, would be more rewarding for quite some patients.

Background:
Lung cancer is the leading cause of cancer mortality worldwide, and metastatic spread of cancer to distant organs is the main reason for lung cancer deaths. They spread to different distant organs, exhibit an outstandingly different situation of clinical characteristics and will be medically and surgically incurable. Thus, there is a clear need for a reliable and efficient in vitro culture model to enable transition to invasion and journey to distant organs of these critical steps in cancer metastatic progression.

Methods:
Here we report a biomimetic multi-organ microfluidic chip system more closely reconstituting the structural tissue arrangements, functional complexity and dynamic mechanical microenvironments and reproducing survival, growth, transition to invasion and journey to distant multi organs in lung cancer metastasis. To reconstitute the actual growth conditions of lung cancer in vivo, we created a thin, porous, flexible membrane, integrated microfluidic chip emulating the in vivo tissue structure and enabling heterotypic cell interactions, while maintaining cell compart-mentalization. The human bronchial epithelial cells and stromal cells were cultured on opposite sides of the membrane. Once the cells were grown to confluence, air was introduced into the epithelial compartment to create an air-liquid interface and more precisely mimic the lining of the lung air space. Then lung cancer cells were cultured on the human epithelial compartment to mimic lung cancer formation and the multi organ chambers were linked with side channels that supply lung cancer cells to the brain, bone or liver cells chamber to mimic lung cancer metastasis. In addition, the system provided analyzing cell physiology and visualizing complex cell behaviors in a more physiologically relevant context.

Results:
A biomimetic multi-organ microfluidic chip system was created. The quick formation of lung cancer cells that grow away from their natural margins and then attack adjacent components and spread to other organs were observed at all times and the cells characterizations were also detected accurately and effectively. In this multi-organ pathogenesis system, it might be possible to provide an ultrahigh level of reproducibility, authenticity and sensitivity.

Conclusion:
This microdevice provides a proof of principlefor this novel biomimetic strategy that is inspired by the integrated chemical, biological, and mechanical structures and functions of the living multi organs. This versatile system enables direct visualization and quantitative analysis of diverse biological processes of the intact lung cancer metastasis in ways that have not been possible in traditional cell culture or animal models.

Background:
Next-generation sequencing (NGS) can be used to interrogate multiple areas of the tumor genome. Several hot-spot panels have been developed to identify variants amenable to targeted therapies and enrollment into clinical trials. Variants of unknown significance (VUS) in the vicinity of hot-spots are routinely discovered. To better understand these obscure VUS, we built a Protein Viewer that displays the relationship of known actionable variant(s) to the VUS.

Methods:
We developed a web-based protein viewer that can be deployed across multiple browsers. The tool supports the visual representation of 23 genes which are interrogated by our NGS platform. We used the longest mRNA transcript (hg19) to define the protein domains. All actionable variants as reported by an knowledge database were included, with the selected VUS differentially highlighted. VUS is defined as a non-actionable variant that is not reported in dbSNP.

Results:
Approximately 50% of all stage III and IV lung cancer patients tested by our NGS platform have one or more VUS. After the variant information is loaded in the Protein Viewer, a two-dimensional image of the full length protein with actionable variants and VUS is displayed (Figure 1). The Viewer is utilized at RPCI to present cases at our molecular tumor board for quick visualization and discussion. Figure 1 Figure 1: Protein Viewer with a PIK3CA VUS harboring a Q546H (pink) in a lung adenocarcinoma. Top panel with PIK3CA exons 2-21 boundaries (vertical lines) with protein domains (blue rectangles along axis). Bottom panel with the zoom feature which allows more discreet visualization of the VUS, a neighboring Q546K actionable variant (green), and additional actionable variants for ovarian cancer (green rectangles).



Conclusion:
Understanding the relationship of VUS to protein domains and proximity to previously known actionable sites is a potentially powerful way to evaluate and determine whether a patient might be a candidate for targeted therapy. Because the exact effect of the VUS on the function of the protein is still impossible to discern (tyrosine kinase inhibitor sensitivity/ resistance/no effect), the next generation of protein viewers should incorporate 3D and protein folding/domain interaction prediction capabilities.

Abstract not provided

Background:
Genomic profiling is a powerful method for identifying mutations that drive tumors and matching patients to targeted therapies. However, this may only be a transient solution and resistance commonly emerges as the mechanism of targeted inhibition is overcome. Proteomic profiling of the tumor provides a dynamic tool to survey altered protein expression and deregulated pathways, which in turn may implicate specific treatments or identify novel therapeutic targets. Mass spectrometry offers highly multiplexed proteomic measurements, but extensive sample pre-processing and low sample throughput can lead to extended analysis times of weeks or months. Thus a need exists for a high throughput, sensitive and quantitative platform for proteomic analysis.

Methods:
We used the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV, to analyze protein lysates from 63 lung tumor samples. The assay does not require sample pre-fractionation, and this study (which generated over 142,000 protein measurements) represents less than one day of SOMAscan throughput. The study consisted of matched tumor/non-tumor protein lysates prepared from 18 squamous cell carcinoma and 45 adenocarcinoma fresh-frozen resected specimens, 86% of which were Stage I/II. The paired log~2~ tumor/non-tumor ratio was calculated and hierarchical clustering heat maps and dendrograms were constructed to identify related protein regions and tumor phenotypes.

Results:
Common proteomic changes and unique tumor phenotypic groups were identified by unbiased clustering algorithms. Large, consistent tumor/non-tumor differences of at least 4-fold were observed for 35 proteins in at least 20 (32%) of the tumors. Some of these proteins were more than 100-fold higher in individual tumors. The two most commonly elevated proteins were thrombospondin 2 and MMP12, which were increased in 81% and 61% of the tumors, respectively. We have previously reported higher levels of MMP12 in the serum of lung cancer patients, and the current data supports a tumor-associated origin for circulated MMP12. A second analysis identified sub-phenotypes of tumors clustered by common protein alterations independent of histological classification or mutation status. Many of these tumor subsets had increased expression of known oncology drug targets.

Conclusion:
Broad, unbiased high-throughput proteomic profiling of tumor tissue may reveal individual phenotypes that hold the potential to respond to targeted therapies and to monitor therapeutic efficacy throughout treatment. Measuring proteins complements mutation analysis by enabling therapeutic selection beyond driver mutation targets, including immune modulator therapies, repurposing existing drugs and enriching clinical trials with likely responders. While genomics is a fixed snapshot, blood- and tissue-based serial proteomic measurements respond to change and can lead to the personalized adaptation of treatment and identification of novel therapeutic targets.

Background:
There is a clinical need to establish whether those pathways activated in vitro and in animal models, are also activated in human lung cancer. We established a window-of-opportunity clinical trial platform in lung cancer where novel agents are administered in the preoperative period. Intratumoral drug concentrations are correlated with molecular marker changes. Our four completed window-of-opportunity clinical trials established that optimal intratumoral drug concentrations are needed for the desired pharmacodynamic effects, providing direction for optimal dose and schedule. To further evaluate the value of this window-of-opportunity platform, we investigate the impact on standard postoperative outcome measures.

Methods:
39 consecutive patients enrolled under the window-of-opportunity platform were matched to 39 contemporary patients undergoing the same operation by the same surgeon. Co-morbidities and stage of lung cancer and postoperative complications were compared using univariate and multivariate analysis. Wilcoxon Scores (Rank Sums) for variable data elements and Fisher’s Exact Test was used for analysis.

Results:
When comparing window-of-opportunity patients to control patients, there was no difference in age, pack years of smoking, or incidence of comorbidities including diabetes, coronary artery disease, hypertension, chronic obstructive pulmonary disease, and previous cancer. There was no difference in the stage distribution, (stage I: 28 vs. 22, stage II: 5 vs. 3, stage III: 5 vs. 2 stage IV: 1 vs. 1, p=0.1642). There was also no difference in the incidence of postoperative pneumonia (4 vs. 9, p=0.2235), other infection (2 vs. 3, p=0.8208), atelectasis (2 vs. 4, p=0.6748), myocardial infarction (0 vs. 0, p=1.000), reoperation for bleeding (1 vs. 1, p=1.000), pulmonary embolism (1 vs. 2, p=1.000) or number patients experiencing any complication (14 vs. 8, p=0.131118). There was no difference in the distribution of survival at 2 years (27 vs. 30) or 5 years (10 vs. 15), p=0.2266.

Conclusion:
The window of opportunity platform does not increase the perioperative risk of complications in early stage NSCLC patients undergoing surgery. By evaluating drug effect and the potential toxicities, window-of-opportunity trials validate mechanisms established in the laboratory and facilitate bi-directional translation research.

Background:
Small Cell Lung Cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Response rates to first line chemotherapy are generally high, but progression free survival is short due to development of chemotherapy resistance via mechanisms not well understood. Due to the difficulty in collecting tissue biopsies in SCLC, blood, which can be sampled simply and routinely, provides a means of inferring the current genetic status of a patients tumour via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). These offer a minimally invasive opportunity to study drug resistance mechanisms, evaluate tumour heterogeneity and potentially reveal new drug targets in this disease. However, accurate assessment of both CTCs and ctDNA requires all blood cells be maintained intact until samples are processed, particularly when analytes present are at very low concentrations. Here we describe and validate a blood collection protocol that does not require on-site processing, and which is amenable for analysis of both CTCs and ctDNA following storage at ambient temperature in CellSave vacutainers for up to 96 hours after blood collection.

Methods:
To evaluate the suitability of using CellSave preserved samples for circulating free DNA (cfDNA) analysis, we undertook a 20 healthy normal volunteers (HNV) study and 45 patient sample study, with parallel EDTA and CellSave bloods collected. For each sample cfDNA was isolated between 4 hours and 96 hours post-draw and cfDNA yields determined. A potential issue with using CellSave blood was that the CellSave preservative could act as a DNA damaging agent and effectively increase background sequencing errors. To test this, the EDTA and CellSave cfDNA samples were subjected to next generation sequencing (NGS) to estimate the overall mutation burden. In addition, the utility of CellSave ctDNA for targeted NGS was also determined. Finally, SCLC-specific copy number aberrations (CNA) were analysed in ctDNA and CTCs isolated from the same CellSave blood sample from individual SCLC patients.

Results:
We demonstrate that yields of cfDNA obtained from 96-hour whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 hours of blood draw. Targeted and genome-wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples, thereby validating CellSave blood as a viable source of ctDNA. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA allowing direct comparison of the genetic status of patients’ tumours.

Conclusion:
In summary, we have demonstrated the suitability of whole blood CellSave samples for both CTC and ctDNA molecular analysis in SCLC. The ability to generate informative molecular profiles of both CTCs and ctDNA from a simple whole blood sample, up to 4 days post-draw represents a significant methodological improvement for clinical benefit. We posit that as minimally invasive, liquid biopsies become increasingly employed for cancer patient management, the ability to routinely and simply draw blood and ship samples to accredited biomarker assessment laboratories will greatly facilitate the delivery of personalised cancer medicines.

Abstract not provided

Background:
The National Lung Screening Trial (NLST) established a 20% reduction in lung cancer-specific mortality with low-dose computed tomography (LDCT) in 30 pack year smokers. However, approximately 25% of all lung cancers occur in non-smokers, and screening guidelines are needed for this large cohort. Pre-test probability of lung cancer can be estimated in this group using a validated risk prediction model [Liverpool Lung Project, LLP]. The LLP compares risk in 579 lung cancer cases with 1157 age and sex matched controls.

Methods:
We used the LLP model to illustrate risk profiles for non-smoking females compared to 30 pack year smokers [the NLST target population]. This tool revealed the individual and cumulative effect of risk factors in non-smoking females. The LLP estimates the probability of developing lung cancer within 5 years based on age, sex, smoking history, family history of lung cancer, infectious and occupational exposures, and prior diagnosis of a malignant tumor other than lung cancer. This tool has been validated in a Caucasian population including never and ever smokers up to 79 years of age (cross validation of tool: AUC=0.70).

Results:
We generated risk profiles for female non-smokers between 65-79 years old and no other co-morbidity, and compared the risk against those for women in the same age bracket with 30-pack year smoking history or additional non-tobacco risk factors (i.e. previous pneumonia, asbestos exposure, having a relative with lung cancer < 60years, and the combination of all factors listed). Significant risk with increasing age was predicted by the LLP model for women with 30 pack year smoking history (peak risk at age 75 years 2.2% over next 5 years). This is less than the risk of 6.7% over the next 5 years (at age 75 years) for non-smoking women with the combination of all mentioned risk factors. Relative risk of lung cancer of non-smoking women with all noted risk factors was 3.5 compared to women with no other risk factors other than 30 pack-years smoking history. Relative risk of smoking women compared to non-smoking without other risk factors was 4, while relative risk of non-smoking women with cumulative risk factors was 14 compared to non-smoking women with no other risk factors. Figure 1



Conclusion:
Therefore, the development of lung cancer risk prediction models is a key advance in the assessment of patients at risk. Individual risk assessment can be judged using the LLP model and could encourage refinement of screening recommendations.

Background:
Epidemiological studies consistently show that chronic obstructive pulmonary disease (COPD) is associated with an increased risk of lung cancer among smokers. However, debate exists as to whether there is a linear relationship between the severity of COPD and risk of lung cancer. The National Lung Screening Study (NLST) and it’s sub-study by the American College of Radiology and Imaging Network (ACRIN), provides the means to re-examine these findings. We examined the effect of spirometry-defined COPD (according to GOLD status at baseline), on the risk of lung cancer in the NLST-ACRIN cohort (according to lung cancer incidence), in a large prospective lung cancer screening study of high risk smokers.

Methods:
In the NLST-ACRIN cohort of 18,475 screening participants eligible for the NLST, 6,436 screening participants had COPD (35%) according to baseline pre-bronchodilator spirometry and were followed for a mean of 6.4 years. From this group, 401 lung cancer cases were identified. The 6,436 screening participants with COPD were sub-grouped according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1 (N=1607), 2 (N=3528), 3 (N=1083) and 4 (211). Lung cancer incidence at the end of follow-up was compared between the GOLD subgroups and those with normal spirometry (N=12,039).

Results:
Compared to those with normal spirometry, where the lung cancer incident rate was 4.63/1000 person years, the lung cancer incident rate was 7.58/1000 person years for GOLD 1, 9.43/1000 person years for GOLD 2, 12.7/1000 person years for GOLD 3 and 15.55/1000 person years for GOLD 4 (all P<0.0001). The lung cancer histology was significantly different, with more squamous and non-small cell cancers in those with COPD but more adenocarcinoma and Bronchoalveolar carcinoma in those with normal lung function (P<0.004). Figure 1



Conclusion:
In a large prospective study of unselected high risk smokers with and without COPD, we report a strong linear association between increasing severity of COPD and increasing lung cancer risk (incidence). This suggests that the risk of lung cancer is greatest in those with the most severe COPD and 3-4 fold greater than those with normal lung function. We also report that lung cancers of more aggressive histology were more common in those with COPD. Funding This study was funded by a grant from Johnson and Johnson and grants U01-CA-80098 and U01-CA-79778 to the American College of Radiology Imaging Network

Background:
Chronic obstructive pulmonary disease (COPD) is a major intermediate phenotype for Lung cancer (LC); the presence of COPD conferring a three- to 10-fold increased risk of LC when compared with smokers without COPD. Variability in lung function and risk for COPD in people with similar cigarette smoking histories, together with studies of familial aggregation, support an important role for genetic factors in COPD. Therefore, we employed a targeted sequencing approach to identify variants associated with susceptibility to COPD. We focused on 107 common susceptibility loci identified in recent genome-wide association studies (GWAS) catalog in LC, COPD, lung function and smoking behavior.

Methods:
We employed an extreme phenotype approach in two carefully phenotyped extreme categories of smokers from the COPDGene study: 1) Long-term smokers with normal lung function defined as post-bronchodilator FEV1 ≥ 80% predicted, FEV1/FVC ≥ 0.7, with smoking histories of 15+ pack-years, considered as resistant to the effects of smoking, n = 318.; 2) Susceptible smokers with severe COPD defined as GOLD Stages 3-4 (post-bronchodilator FEV1 < 50% predicted and FEV1/FVC < 0.7), with smoking histories of 10+ pack-years, n = 309. We performed exome sequencing and analyzed rare (minor allele frequency [MAF] < 0.01 in reference exome databases) substitution and indel variants predicted to be functional in susceptibility loci previously identified by GWAS.

Results:
Our analysis revealed eight potentially causative non-synonymous substitution variants, occurring in 3+ susceptible smokers with COPD, and with none in resistant smokers. The two most intriguing associations were TGM5 Thr42Asn and ZBTB9 Leu43Val, that presented in six and four susceptible smokers with severe COPD, respectively. Moreover, we found an additional TGM5 compound heterozygous mutation, Val202Ile, carried by two severe COPD patients with none in the resistant smokers. TGM5 is located on 15q15.2, a susceptibility locus only reported in LC GWAS, and the p.Thr42Asn in exon 2 is only 1563bp away from the LC GWAS hit (rs504417) in intron 1. ZBTB9 is located on 6p21.31, a locus common to LC, lung function and smoking behavior. Table 1. List of top candidate deleterious mutations in susceptible smokers

Marker	Gene	Protein	# Mutated COPD	# Mutated Control	MAF in KG
rs148913728	TGM5	Thr42Asn	6	0	0.0012
rs144575810	TGM5	Val202Ile	2	0	0.0002
rs41267651	ZBTB9	Leu43Val	4	0	0.0008
rs147018937	NID2	Lys1296Arg	3	0	0.0004
rs147278493	SLC6A18	Gly496Arg	3	0	0.0002
rs116926108	IFIT3	Leu390Arg	3	0	0.0002
rs142934543	LAMA1	Lys2086Thr	3	0	0.0002
rs41316996	DBH	Gly482Arg	3	0	0.0004
rs41298243	MYOF	Phe1400Leu	3	0	0.0002

LC, lung cancer; COPD, chronic obstructive pulmonary disease; SM, smoking behavior; PF, pulmonary function; MAF, minor allele frequency; KG, thousand genome.

Conclusion:
Our targeted exome sequencing results demonstrate highly disruptive COPD risk-conferring TGM5 and ZBTB9 rare mutations that are associated with susceptibility to lung cancer in smokers, and strengthen the concept of a shared genetic link between COPD and LC.

Background:
The National Lung Cancer Screening Trial found, after 6.5 years, a 20% reduction in LC mortality in high-risk patients (pts) screened with low-dose computed tomography compared to chest x-ray. However, LC screening programs (SP) result controversial due to potential cost-effectiveness issues. Familial LC aggregation (fLCa) has been described previously. The estimated relative risk of LC is ∼1.8 for offspring of parents with LC. Linkage analysis has mapped a dominant locus to chromosome 6 in LC pedigrees. Therefore, in this high-risk subpopulation, SP may have clear advantages. This is the first study to investigate the incidence of fLCa conducted in a sEp.

Methods:
Overall, 509 cancer pts of Spanish (n = 473) or Portuguese (n = 36) origin were included in the analysis. A cohort of 236 consecutive pts (cases) diagnosed with LC was studied for family history (FH) of any type of cancer including LC. Another cohort of 273 pts (controls) with similar demographic characteristics diagnosed with cancer types other than LC was also studied for FH of cancer. We investigated whether LC pts show a higher incidence of fLCa than subjects with other solid tumors.

Results:
Among LC pts with a positive FH for LC, 36.7% showed one of their parents as the only LC relative, 26.5% showed one or more siblings, 18.4% one or more either uncle or aunt, 6.1% their grandfather/grandmother and 12.2% other combinations. Regarding the number of relatives affected, in our LC cohort one relative was the most frequent finding with 42/49 pts (85.7%), 2 in 3 cases (6.2%) and > 3 relatives in 4 subjects (8.1%). We studied the overall incidence of any type of family cancer among cases and controls. No differences were found between groups (72.9% vs 67.4%; p = 0.18). However, in our cohort of LC cases, 49/236 pts (20.8%) had a FH of LC in first or second degree whereas among cancer controls only 29/273 pts (10.6%) showed a LC FH (p = 0.002).

Conclusion:
This is the first estimation of LC FH in a non-selected sEp with LC. 20.8% of LC cases showed a positive FH for LC, being significantly higher (twofold) compared to other cancer pts. Therefore, the usefulness of directed SP for subjects with positive FH of LC should be prospectively evaluated and potential genomic drivers studied.

Table 1. Comparison of incidence of any type of familial cancer and fLCa between a cohort of LC patients and a cohort of subjects with other solid tumors
	LC patients	Other solid tumor patients		p value
	N= 236	N=273
Familial cancer (any type) (n (%))
Yes	172 (72.9)	184 (67.4)		0.18
No	64 (27.1)	89 (32.6)
Familial Lung Cancer (n (%))
Yes	49 (20.8)	29 (10.6)		0.002*
No	187 (79.2)	244 (89.4)
*Statistically significance at p < 0.05
fLCa: familial lung cancer; LC: lung cancer

Background:
Lung cancer with different molecular profile behaves in different ways in terms of etiology, clinical characteristics, and prognosis. It implies that fighting against lung cancer should be moving forward in the direction of modifying the screening strategies on genetically-defined high risk groups, initiating early chemoprevention trials on selected high-risk subjects, and developing personalized cancer treatments. Lung adenocarcinoma in never-smokers, more often harboring epidermal growth factor receptor (EGFR) mutations especially in Asians, has a remarkable therapeutic response to specific tyrosine kinase inhibitors. Most single nucleotide polymorphisms (SNPs) identified by candidate gene and genome-wide association studies are of genes involved in carcinogen metabolic pathway, DNA repair pathway, inflammatory pathway and tumor suppressor pathway. Conflicting results are likely due to heterogeneity of the study population and lack of focus on specific molecular subgroups (e.g. EGFR-mutants). We therefore embark on the current study to identify susceptibility genes in a molecularly-defined (EGFR-mutated) subgroup of never-smoking lung adenocarcinoma in Hong Kong population.

Methods:
Eligible patients with confirmed primary lung adenocarcinoma were recruited from Queen Mary Hospital, Hong Kong. Voluntary healthy controls were recruited from blood donors in Hong Kong Red Cross. 10mL venous blood samples were taken from both cases and controls for DNA extraction and SNP assays. 51 SNPs of 14 genes involved in four different pathways were tested using MassARRAY. A structured questionnaire including the information of environmental exposures at home and workplace, family history of lung cancer and other cancer for all subjects, and clinical characteristics (cell type, EGFR mutation status, staging and treatment etc.) for lung cancer patients were administered to cases and controls. Using SNPstats package, logistic regression analysis adjusted for age and gender was performed to evaluate the association between the studied SNPs and lung cancer development.

Results:
From September 2006 to February 2015, a total of 614 lung cancer patients regardless of histological type and smoking status were recruited. Out of which, 267 never-smoking lung adenocarcinoma patients (72% females, mean age 61.6+/-12.6 years) were regarded as cases in the study. From July 2013 to August 2014, a total of 453 healthy controls (40% females, mean age 53.8+/- 8.3 years) were recruited. Most cases (69%) were at advanced stage with chemotherapy treatment (67.8%). Higher proportion of cases (41.7% at home; 35.4% at workplace) than controls (24.9% at home; 26.9% at workplace) had been exposed to second-hand smoke. Genetic analysis was restricted to 184 pairs of age and gender well-matched cases and controls. Two of the 51 SNPs showed a significant association with lung adenocarcinoma, which were rs2069840 of IL-6 gene in inflammatory pathway (OR: 5.80; 96%CI: 1.60-20.99) and rs1106087 of XPC gene in DNA repair pathway (OR: 3.72; 95%CI: 1.40-9.83).

Conclusion:
Our results suggest that IL-6 rs2069840 and XPC rs1106087 are susceptibility genes for development of EGFR-driven lung adenocarcinoma in never-smoking Hong Kong population.

Abstract not provided

Background:
Lung SCC arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. Our group is using high-throughput genomic techniques to characterize the process of premalignant progression by examining PMLs and non-lesion areas of individuals with PMLs (“field of injury”) to identify events that lead to the development of SCC. Pathway analysis revealed enrichment oxidative phosphorylation (OXPHOS) /respiratory electron transport among genes up-regulated in the airways of subjects with PMLs. OXPHOS is the most efficient metabolic pathway that generates energy in the form of ATP by utilizing the structures and enzymes of the mitochondria. OXPHOS is often elevated during epithelial tissue repair and is superseded by glycolysis in the development of cancer.

Methods:
mRNA-Seq was conducted on cytologically normal airway epithelium collected from indviduals with (n=50) and without (n=25) PMLs. Linear modeling strategies were used to identify genes altered between subjects with and without PMLs (n=206 out of 13,900, genes at FDR<0.001). Pathway analysis by GSEA revealed enrichment (FDR<0.05) of oxidative phosphorylation (OXPHOS)/respiratory electron transport genes among genes up-regulated in subjects with PMLs. To validate these findings, we examined oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in primary airway epithelial cells cultures from PMLs and non-lesion areas and cancer cell lines that have high OXPHOS/ moderate glycolytic (H1299), moderate OXPHOS/ high glycolytic (HCC4006) or low OXPHOS/ low glycolytic (H2085) gene expression. In addition, protein expression of genes elevated in the field of injury including, translocase of the outer mitochondrial membrane (TOMM 22) and cytochrome C oxidase (COX-IV) were measured in FFPE sections of human PMLs and PMLs from the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC.

Results:
OCR and ECAR values in the lung cancer cell lines were consistent with gene expression patterns. Perturbations of OXPHOS resulted in 3 fold (H1299) and 2 fold (HCC4006) higher OCR vales than those in H2085 cells (p<0.05) reflecting higher OXPHOS activity. Whereas the ECAR values were 2.5 fold (HCC4006) and 1.5 fold (H1299) higher than those in H2085 cells (p<0.05), reflecting higher glycolytic metabolism. The OCR and ECAR patterns in the primary premalignant cultures also supported the computational findings in the field of injury of PMLs. The baseline OCR/ECAR values were 1.5 fold higher in the cultures from PMLs compared to non-lesions controls (p<0.001). Additionally the OCR and ECAR values were elevated in response to perturbations in OXPHOS in the PMLs compared to controls. Protein levels of TOMM 22, and COX-IV were found to be elevated in dysplastic lesions compared to controls.

Conclusion:
Together these data suggest that metabolism-associated gene expression is correlated with cellular metabolism and there is an increase in OXPHOS associated with the development of PMLs. Furthermore, there is potential that therapeutically increasing or maintaining OXPHOS in premalignant lesions or the field of injury may be a mechanism of prevention for lung cancer.

Background:
Recently, SPNs have become more frequently encountered in bronchology. Therefore, an efficient and reliable method for detecting SPNs based on their morphological characteristics is needed The aim of this study was to compare the diagnostic value of near infrared (NIR) spectroscopy, in vivo Raman spectroscopy and radial endobronchial ultrasound (EBUS) for solitary pulmonary nodule (SPN). Fluoroscopic guidance with transbronchial biopsy and needle biopsy was performed in all patients.

Methods:
Between February 2014 and February 2015 we examined 22 male and 29 female patients having a median age of 68 years with positron emission tomography-computed tomography findings of metabolically active SPN between 1, 5 to 3 cm in diameter. We used tree types of point monitoring systems. Fluoroscopic guidance (with guide- sheath) was combined with a radial EBUS. In the case radial EBUS conclusively showed catheter position in the centre of SPN (41 cases) than in-vivo Raman Spectroscopy and NIR spectroscopy probes were placed into the guide sheath in order to gather tissue information. Mean measurement time was less than five minutes after establishing ideal position of guide -sheath. Results of in spectroscopy measurements from both Raman spectroscopy and NIR spectroscopy were obtained as differences between spectral characteristics of normal tissue (same side, different lobe) to SPN tissue.

Results:
The results are expressed as sensitivity of RAMAN spectroscopy and NIR spectroscopy towards EBUS navigated biopsies. Statistical analysis of the results showed comparable very high sensitivities for NIR spectroscopy and Raman spectroscopy in confirmation of SPN tissue. From 41 EBUS positive visualisations of SPN there were 38 conclusive histological findings. Both Raman spectroscopy (positive differences in 38 cases) and NIR spectroscopy (positive differences in 36 cases) showed good performance in tissue discrimination.

Conclusion:
Every confirmatory method brings different information about tissue. EBUS describes volume of the SPN and gives valuable information about the position of catheter in the SPN. Raman spectroscopy and NIR spectroscopy bring information about biochemical/ optical characteristics of the tissues.

Background:
Probe-based confocal laser endomicroscopy (pCLE) allows for real-time noninvasive histological imaging via bronchoscopy. Interpreting pCLE images and correlating with pulmonary disease remains challenging. We performed an in vivo study to evaluate the correlation between pathological diagnosis and pCLE imaging of pulmonary disease.

Methods:
We sequentially enrolled the patients with undiagnosed lung lesion, and randomly grouped into control group (TBLB and peripheral EBUS) and pCLE group (TBLB + pCLE and peripheral EBUS + pCLE). pCLE was performed with Cellvizio system (Mauna Kea Technologies, Paris, France). All patients were consent to the procedure. Pathologists and pulmonologists reviewed the images by the Columbus Classification (CC). Questionnaires were applied post the procedure to collect patients’ condition. We developed a computer assistant diagnostic (CAD) system to calculate alveolar diameter, vessel diameter and optical density percentage and compare the CAD diagnostic accuracy with CC standard. The CAD system involved image processing methods to calculate the diameters in pixel domain and then transformed them into the real value. Pseudo-color processing was used to show the density percentage of different tissues. And the histogram was also calculated to figure out the distribution alone gray scale.

Results:
258 patients enrolled in the study, 98 under pCLE examination, while 160 under control group. Among them 128 lesions were diagnosed as malignant tumor by pathological diagnosis, 87 cases were diagnosed as benign disease. Primary features were observed in the samples using pCLE in the lesion of cancer: The normal alveolar in malignant nodules is smaller than benign nodules. While, the vessel in the malignant nodules is thicker than the benign ones. The cellular structure and vessel domination in various subtypes of lung cancer is different. There was no significance on procedure time between control and pCLE group, as well as patients’ secretion, tolerance and willing for repeat examination.

Conclusion:
pCLE can identify lung carcinoma in in vivo procedure with well tolerance and with limit procedure time. As a non-invasive method, pCLE could improve accuracy and avoid unnecessary biopsy. The Computer Assist Diagnosis system could help pulmonologists to better acquire the right image and to differentiate diseases on the site.

Background:
Lung squamous carcinoma is believed to arise from premalignant bronchial epithelial dysplasia, which demonstrates progressive histologic changes leading up to invasive cancer. However, only a small subset of these lesions progress to carcinoma. Recent studies have shown that somatic chromosomal alterations (SCAs) status is a better biomarker than premalignant histology alone. Single-nucleotide polymorphism microarray (SNP array) has been frequently used to delineate these genomic alterations across the whole genome. However, the cellular heterogeneity, from clinical samples such as endobronchial specimens, is a basic obstacle to perform sensitive and accurate detection of SCAs.

Methods:
We used: 1) a lung cancer cell line (NCI-H1395) and its matched lymphoblastoid (NCI-BL1395) cell line; 2) frozen lung tissues containing different percentage of invasive cancer cells surgically resected from a patient; and 3) biopsies and brushings obtained at the visually concerning areas during bronchoscopy. The histology of the clinical samples were graded by the study pathologist. Genomic DNA was isolated from each sample, quantified, and labeled for Illumina SNP array (HumanOmni 2.5-Quad BeadChip). Data analysis and visualizations were performed using Partek Genomic Suite 6.6 software.

Results:
Our study focused on the detection of SCAs by the comparison of genomic DNAs from cancer/premalignant cells (subject) to blood/normal cells (reference) from the same individual. We tested a B allele frequency metric, the subtraction of allelic fractions (delta-θ), on a standardized mixture of genomic DNAs from a lung cancer cell line and its matched lymphoblastoid cell line. Delta-θ proved to be a sensitive parameter to clearly delineate SCAs present in the tumor cell line even with a large proportion of normal cells (up to 90%). To explore the utility of using delta-θ for heterogeneous samples, we used clinical lung cancer specimens with known cancer cell content. In comparison to the other publicly available analytical metrics/algorithms (conventional Log R Ratio plot, mirrored B Allele Frequency plot, and GAP algorithm), delta-θ performed as well or better (with lower computational power needed), and enabled the detection of SCAs even in highly heterogeneous clinical samples (<30% tumor cell content). In addition, we completed a study using a number of bronchial biopsies and brushings with histologic grade ranging from normal to squamous cell carcinoma. SCAs were rarely detected in those of low to mild dysplasia, while they were detected in approximately 25% of moderate or severe dysplasia, and in all carcinoma in situ (CIS) and squamous cell carcinoma specimens. Longitudinal, repeated samplings from a high risk patient who persistently showed high grade dysplasia across the bronchus, revealed that delta-θ could identify SCAs continuously across the whole genome. The fact this individual had highly overlapping SCAs between different bronchial locations indicates genomic field cancerization may occur, along with the histological field effect in premalignant epithelium.

Conclusion:
In SNP microarray studies, delta-θ is a highly sensitive metric for detecting SCAs even in heterogeneous dysplastic bronchial specimens. SNP array may be a powerful tool to understand premalignant genetic alterations and field cancerization.

Abstract not provided

Background:
Lung cancer is the result of a multi-step accumulation of genetic and/or epigenetic alterations; therefore, a better understanding of the molecular mechanism, by which these alterations affect lung cancer pathogenesis, would provide new diagnostic procedures and prognostic factors for early detection of recurrence. In this regard, many have studied molecular or other markers in pre-neoplastic and neoplastic lesions to discover what might relate to tumor recurrence and shortened survival.

Methods:
A series of 136 lung/bronchial and lung parenchyma tissue samples from 136 patients consisting of basal cell hyperplasia, squamous metaplasia, moderate dysplasia, adenomatous hyperplasia, severe dysplasia, squamous cell carcinoma and adenocarcinoma were analyzed for the distribution of hyaluronidase 1 (HYAL1) and 3 (HYAL3), and hyaluronan synthases 1 (HAS1), 2 (HAS2) and 3 (HAS3) by immunohistochemistry.

Results:
HYAL 1 was significantly more expressed in basal cell hyperplasia compared to moderate dysplasia (p=0.01), atypical adenomatous hyperplasia (p=0.0001) and severe dysplasia (p=0.03). A lower expression of HYAL 3 was found in atypical adenomatous hyperplasia compared to basal cell hyperplasia (p=0.01) and moderate dysplasia (p=0.02). HAS 2 was significantly higher in severe dysplasia compared to basal cell hyperplasia (p=0.002), and equally higher in squamous metaplasia compared to basal cell hyperplasia (p=0.04). HAS 3 was significantly expressed in basal cell hyperplasia compared to atypical adenomatous hyperplasia (p=0.05) and severe dysplasia (p=0.02). A lower expression of HAS 3 was found in severe dysplasia compared to squamous metaplasia (p=0.01) and moderate dysplasia (p=0.01). Epithelial HYAL 1 and 3 and HAS 1, 2 and 3 expressions were significantly increased in pre neoplastic lesions compared to neoplastic lesions. Comparative Cox multivariate analysis controlled by N stage and histologic types of tumors showed a significant association between poor survival and high pre neoplastic cell associated to HAS3 (HR=1.19; p=0.04).

Conclusion:
We concluded that localization of HYALs and HASs in lung/bronchial pre-neoplastic and neoplastic lesions was inversely related to malignancy, these factors emerging as potentially important diagnostic markers in patients with suspicion of lung cancer.

Background:
The extracellular sulfatases (SULF1 and SULF2) are overexpressed in a wide assortment of human cancers. SULF2, in particular, has been shown to drive carcinogenesis in non-small cell lung cancer (NSCLC), malignant astrocytoma, and hepatocellular carcinoma. As extracellular enzymes that are both tethered to the cell membrane and secreted, the SULFs and their heparan sulfate proteoglycan (HSPG) substrates are present in the extracellular environment. We hypothesize that the blood levels of SULF2 can serve as biomarkers for the early detection of NSCLC and malignant astrocytoma. The primary goal of this study is to evaluate the patient tumor and blood samples for the presence of the SULF2 in order to develop novel biomarkers for the early detection of NSCLC.

Methods:
We identified patients who underwent lung resection for adenocarcinoma (ADC) (41 patients) or squamous cell carcinoma (SCC) (51 patients) at our institution from 2000 to 2006. We excluded patients with recurrent lung cancer, or less than 3 mm of invasive tumor on H&E slide. A section from each paraffin-embedded tissue specimen was stained with a monoclonal antibody to SULF2. A pathologist determined the percentage (0-100%) and intensity (0-3) of tumor cells staining. Survival analysis was performed using a multivariate Cox proportional hazards model. We developed an ELISA to detect SULF2 in human blood. After testing a number of different strategies including using different combinations of our anti-SULF2 mAbs, we determined that a sandwich ELISA with capture mAb 5C12 followed by detection with biotinylated mAb 8G1 was best for the most sensitive detection of SULF2.

Results:
SULF2 staining (either tumor or stroma) was positive for 82% of the samples The SCC samples had a higher mean percentage of tumor staining compared to the ADC samples (100% vs. 60%; p<0.0005). However, after adjusting for age, sex, race, histologic type, stage, and neoadjuvant therapy, there was no significant association between percentage of SULF2 tumor staining and overall survival. Nonetheless, these initial findings are very encouraging, because the vast majority of ADC samples, including early stage disease, and all of the SCC tumor samples have some degree of staining for SULF2 protein. Using our SULF2 ELISA assay, we analyzed plasma samples from 54 healthy donors and 85 patients with newly diagnosed early stage NSCLC before surgical resection. The level of SULF2 protein is significantly higher in patients with NSCLC compared with healthy controls (738.4 ± 55.17, vs. 439.4 ± 40.88 pg/mL; p<0.0001).

Conclusion:
SULF2 protein was detected in the vast majority of tumor and blood samples of patients with lung cancer. Although additional studies are required, these data provide the first indication that SULF2 blood level may be a useful biomarker for the early detection of lung cancer.

Background:
Long non-coding RNAs (lncRNAs) are new-founding RNAs which could regulate many biological processes. Our previous study shown that lncRNA-GAS5 was decreased in lung cancer tissue, which contributed to the proliferation and apoptosis of non-small lung cancer (NSCLC). GAS5 was also associated with the prognosis of lung cancer patients. However, the plasma samples were more easily available than the tissue sample in the clinic. And the expression of GAS5 in the plasma of NSCLC patients was unknown.

Methods:
90 patients with NSCLC and 33 health controls were included in our study. Blood samples were collected before surgery and therapy. We extracted the free RNA in the plasma and analyzed the expression of GAS5 with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Suitable statistics methods were used to compare the plasma GAS5 levels between the NSCLC patients and health controls, preoperative and postoperative plasma samples. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic sensitivity and specificity of plasma GAS5 in NSCLC.

Results:
The 2[-][△][CT ]of GAS5 in the plasma of NSCLC patients and health controls are 1.053774 and 3.019817, respectively. GAS5 in NSCLC plasma was down-regulated compared with health controls (P=0.001), which was significantly correlated with TMN stage (P=0.024). Furthermore, plasma GAS5 increased markedly on day 7 after surgery compared with preoperative levels in NSCLC patients (P=0.003). The CT values of preoperative and postoperative are 2.225909 and 1.050455, respectively. The area under the ROC curve of GAS5 was up to 0.832. The combination of the GAS5 and CEA could produce 0.909 area under the ROC curve in distinguishing NSCLC patients from control subjects (95% CI 0.857–0.962,p=0.000).These results indicated that lncRNA GAS5 may be a more precise biomarker in NSCLC.

Conclusion:
We have demonstrated that GAS5 was decreased in NSCLC plasma expression and the plasma samples were more easily available than the tissue sample in the clinic. So GAS5 could be ideal biomarkers for the early diagnosis of NSCLC.

Abstract not provided

Background:
Malignant Mesothelioma (MM) is an aggressive neoplasm affecting the pleura and more rarely the peritoneum. Occupational exposure to asbestos is the most common cause, but domestic exposure through cohabitation with an asbestos worker and environmental exposure by living in proximity to an asbestos emitting industry are acknowledged risk factors. The Region of North Jutland in Denmark hosted two large shipyards and the only Danish asbestos cement factory in the period 1928-1986, using mainly chrysotile asbestos and resulting in high MM incidence (5/100,000 in 2011). In our patient cohort the proportion of MM cases in peritoneum (MAM) is larger for females than males, also noted by others. We examined whether this could be related to the source of asbestos exposure

Methods:
A retrospective investigation of medical records (Departments of Pathology and Medicine, Aalborg University Hospital) was performed, concerning females diagnosed with pleural MM (MPM) and MAM between January 1992 and February 2015. We focused on the correlation between the source of asbestos exposure and the development of either pleural or peritoneal disease. The asbestos exposure source was divided in two categories; primary, representing occupational asbestos exposure and secondary, including domestic and environmental exposure. Patients with unknown asbestos exposure or habitation history were excluded. We hypothesized that the site of MM development, pleura or peritoneum, is independent from primary or secondary asbestos exposure. Fischer´s exact test was applied to test the hypothesis.

Results:
Out of 67 women with MM, 27 were excluded due to insufficient information about asbestos exposure or habitation (data not shown). Of the remaining 40 females, 33 were diagnosed with MPM (83%) and 7 with MAM (17%). The median age for MAM and MPM diagnosis was 60 and 72 years respectively. Among the 40 MM patients, 25% (n=10) had a history of occupational asbestos exposure, 57.5% (n=23) had domestic and 17.5% (n=7) had environmental exposure. Importantly, secondary asbestos exposure was documented for 85% of the MPM patients (n=28) while primary in only 15% of them (n=5). On the contrary for the MAM patients, secondary asbestos exposure was reported for 29% (n=2) and 71% of them had primary exposure (n=5). The correlation between the source of asbestos exposure and the MM site was significant (p= 0.006, OR= 0.078).

Conclusion:
The majority of female MM patients have a non-occupational asbestos exposure, with a considerable rate of environmental exposure. Furthermore, the source of asbestos exposure seems to play an important role in determining the site of MM development. Primary asbestos exposure, inferring more intense exposure through occupation, may predispose to peritoneal while secondary, lighter asbestos exposure to pleural disease. This may also be indicated by younger age at MAM diagnosis and is in line with previous reports. Anatomical, biological or other genetical differences related to the site of MM cannot be excluded, as some studies indicate that asbestos exposed women develop MAM more often than men. Our study of large Danish population cohorts is in progress and aims to elucidate these questions (updates will be presented at the conference).

Background:
Turkish Mesothelioma Working Group; Drs. Abdurrahman Abakay, Sedat Altın, Güntülü Ak, Şule Akçay, Hasan Bayram, Mehmet Bayram, Hasan Fevzi Batırel, Serdar Berk, Mehmet Bilgin, Nilgün Yılmaz Demirci, Figen Deveci, Levent Elbeyli, Ahmet Erbaycu, Dilek Ernam, Sebahat Genç, Murat Gültekin, Ezgi Hacikamiloğlu, Hüseyin İlter, Selahattin Kadir, Hasan Kahraman, Mehmet Karadağ, Özkan Kaan Karadağ, Talat Kılıç, Gamze Kırkıl, Berna Kömürcüoğlu, Muzaffer Metintaş, Selma Metintaş, Arzu Mirici, Tevfik Pınar, Ömer Özbudak, Sibel Özkurt, Önder Öztürk, Mutafa Taşdemir, Dursun Tatar, Engin Tutkun, Umran Toru, Toros Selçuk, Zehra Seyfikli, Nazan Şen, Abdurrahman Şenyiğit, Gaye Ulubay, Ülkü Yılmaz, Adil Zamani In this study, we aimed to detect the current and future mesothelioma risk for Turkey to guide the studies for the elimination of asbestos exposure in the rural areas.

Methods:
Thirty-eight faculty members who were members of the Turkish Mesothelioma Working Group and and 4 foreign consultants took part in this study. In hospitals of 30 provinces where mesothelioma cases were most diagnosed, the patients diagnosed with "mesothelioma" under the code of C45 between 2008 and 2012 (for five years) were identified. The cases were checked one by one according to their identity, name, age, birth place, register and address information with their national identification numbers from the Central Register System (MERNIS). The deceased cases were identified; their dates of death and age were determined and these were also verified by the national registers. Following the identification of all deceased cases, the median survival was determined according to their diagnoses dates. After obtaining the final records of the cases with mesothelioma, the cases born in villages/rural areas were determined. Finally, "population exposed to asbestos in rural areas for a risky period of time" was determined. The number of mesothelioma to develop in the risky populations for the next 20 years was estimated based on those findings.

Results:
7,789 cases with C45 code between 2008 and 2012 in Turkey was collected and the demographic information was collected from 5,617 mesothelioma cases whose data were reliable. Out of these cases, 3,718 were born/lived in villages. 3,495 of the 5,617 mesothelioma cases had died until July 2014. The median survival was 8 months. 1,879 cases were not born and/or lived in a village with known asbestos exposure. These cases were most possibly related with occupational exposure. Between 2013 and 2033, it was projected that 15,450 mesothelioma will emerge in the population exposed to asbestos in the above mentioned rural areas. Moreover, it was projected that 2,511 new mesothelioma cases will emerge in the population who are still being exposed to environmental asbestos after 2013.

Conclusion:
We determined that mesothelioma caused by environmental and occupational asbestos exposure in Turkey is a more serious problem than previously anticipated. This is a unique epidemiological study that has determined the magnitude of an environmental/occupational carcinogen induced cancer problem through data collection from patients. Country wide measures are being implemented by the Public Health Institute of Turkey and Turkish Mesothelioma Working Group.

Background:
Asbestos is an important carcinogen and people can be exposed to asbestos either occupationally or environmentally. There is naturally occurring asbestos in some regions of Turkey. The soil including asbestos was commonly used for plastering, whitewashing and roofing to build village house in these regions. The aim of this study is to determine knowledge and awareness level of people about asbestos in a region of Turkey where asbestos related diseases is endemic due to environmental exposure to asbestos in the rural area.

Methods:
The study is a questionnaire-based cross-sectional study included patients and their relatives admitted to a tertiary hospital in Eskisehir located central part of Turkey, in 2015. The questionnaire was applied by face-to-face interview after verbal consent from each participant. The questionnaire included 19 questions. The data was determined by percent and confidence interval. Chi-square test was used to compare the groups and multiple logistic regression analysis was used to determinate variables affecting knowledge level of asbestos.

Results:
A total number of 505 participants were included in this study. The mean age of them was 52.6±15.2 years (15-89) and 51.9% of them were male. About 41.2% of participants were born in the rural area and 14.5% of them still live in the villages. The educational status: 8.3% never, 55.7% elementary school; 19.0% secondary school; 17% university. About 4.4% of the participants were unemployed, 13.5% official, 20.8% worker, 2.8% farmer, 34.5% housewife and 24.2% others including retired, barber etc. About 76.4% of the participants who live in the villages had history of asbestos-contaminated soil in their villages. Only 20.5% of the group knew that white soil included asbestos. About 3.5% of the group mentioned that asbestos was useful, 44.1% harmful, 3.5% ineffective and the rest of them had no idea. Regarding the knowledge about asbestos related diseases among them, 34.9% stated that asbestos can cause lung cancer, 18.3% benign pleural or lung diseases and 23.1% mesothelioma. Most of the participants who never live in the village or moved to the city (62.9%) stated that asbestos was harmful and 30.9% had no idea. About 53.1% of them mentioned that asbestos can cause lung cancer, 26.9% benign pleural or lung diseases and 33.5% mesothelioma. In this group, the rate of true answers regarding usage of asbestos at workplace was 12.9% (8%-20%). Their knowledge about environmental usage of asbestos was low (16.6%) in this group. More than half of the participants (53.5%) pointed out that it is necessary to avoid from asbestos. The factors affecting knowledge level of asbestos were found to be young (p=0.016), live in the city (p=0.016) and high education level (p<0.001) by multiple logistic regression analysis.

Conclusion:
Although approximately 30 years passed after the first scientific report about hazards of environmental asbestos exposure on the respiratory system in this region, knowledge and awareness level of asbestos was quite low by sampling of this population. Health and environment authorities should inform the people as quickly and intensely about asbestos.

Background:
Exposure to asbestos fibers is the predominant causal factor in malignant mesothelioma (MM). The higher than expected incidence of MM in Louisiana, particularly among males, has been attributed to occupational exposure at facilities engaged in the shipping, ship construction and manufacturing industries that utilized substantial quantities of materials containing asbestos fibers. The primary aim of this study was to investigate the locations and demographic characteristics of spatial clusters of malignant mesothelioma (MM) cases reported in Louisiana between 1992 and 2011.

Methods:
Incident case data reported to the SEER Louisiana Tumor Registry (LTR) were geocoded by census tract and stratified into four categories based on gender and age at diagnosis - Males under 60 years, Males 60 years and over, Females under 60 years and Females 60 years and over. Investigation of probable spatial clusters for each gender/age group combination was conducted using a Poisson-based model for the LTR incidence data. The ratio of male to female cases in the probable clusters was utilized to assess environmental versus occupational exposure. Locations of possible spatial clusters and locations of facilities processing, manufacturing and utilizing asbestos materials were illustrated graphically using geographic information system software.

Results:
The most likely clusters of MM for each age/gender group were located in the Greater New Orleans area and were statistically significant. There was substantial overlap of the boundaries for the four clusters located in Jefferson Parish with 15 census tracts included in all four clusters. The overall ratio of male to female cases in the most likely clusters was 1.35:1. A statistically significant secondary cluster was identified for males under 60 years of age northwest of Baton Rouge, LA. A pair of spatial clusters was identified for Males under 60 years and Females 60 years and older south of Lafayette.

Conclusion:
Our results of a most likely cluster of MM for males 60 years and older in Jefferson Parish was expected given the presence of large business enterprises known to have processed, manufactured and used asbestos materials since the 1930s. The presence of probable spatial clusters of MM cases of females in both age groups and males under 60 years of age was unexpected. The spatial clusters reported for Males under 60 years and Females 60 years and older near Lafayette and no cluster for Males 60 years and older in the area also suggested possible environmental exposure in the area. These results, along with the ratio of male to female cases among the four combined spatial clusters in the Greater New Orleans area suggested that exposure to asbestos was likely a combination of occupational and environmental exposure. Spatial analysis of MM incidence is an effective approach for investigating geographic areas with elevated rates of the disease, especially where exposure to asbestos has not been investigated. Further research is needed to evaluate occupational and environmental exposure sources in these populations by gathering detailed asbestos exposure measurements, demographic and health data among cases by gender and age group.

Abstract not provided

Background:
Malignant pleural mesothelioma (MPM) is associated with high morbidity and poor prognosis. We evaluated in population-based analysis the impact of enhanced diagnostics like positrone emission tomography (PET) and thoracoscopy. Also introduction of new medical therapy, Pemetrexed in 2004 could also benefit overall survival.

Methods:
Complete national data on 763 patients from the Finnish Cancer Registry sampled from 2000 to 2009 are presented. Survival for 1 year and 2 years and median survival were calculated. Survival data was acquired in February 2013.

Results:
During study period the incidence of MPM has been significantly and steadily increasing. The incidence was highest among men in all time periods (fig 1). Median age at the diagnosis was 68 years (range: 25-94 years). Reported histology was epitheloid in 211 (27.7%), sarcomatoid in 79 (10.4%), biphasic 33 (4.3%) and non-specified in 437 patients (57.3%). Median survival was 10 months (range 0 - 150 months), Fig 2. Between two 5-year cohorts (2000-2004 and 2005-2009) no significant difference in overallsurvival was observed (0.537). One year survival was 43.1% and two year 18.7%, with no differences were noted in the 5-year cohorts (0.826 and 0.402 respectively). Primary diagnosis was made in autopsy in 50 (6.6%) patients. A total of 27 patients (3.7% of deceaced) died for other reasons than MPM. 34 patients (4.5%) lived over 5 years and 16 (2.1%) of these are still alive. Figure 1Figure 2





Conclusion:
Despite significantly increased availability of new diagnostic tools like thoracoscopy, PET, and pemetrexed therapy during this cohort, no significant difference in overall survival was noted in this population based analysis. In order to facilitate better outcomes nationaly, new means are needed to coordinate both diagnostic and therapy of MPM.

Background:
Malignant pleural mesothelioma (MPM) is an aggressive and rare form of cancer which arises from mesothelial cells lining pleural cavity. Since it is difficult to differentiate the benign pleural thickenings from carcinomas, MPM can only be diagnosed in the advanced stage. To decrease the mortality rate of this disease highly sensitive and specific diagnostic methods are required. In the current study we propose Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy coupled with chemometrics as a novel approach in diagnosis of MPM from pleural fluids with better sensitivity and specificity values than the other biomarker analysis methods that are currently used.

Methods:
The pleural fluid samples from MPM (n=24), lung cancer (LC, n=20) and benign transudate (BT, n=20) patients were collected to perform FTIR spectroscopic experiments. Recording and analysis of the spectral data were performed by using Spectrum One software. Unsupervised chemometric analysis methods including hierarchical cluster (HCA) and principal component analysis (PCA) were applied to discriminate MPM from BT and LC groups. To develop a diagnostic method, SIMCA, a supervised chemometric method was also performed.

Results:
Quantitative spectral analysis indicated that lipid, triglyceride, cholesterol ester, protein and nucleic acid contents of MPM group differ from BT and LC groups. The score plots obtained from PCA analysis of pleural fluid at whole (4000-650 cm[-1]), lipid (3000-2800 cm[-1]) and fingerprint (1800-650 cm[-1]) spectral regions showed that BT, LC and MPM groups are successfully discriminated from each other (fig 1.).Figure 1 Figure 1. PCA scatter plots for all BT, LC and MPM pleural fluid samples in the 4000–650 cm-1 spectral region. Moreover, the loading plots obtained from these spectral regions supported the differences in molecular content of all three groups. SIMCA results performed at whole and fingerprint regions also revealed high accuracy values for the diagnosis of MPM and LC.



Conclusion:
The results demonstrated that ATR-FTIR spectroscopy together with chemometric tools can be used for successful and rapid differential diagnosis of MPM from LC and BT groups . *This work was supported by the Scientific and Technical Research Council of Turkey (TUBITAK), SBAG-113S294 Research Fund.

Background:
Despite the use of asbestos has been banned in many countries, 125 million people worldwide are still exposed to asbestos and at risk for developing malignant pleural mesothelioma (MPM). Since MPM is a lethal tumor, with diagnosis mainly at advanced stage due to non-specific symptoms and investigations, it is thought that only an early diagnosis will improve patient’s outcome (van Meerbeeck et al., 2011). Breathomics has emerged as a new research field, allowing to detect volatile organic compounds (VOCs) in breath which can be used as non-invasive markers for disease (Lamote et al., 2014). We investigated if asbestos induces VOCs and how breath VOCs could help discriminating MPM patients from occupational asbestos-exposed and non-exposed controls.

Methods:
Twenty-three MPM patients, ten healthy asbestos-exposed (AEx) individuals and twelve healthy non-exposed (HC) persons were included. After a fasting period of 2 hours before the breath sampling, participants breathed tidally for 3 minutes through a mouthpiece connected to a bacteria filter. Subsequently, ten ml alveolar air was sampled via a CO~2~-controlled ultrasonic sensor and analyzed using the BioScout Multicapillary Column/Ion Mobility Spectrometer (MCC/IMS, B&S Analytik, Dortmund, Germany). Per subject a background sample was taken. VOCs were visually selected and their intensity (V) was calculated via on-board VisualNow 3.7 software. After calculating the alveolar gradient, we performed a lasso regression in R to search for peaks that have the most discriminative power to distinguish MPM patients from controls. Predictions were made by leave-one-out cross-validation. The use of breath VOCs on the diagnostic performance was investigated by ROC-analysis.

Results:
Eighty-nine VOCs were selected in breath and background samples. The VOCs P25, P8 and P7 were able to discriminate HC from AEx controls with 91% accuracy (AUC~ROC~=0.95), yielding a sensitivity, specificity and positive (PPV) and negative predictive value (NPV) of respectively 90%, 92%, 90% and 92%. MPM patients were discriminated from AEx by the VOCs P5, P3, P30, P1 and P54 with 82% accuracy (AUC~ROC~=0.73), yielding a sensitivity, specificity and PPV and NPV of respectively 91%, 60%, 84% and 75%. When discriminating MPM patients from pooled HC and AEx controls, the VOCs P5, P3 and P1 were found to be important, yielding 73% accuracy (AUC~ROC~=0.71) and a sensitivity, specificity and PPV and NPV of respectively 70%, 77%, 76% and 71%.

Conclusion:
Breath analysis can discriminate MPM patients from healthy asbestos-exposed persons with 82% accuracy and from combined asbestos-exposed and non-exposed controls with 73% accuracy while healthy asbestos-exposed persons can be discriminated from non-exposed persons with 91% accuracy. The VOCs P25, P8 and P7 seem markers for asbestos-exposure while VOCs P5, P1 and P3 seem linked to MPM pathogenesis after exposure. For screening and to rule out diagnosis, a high sensitivity and NPV are mandatory and to rule in diagnosis, a high specificity and PPV are mandatory, which can enrich a population at risk for follow-up with (annual) CT scans or chest radiography. Hence, our results hold promise to use the breath test for screening of asbestos-exposed healthy seniors.

Background:
In most instances definitive diagnosis of malignant pleural mesothelioma (MPM) requires a tissue biopsy of sufficient size. As a biopsy is not always feasible, the identification of an accurate biomarker easily measured in blood would represent an important step forward. A recent study indicated that microRNA miR-625-3p was present in elevated concentration in plasma or serum of MPM patients compared to healthy controls and asbestosis patients. (Kirschner et al, JTO; 7:1184). In this study, we have further investigated the diagnostic potential of miR-625-3p.

Methods:
MiR-625-3p and other microRNAs were measured by RT-qPCR in two independent series of MPM patients and controls. After exclusion of haemolysed samples and those yielding RNA of insufficient quality, series 1 consisted of serum samples from 73 MPM patients, 69 healthy volunteers and 64 patients with non-small cell lung cancer (NSCLC) collected at the Netherlands Cancer Institute (NKI) between 1994 and 2013. The second series consisted of plasma samples from 29 MPM patients and 35 healthy volunteers collected in Vienna and Hungary (V/H) between 2011 and 2013. Additionally levels of soluble mesothelin-related protein (SMRP) were assessed (ELISA) in the NKI series.

Results:
Analyses of samples from patients and controls in the NKI series revealed that serum miR-625-3p concentrations were on average 5.35-fold higher (p=0.0054) in MPM, and 3.47-fold (p=0.003) in NSCLC than in control samples. Levels in MPM patients were 1.54-fold higher than in NSCLC patients but this did not reach statistical significance (p=0.273). Compared to healthy controls, the areas under the ROC curve (AUC) were 0.82 (95% CI: 0.75-0.89) for MPM and 0.75 (95% CI: 0.67-0.84) for NSCLC. In the samples of the V/H series, plasma miR-625-3p concentrations were on average 1.98-fold (p<0.001) higher in MPM patients than in healthy volunteers, with an AUC of 0.80 (95% CI: 0.69-0.91). Assessment of SMRP in the NKI series revealed AUCs of 0.69 (95% CI: 0.59-0.78) differentiating MPM from healthy individuals and 0.65 (95% CI: 0.54-0.75) separating MPM from NSCLC, comparable to AUC values reported earlier.

Conclusion:
Data from two independent validation series confirms the previously observed increased abundance of miR-625-3p in blood from MPM patients. However, the miR-625-3p levels observed in NSCLC patients show that elevation of the level of this microRNA in plasma/serum is not restricted to MPM. Further studies into combinations of microRNAs and SMRP (diagnostic signature) in MPM are warranted.

Abstract not provided

Background:
The discovery of immune checkpoint receptors as cytotoxic T lymphocyte antigen-4 (CTLA-4) and more recently programmed death-1 (PD-1) introduced a new era in cancer immunotherapy. Immune checkpoints are responsible for controlling and inactivating the immune system in order to avoid autoimmunity and prevent tissue damage. PD-1 is expressed primarily on activated effector T lymphocytes. Its natural ligands are programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). Expression of PD-L1/PD-L2 on tumor cells or in stroma impairs effector T lymphocyte activity within the tumor microenvironment. Trials with antibodies that block the ligand-immune checkpoint interaction have shown promising results in several cancer types.Data on few mesothelioma patients suggest that blocking immune checkpoints could offer new opportunities for treatment of this very aggressive tumor.. We investigated PD-1, PD-L1 and PD-L2 expression in MPM. Furthermore the effect of interferon-gamma (IFNg), an important cytokine for immune-mediated tumor control, on their expression pattern was analyzed.

Methods:
Flow cytometry and immunohistochemistry (IHC) were used for the expression of PD-1, PD-L1 and PD-L2 on human primary MPM and T cells and on MPM cell lines that cover the three major histological subtypes of of MPM, i.e. epitheloid (M28, H2795, H2818), sarcomatoid (VAMT-1, H2731, H-Meso-1) and mixed (NKI04, MSTO-211H) mesothelioma cells. The effect of stimulation with IFNg on expression of PD-1 and its ligands was measured.

Results:
PD-1 surface expression was found on T cells and not on MPM tumor cells, corresponding to literature showing that PD-1 is only expressed on T cells, B cells and macrophages. Different expression patterns were observed regarding PD-L1 and PD-L2. Flow cytometry showed significant PD-L1 expression on all the epitheloid and sarcomatoid mesothelioma cell lines. Two out of three cell lines tested positive for PD-L2, both for the epitheloid and the sarcomatoid subtype. The mixed cell lines were negative for PD-1 and its ligands. Following IFNg stimulation, PD-L1 and PD-L2 expression was induced or upregulated on all cell lines. Primary MPM cells showed variable expression of PD-L1. IHC data for PD-1 and PD-L1 expression correspond to the flow cytometry results.

Conclusion:
Taken together, these data on PD-1, PD-L1 and PD-L2 expression on human MPM cells and T cells support further investigation of the expression profile of the immune checkpoint PD-1 and its ligands in MPM patients samples. We are currently performing this using multicolor flow cytometry and IHC.

Background:
“Avoiding immune destruction” is one of the emerging hallmarks of cancer, as proposed by Weinburg and Hanahan. High expressions of immunosuppresive proteins strongly links to prognosis and cancer treatment. This study aimed to exam the expressions of immunosuppressors programmed death receptor ligand-1 (PD-L1) and transforming growth factor –β (TGF-β), and CD8+ tumor-infiltrating lymphocytes (TILs) in pre-treatment specimens from patients with advanced thymic epithelial tumors (TETs) including advanced thymic carcinoma and advanced invasive thymoma. To our knowledge, this is the first report to demonstrate the expression of PD-L1, TGF-β and CD8 and their clinical relevance in advanced TETs in Chinese population.

Methods:
Retrospective analysis was performed using tumor specimens from 20 patients with stage IV thymic carcinoma and 13 patients with stage III/IV invasive thymoma. Tissue biopsies were obtained before the first-line chemotherapy with (or without radiotherapy). The expression level of PD-L1, TGF-β and the prevalence of CD8+ TILs were assessed using immunohistochemistry (IHC). Their prognostic value for predicting overall survival (OS) and progression-free survival (PFS) were statistically analyzed using the SPSS software.

Results:
Higher expression levels of PD-L1 and TGF-β were detected in advanced thymic carcinoma than in advanced invasive thymoma (65.0% vs. 46.2%, 65.0% vs. 15.4%, respectively). Low level of CD8+ TILs was presented in 45.0% cases with advanced thymic carcinoma. In advanced thymic carcinoma, higher TGF-β expression was strongly associated with worse OS, with a p-value almost reaching statistical significance (p = 0.052). Median OS of patients with TGF-β high and low expression was 29.5 ms (95%CI: 18.6-40.4) and 62.9 ms (95%CI: 15.6-110.1), respectively. Higher PD-L1 expressions significantly predicted worse PFS after firs-line chemotherapy with (or without) radiotherapy (p =0.043). Median PFS was not estimable in PD-L1 low expression group. Mean PFS of patients with PD-L1 high and low expression was 13.3ms (95%CI: 8.0-18.6) and 23.5ms (95%CI: 13.9-33.2), respectively. An additional radiation treatment was particularly needed for CD8 low expression patients, in which first-line treatment with “chemotherapy + radiotherapy” significantly prolonged PFS compared to “chemotherapy-alone” (median PFS = 6.8ms, 95%CI: 0.0-2.7 vs. 3.5ms, 95%CI: NE, p = 0.015).

Conclusion:
Our results documented the clinical relevance of PD-L1, TGF-β, and CD8 in advanced TETs, with the prognostic value of predicting OS and PFS, as well as a potential association of immune conditions with therapeutic benefits.

Background:
Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour, characterized by a higher resistance to systemic treatments, more frequent distant spread and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking

Methods:
The present study analyzed the expression levels of MDM2 and HIF1alpha and the presence of inflammation, necrosis and proliferation in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2, and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. A pathological assessment of necrosis, inflammation and proliferation index (through Ki67 immunostaining) was also performed. Molecular markers, pathological features and clinical characteristics were related to overall (OS) and progression free survival (PFS).

Results:
Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p<0.001), HIFalpha (p=0.013), necrosis (p=0.013) and proliferation index (p<0.001) were significantly associated with sarcomatoid/biphasic subtypes, while higher levels of inflammation were significantly associated with epithelioid subtype (p=0.044). MDM2 expression levels were correlated with HIF1alpha (p=0.0001), necrosis (p=0.008) and Ki67 (p=0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p=0.04), high levels of necrosis (p=0.037) and proliferation index (p=0.0002) with shorter PFS. This finding, however, was not confirmed by the multivariate analysis.

Conclusion:
Sarcomatoid/biphasic and epithelioid mesotheliomas show different MDM2 and HIF1alpha expression levels and are characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted in order to confirm a prognostic and predictive role of such markers and features.

Background:
Immunotherapy of pulmonary tumors is now a clinical reality, however most patients do not respond. To convert non-responders into responders one potential approach is to identify the tumor‐specific ‘neo‐antigens’ that arise from DNA mutations in order to follow tumor-specific responses and to design therapeutic vaccines to try to ‘enforce’ a response against these resistant tumors.

Methods:
First, in order to identify tumor neo-antigens we performed RNAseq and exome analysis to identify single nucleotide variants (SNV) in murine pulmonary tumors. An average of 485 SNVs was found. We focused on AB1 and AB1-HA (asbestos-induced mesotheliomas, which mimic human mesothelioma) and Line 1 (lung cancer). We used the NetMHCpan 2.8 algorithm to identify candidate mutation‐carrying peptides and screened them in an interferon‐γ ELISPOT assay. Second, to determine if more neo-antigens could be ‘unmasked’ by therapy, we tested three candidate therapies in our murine model then reanalyzed neo-antigen responses a) Treg depletion using Foxp3-DTR mice, b) gemcitabine, an immunogenic cytotoxic chemotherapy commonly used for pulmonary malignancies, and c) antiCTLA4 (a checkpoint blockade therapy).

Results:
We identified 20 candidate mutation‐carrying peptides in the ELISPOT assay. A strong spontaneous endogenous pre-treatment immune response was demonstrated to DUqcrc2, a component of the respiratory chain protein ubiquinol cytochrome complex. It was found to stimulate a strong response at a similar magnitude to the model neo-antigen viral haemagglutinin (HA). The DUqcrc2 peptide sequence (amino acid 405-413) is predicted to bind the H-2Kd, and the mutant has a proline to alanine substitution mutation at position 408. Treg depletion unmasked a second neo-antigen, DGANAB. GANAB is an alpha glucosidase which cleaves the 2 innermost alpha-1,3-linked glucose residues from the Glc-2-Man-9-GlcNAc-2 oligosaccharide precursor of immature glycoproteins. There is an arginine to glutamine substitution mutation at position 969 of DGANAB (965-972) sequence. This observation supports the theory that removing Treg cells may broaden the immune response to a greater number of neo-antigens, a response presumably otherwise restrained by Treg suppression. Gemcitabine and antiCTLA4 checkpoint blockade did not unmask any additional neo-antigens.

Conclusion:
Thus, removing some immune restraints may expose a greater number of neo-antigens as potential clinical targets. The results from these approaches suggest novel ways to improve the immunotherapy of lung tumor and are the basis for planning current clinical trials.

Abstract not provided

Background:
Following failure of a platinum-antifolate combination regimen, there is no standard therapy for advanced malignant pleural mesothelioma (MPM). The fibroblast growth factor receptor (FGFR) signaling pathways may be a relevant target in MPM. Dovitinib inhibits multiple tyrosine receptor kinases, predominantly the vascular endothelial growth factor receptors (VEGFR), but also FGFR.

Methods:
This open-label multicentre phase II trial enrolled consenting adult patients with advanced, histologically-confirmed MPM who had previously received platinum-antifolate combination chemotherapy and up to one additional line of systemic therapy. Patients were ECOG PS < 2 and had adequate end-organ function. Dovitinib was administered orally at 500 mg/day for 5 days on, 2 days off; cycle length was 28 days. Two dose reductions (to 300 mg) for toxicity were permitted. Response was assessed every 2 cycles using RECIST 1.1 criteria modified for MPM. Correlative studies included FGFR-1 amplification on archival tumour and serum samples for circulating angiogenesis factors. Pre- and cycle 1 day 15 on-treatment diffusion-weighted pleural MRI was evaluated for its potential as an early marker of drug effect. The primary end-point was the proportion of patients progression-free at 3 months (PF3). A two-stage design was used: H0: 3-month PFS=40% versus HA: 3-month PFS=65% (roughly corresponding to a median PFS of 4.5 months), with α=0.05, β=0.20. If 6 of 12 PF3 in stage I, an additional 14 patients would be enrolled, with dovitinib of interest if > 15 of 26 PF3.

Results:
12 patients (10 males, median age 67) were enrolled. The median number of cycles administered was 2.5 (range 1-8). Commonly observed and / or grade 3 at least possibly related adverse events (any grade / grade 3, %): diarrhea (67 / 0%) vomiting (50 / 0%) fatigue (42 / 8 %), nausea (42 / 8 %), rash (0 / 17 %), syncope / generalized muscle weakness / elevated ALT (0 / 8 % each). No hyperphosphatemia was observed. 7 patients had at least one dose interruption (5 in cycle 1) and 5 had a dose reduction (1 to 300 mg); median dose intensity during cycles 1 and 2 was 80 %. 3 patients discontinued due to clinical progression by day 1 cycle 2. Best response: 1 unconfirmed PR, 4 SD, 2 PD and 4 inevaluable (3 with clinical PD; 1 intercurrent illness). The median PFS was 2.6 months and the median OS was 4 months. PF3 was 50%; although the criterion for proceeding to stage II accrual was met, the trial was halted due to a combination of minimal activity with several early progression events and poor tolerability in this patient population.

Conclusion:
Dovitinib has minimal activity and a toxicity profile comparable to other VEGFR inhibitors in previously-treated MPM; it is not clear if FGFR is effectively targeted. Correlative studies are ongoing and may help to clarify the role of the FGFR in MPM. [NCT01769547].

Background:
To observe the efficacy and safety of intra-thoracic chemotherapy of recombinant human endostatin (Endostar) combined with cisplatin in the treatment of malignant pleural effusion.

Methods:
A total of 84 patients with malignant pleural effusion were randomly divided into intra-thoracic chemotherapy of Endostar combined with cisplatin group (combination group) and single cisplatin group (single group). Before treatment, pleural effusion was completely resolved. Combination group was treated with intra-thoracic injection of 40～50 mg cisplatin and 60 mg Endostar twice a week, and 4 times were as a cycle at most. Single group was only treated with cisplatin, and other operations were the same as the combination group. RECIST1.0 hydrothorax evaluation criteria and NCI-CTC AE 3.0 version classification criteria were applied to evaluate the efficacy and adverse reactions, respectively.

Results:
The response rates of initially-treated patients in combination group and single group were 63.6% and 40.6%, respectively, and significant difference was presented (X[2]＝2.737, P＝0.022). The response rates of all patients in combination group and single group were 58.1% and 36.6%, respectively, and the difference was significant (X[2]＝4.877, P＝0.019). The progression-free survival (PFS) in combination group was dramatically longer than in single group (95 d vs. 53 d; X[2]＝3.872, P＝0.039). No adverse reactions at degree Ⅳ were observed in all groups. Incidences of adverse reactions including neutropenia, anemia, fatigue and increase of blood pressure in combination group were all higher than in control group, but there was no statistical significance (P＞0.05).

Conclusion:
Intra-thoracic injection of cisplatin alone is effective for treating patients with malignant pleural effusion, and its efficacy is better in combination with Endostar. Cisplatin combined with Endostar has a synergistic effect and better safety, being worthy of further popularization in clinic.

Background:
Malignant pleural mesothelioma (MPM) is usually a fatal neoplasm, and current therapeutic interventions are far from satisfactory. Naftopidil, an α1-adrenoceptor antagonist, is used clinically for the treatment of benign prostate hypertrophy, and has been found to reduce the incidence of prostate cancer and to inhibit prostate cancer cell proliferation via G1 cell cycle arrest. Recently, naftopidil has been demonstrated to induce apoptosis in mesothelioma cells by activating caspase-8 and the effector caspase-3 independently of α1-adrenoceptor suppression. Hence, a more potent naftopidil analogue, HUHS1015, was synthesized. The current study evaluates the inhibitory effect of HUHS1015 on malignant mesothelioma cell proliferation in preclinical models and assesses whether HUHS1015 can be the basis for new drug for the treatment of MPM.

Methods:
We treated the human MPM cell lines MSTO-211H, NCI-H28, NCI-H2052 and NCI-H2452 with HUHS1015, and evaluated cell viability using the MTT method. Additionally, NCI-H2052 tumor xenograft models in BALB/c-nu/nu mice were utilized to investigate anti-mesothelioma activity in vivo.

Results:
HUHS1015 reduced the viability of MPM cells more potently than cisplatin or paclitaxel at concentrations higher than 30 μM, and the drug induced both necrosis and apoptosis of MSTO-211H and NCI-H2052 cells. The effect of HUHS1015 on the expression of Bcl-2 family mRNAs in MSTO-211H and NCI-H2052 cells was tested using real-time RT-PCR. Puma, Hrk, and Noxa mRNAs were up-regulated in both cell lines. In the NCI-H2052 mouse xenograft models, HUHS1015 strongly suppressed tumor growth.

Conclusion:
These results indicate that HUHS1015 may be an effective anticancer drug candidate for the treatment of MPM. HUHS1015 induces apoptosis of MPM cells through modulation of a mitochondrial pathway, and future clinical investigations with this drug are warranted for mesothelioma.

Background:
Sarcomatoid lung cancer (SLC) is an aggressive subset of poorly differentiated non-small cell lung carcinoma (NSCLC), comprising just one percent of all NSCLC. Further elucidation of this unique histological entity has been hampered by a lack of large-scale clinical trial evidence. The National Comprehensive Cancer Network (NCCN) contains no clear direction regarding optimal management. The purpose of this study, therefore, is to identify potential therapeutic options for this disease using a multiplatform, biomarker-directed approach.

Methods:
In total, 48 SLC specimens analyzed via a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ) consisting of gene sequencing (Sanger or next generation sequencing [NGS]), protein expression (immunohistochemistry [IHC]) and gene amplification (CISH or FISH) were retrospectively evaluated.

Results:
High rates of PD-L1 (83.3%, 5/6) and PD-1 (80.0%, 4/5) protein expression by IHC imply benefit to recently-approved compounds. EGFR amplification by ISH (18.8%, 3/16) and MET amplification (9.5%, 2/21) were independent of EGFR mutation in this group. No ALK, HER2 or ROS1 ISH abnormalities were detected. Mutational analysis shows the highest mutation rates in TP53 (50.0%, 7/14), KRAS (44.4%, 16/36), cKIT (5.3%, 1/19), EGFR (5.1%, 2/39) and BRAF (4.8%, 1/21). The two EGFR mutations detected were L858R and exon 20 insertion.

Conclusion:
Multiplatform profiling identified multiple potential actionable targets with various approved therapies. PD-1 and PD-L1 overexpression rate was comparable to that published in sarcomatoid renal cell carcinoma. Therefore, new immunotherapies should be prospectively tested in sarcomatoid disease specific trials based on high PD-1/PD-L1 overexpression. Our finding of low EGFR mutational frequency is consistent with previous, published findings in this disease. Lower rates of ALK, ROS1, and EGFR are not surprising given SLC’s association with smoking. Clinical trials evaluating the benefit of imatinib and vemurafenib in subgroups with cKIT or BRAF mutations may be worthwhile.

Abstract not provided

Background:
Controversy over adjuvant radiation of thymoma has raged on among experts for decades. 20-30% thymoma patients present with myasthenia gravis (MG). The co-existence of MG and thymoma makes the surgical treatment and adjuvant radiation more complicated. The aim of this article is to investigate whether patients with MG and thymoma should receive mediastinal radiation therapy and when after extended thymectomy.

Methods:
Between 2002 and 2012, 159 patients with MG and thymoma underwent extended thymectomy. These patients were subdivided into 3 groups: Group 1 (n=89), patients having mediastinal radiotherapy within one month after surgery; Group 2, having mediastinal radiotherapy over three month after surgery (n = 49); and Group 3, without adjuvant radiation (n = 21).

Results:
152 patients underwent extended thymectomy by VATS, and 7 undergoing the trans-sternal approach due to thymoma invading great vessels. The resection was extended to the pericardium in 23 patients, the lung in 17 patients, and the innominate vein in 11 patients. There were no inoperable cases. The proportions of type A, AB, B1, B2, B3, and thymic carcinoma in this data were 0.6%, 19.5%, 25.8%, 32.7%,21.4%, and 0%, respectively. 146 patients were followed for 15 months to 12 years: 82 in Group 1, 45 in Group 2, and 19 in Group 3. Postoperative myasthenic crisis occurred in 38 cases: 16 cases in Group 1, 14 in Group 2, and 8 in Group 3. There was a significant difference in occurrence of postoperative myasthenic crisis between Group 1 and Group 3 (P=0.045). The rates of reaching CSR were 31.7% in Group 1, 22.2% in Group 2, and 21.1% in Group 3, respectively. The overall survival of Group 1, Group 2, and Group 3 were 90.2%, 86.7%, and 78.9%, respectively. 6 patients in Group 1 recurred, while 4 patients in Group 2 and 4 in Group 3 recurred. There was no lymph node metastasis detected. Kaplan-Meier survival curves demonstrate that there is no significant difference in overall survival among 3 groups. However, Cox regression analysis made by entering some factors including sex, age, and adjuvant radiation, revealed that adjuvant radiation might have significant influence in prognosis in thymoma patients ( P = 0.047). Figure 1



Conclusion:
Adjuvant radiation within one month after extended thymectomy may help decrease possibility of postoperative myasthenic crisis, raise the cumulative probabilities of reaching CSR, and might have significant influence in prognosis in thymoma patients with MG. In recurrence cases, no lymph node metastasis was detected

Background:
Thymic epithelial tumours are rare malignancies for which there is no standard treatment for patients with advanced disease progressing on or after chemotherapy. Despite the lack of identified targets in thymic malignancies, several studies demonstrated that VEGFR and KIT pathways are the most relevant targets for therapeutic intervention. Lucitanib is an oral, potent, selective inhibitor of the tyrosine kinase activity of FGFR1-3, VEGFR1-3, and PDGFR α/β, all key targets involved in pro-angiogenic and proliferative pathways leading to tumour progression. Therefore, lucitanib could be a potential therapeutic alternative for patients with recurrent or refractory disease.

Methods:
This first in human study is currently evaluating oral lucitanib as monotherapy in various solid tumours. The escalation phase used a 3+3 design in patients with advanced solid tumours to establish the recommended phase II dose. Safety and efficacy were further evaluated in patients whose tumours were determined to be FGF aberrant (FGFR1 and/or 11q amplification) or in patients with tumours known to be anti-angiogenesis-sensitive such as thymic epithelial tumours. In addition, different doses and administration schedules were investigated.

Results:
Of the 134 patients treated in the study, 3 had B-type Thymoma (T) and 12 had Thymic Carcinoma (TC). Among these patients, median age was 54 years [range 37-72], 7 were males and 8 females. Twelve patients (80%) were treated at 12.5mg on daily basis. The other 3 patients (T) received 5, 15 and 20mg respectively. Patients had received a median of 2 previous anti-cancer treatments [range: 0-6]. Median duration of treatment with lucitanib was 7 cycles [range 2-44]. All patients were evaluable for anti-tumour activity according to RECIST v1.1. Two patients had confirmed partial response (1T / 1TC) lasting at least 7 months (TC patient is still ongoing) and 10 patients had a stable disease with 6 of them lasting at least 6 months. To date, 4 patients are still ongoing and receiving benefit from lucitanib independently of the number of previous regimens. The most common adverse events related to lucitanib in this population (all grades, all doses) were hypertension (80%), hypothyroidism (53%), proteinuria (53%) and diarrhoea (40%). There was no major bleeding event reported. These findings were in line with the overall safety profile of lucitanib already described.

Conclusion:
The results of this tumour cohort analysis suggest that lucitanib has signs of clinical activity in patients with advanced thymic epithelial tumours, and should be further investigated in dedicated studies.

Background:
RYTHMIC (Réseau tumeurs THYMiques et Cancer) is the French nationwide network for thymic malignancies. Starting 2012, all patients diagnosed with thymic tumor had to be enrolled, as recommended by the French National Cancer Institute, part of good clinical practice.

Methods:
RYTHMIC prospective database is hosted by the French Thoracic Cancer Intergroup (IFCT), and collects clinical, imaging, treatment, and follow-up data of patients discussed at the reference national multidisciplinary tumor board (MTB). Data cutoff was April 1[st], 2015 for this analysis.

Results:
1089 questions were raised at the MTB about the management of 888 patients with thymic epithelial tumor. Among assessable cases, Masaoka-Koga stage III-IV tumors accounted for 42% of cases; histology was thymoma in 82% of cases, and thymic carcinoma in 18% of cases. First-line treatment of locally advanced disease, and management (diagnosis and treatment) of recurrent disease led to raise 227 (21%), and 234 (21%) questions at the MTB, respectively, 312 (68%) of which were about the modalities of systemic treatment. Figure 2 shows the proposed regimens for primary (A) and exclusive (B) chemotherapy in treatment-naïve patients, and chemotherapy (C) and targeted agents (D) for recurrent tumors. Combination of cisplatin, adriamycin, and cyclophosphamide and carboplatine, paclitaxel were the most frequently proposed regimens as first- and second-line treatment, respectively. Figure 1 Figure 2





Conclusion:
RYTHMIC is an exhaustive registry of thymic malignancies, which provides unique insights in the management of advanced and recurrent tumors with systemic agents. Meanwhile, limited data have been made available in the literature so far, as clinical trials were conducted in small numbers of patients, and existing databases enrolled a majority of surgically resected, early-stage tumors. Through the use of targeted agents, RYTHMIC allows the rapid implementation of new results in clinical practice, while ensuring patients an equal access to therapeutic innovation. Supported by Institut National du Cancer

Background:
Percutaneous cryoablation (PC) is an ablative technique, being used for local treatment of recurrent mesothelioma in patients following surgical lung sparing decortication and pleurectomy, and occasionally for palliative control of tumor extension to vital structures or pain control. The purpose of this study was to evaluate the safety and efficacy of PC in local control of recurrent mesothelioma.

Methods:
With IRB approval, patients with recurrent mesothelioma following lung sparing pleurectomy and decortication with at least one PC were identified from a database containing ablation information. Intra procedural and immediate post procedural hospital information was assessed for complications and follow up imaging was used to asses for late complications and recurrence. Patients were followed with CT and and PET/CT scans for 6 and some up to 12 months. Local recurrence determined by increased regional metabolic activity or increased size of post ablation zone at 6 months. A stepwise multiple logistic regression model was used to assess predictors of local recurrence after ablation, considering clinical variables including: stage at diagnosis, chemotherapy, radiation, recurrence time lag following surgery, and number of lesions at time of recurrence presentation, And PC variables including: size of the lesion, edge of ice ball beyond the tumor, number of probes, size of probes, number of cryo cycles, maximum and total freeze and thaw time.

Results:
From the database, 25 patients were identified who underwent a total of 117 outpatient cryoablations (range of 1-25). 4 ablations in 3 patients were performed for palliative and pain control indications. Lesions measured a mean of 32.5 mm (range 9-113) by 18.0 mm (range 6-60) in diameter. At 6 months 110/117 (94.0%) of ablations showed no recurrence. No major, but minor complications including hematoma, small pneumothorax and hemoptysis in one patient each and erythema in 3 chest wall subcutaneous lesions (5/117 =4.2%). Late complications in 4/117 (3.4%) ablations. Considering the clinical and cryoablation variables no recurrence was seen in patients having the edge of iceball more than 7 mm beyond the tumor.

Conclusion:
PCT can be used for management of recurrent mesothelioma following surgery with low recurrence rate of 6%, and limited procedural complications 4.2% and late complications of 3.4%. When performing PCT, at least 7 mm of the of iceball is needed to extend beyond the edge of tumor to limit local recurrence.

Abstract not provided

Background:
High dose lung-sparing pleural radiotherapy for malignant pleural mesothelioma (MPM) is difficult. Given the steep dose gradient with volumetric modulated arc therapy (VMAT), accurate target delineation is critical. The optimal imaging modality to define radiotherapy target volumes has not been studied in depth. This is the aim of the present study.

Methods:
Twelve consecutive patients with a histopathological diagnosis of stage I-IV MPM (6 left-sided and 6 right-sided) were included. CT scans with intravenous (IV) contrast, [18]F-FDG PET/CT scans, MRI scans (post-contrast T1-weighted and T2-weighted) and diffusion-weighted images (DWI) were obtained and downloaded from the institutional database onto a standalone image fusion workstation (MIM Software Inc., Cleveland, OH, USA) for image registration and contouring. CT scans were rigidly co-registered with ~18~FDG-CT-PET, with MRI scans and with DWI scans. Four sets of pleural GTVs were defined: 1) a CT-based GTV (GTV~CT~); 2) a PET/CT-based GTV (GTV~CT+PET/CT~); 3) a T1/T2-weighted MRI-based GTV (GTV~CT+MRI~); 4) a DWI-based GTV (GTV~CT+DWI~). Only the pleural tumor was contoured; mediastinal nodes were excluded. In each of the 4 co-registrations, a “quantitative” and a “qualitative” (visual) evaluation of the volumes were performed. “Quantitative” evaluation was carried out through the coefficient of variation (COV; the ratio between the standard deviation [SD] and the mean: a measure of the dispersion of a distribution) and the Jaccard index (the ratio between the union and the intersection between two volumes: a measure of overlap). “Qualitative” evaluation consisted of a visual identification of any additional tumor site in each of the 4 obtained co-registrations.

Results:
Compared to CT-based GTV definition, PET/CT identified additional tumour sites in 12/16 patients. Compared to either CT or PET/CT, MRI and DWI identified additional tumour sites in 15/16 patients. Additional tumour sites were mainly the parietal pleura, the diaphragm and the chest wall. Mean GTV~CT~, GTV~CT+PET/CT~, GTV~CT+MRI~ and GTV~CT+DWI~ (+SD) were respectively 630.1 mL (+302.81), 640.23 (+302.83), 660.8 (+290.8) and 655.2 mL (+290.7). Mean Jaccard index was lower in MRI-based contours versus all the others.

Conclusion:
To the best of our knowledge, this is the first study showing that the integration of the MRI (T1/T2-weighted) and DWI into the target volume definition in lung-sparing hemi-thoracic VMAT in MPM may allow to improve the accuracy of target delineation and reduce the likelihood of geographical misses.

Background:
Although extrapleural pneumonectomy (EPP) and pleurectomy/decortication (P/D) provide similar survival in malignant pleural mesothelioma (MPM), we sought to compare the two procedures in terms of another important outcome" hospital resource utilization (RU).

Methods:
With IRB approval, we retrospectively reviewed our prospective database to determine RU (ICU and hospital stay, mechanical ventilation, and central line use, etc) and Kaplan-Meier median survival (MS) for patient undergoing P/D. Our results are compared with similar findings for EPP reported in the literature.

Results:
We identified 121 pts on an "intent to treat" basis from 1997-2011. 94 (77.7%) were male. Mean age was 65.9 yrs (range 27-84). Comorbidities included hypertension 45.5%, coronary artery disease 11.6%, diabetes 10.7%, and vascular disease 6.2%. Mean surgical time was 7 hrs 57 mins (range 3 hrs 15 min–14 hrs 21 min). R1 resection was achieved in 116 (95.9%). Microscopic "margins" were assessed in 63 with 40 (63.5%) positive. Pathologic T- and N-staging is shown in Table 1. Morbidity was mostly limited to air leaks >10 days 41 (33.9%) and atrial arrhythmias 38 (31.4%). Three patients (2.5%) died. Relevant RU data included: intraoperative CVP lines 3 (2.5%), OR extubation 113 (93.4%), no ICU stay 99 (81.7%), and mean hospital stay 10 (range 5-103) days. RU data with P/D + RTx is compared to EPP as reported by others (figure 1). MS was 13.8 mos for all patients and 17.8 mos for epithelioid histology, which was better than biphasic (10.3 mos) and sarcomatoid (2.1 mos) subtypes (p<0.01). MS for 85/121 patients (70.2%) who completed P/D + RTx was 19.7 mos. MS for similar groups of EPP patients is reportedly 16.8-19 mos (eg, Thorac Cardiovasc Surg 1999;117(1):54-65 and J Clin Oncol 2009;27(18):3007-13).

Conclusions: P/D +RTx provide essentially the same outcomes as EPP with less use of hospital resources

	T Stage	N Stage
0	0	57(47.1%)
1	0	3(2.5%)
2	24(19.8%)	58(47.9%)
3	70(57.9%)	0
4	27(22.3%)	-

Figure 1



Conclusion:
P/D provides essentially the same outcomes as EPP with less use of hospital resources.

Background:
Surgical resection for pulmonary metstasis of osteosarcoma has been considered as the treatment of choice, however, it was not feasible to predict the benefit of metastasectomy for patients with multiple poor prognostic parameters. Survival prediction model can be very helpful for this purpose, so we made a nomogram based on parametric survival model(PSM) and regression trees from recursive partitioning analysis(RPA).

Methods:
We reviewed the clinical variables of patients who underwent single or multiple surgical resection for pulmonary metastasis of osteosarcoma between 1994 and 2012. Prognostic parameters were incorporated into PSM and RPA to build a nomogram and regression trees for the prediction of survival after single or multiple metastasectomy. The ‘rms’ and ‘rpart’ package of R(version 3.2.0) were used for this procedure. PSM was validated with C-index calculated by bootstrap method and then the parameters of PSM were used for RPA.

Results:
We analyzed 186 patients who received 294 metastasectomies. The number of second, third, and forth metastasectomy cases were 62, 28, and 11 respectively. Overall 5-year survival rate after first metastasectomy was 47%. Age, gender, number of metastatic nodules, frequency of metastasectomy, disease free interval before metastasectomy, size, subtype and resection margin of primary tumor were affecting overall survival. Nomogram and regression trees were displayed in figures. C-index of PSM was 0.71. Figure 1 Figure 2





Conclusion:
Our prediction model using a nomogram and regression trees can be easily employable for calculating survival benefits. Nomogram and RPA are complementary to each other. RPA displays comprehensive grouping of patients who have similar prognosis, while nomogram is useful for predicting hazard ratio of individual patient. In this study, our combined model constitutes a useful tool for predicting prognosis of patients who undergo repeated metastasectomy for osteosarcoma.

Background:
Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) is a rare disorder characterized by proliferation of neuroendocrine cells in the bronchial wall and considered to be pre-invasive lesion for lung carcinoid tumors [1]. There is increasing rate of diagnosis of this condition due to widespread availability and use of cross sectional imaging. DIPNECH is reported as an incidental finding in approximately 5.4% of patients undergoing resection for lung neoplasms [2]. The optimal management of this condition is currently not well-established. The limited data regarding the clinicopathologic characteristics and long term outcome for patients with DIPNECH provided a strong rationale for this study.

Methods:
We employed medical records to obtain demographic, clinical characteristics and survival for patients diagnosed with DIPNECH at our institution between January 1990 to December 2014. A review of archival diagnostic material was conducted by expert pulmonary pathologists to confirm the original diagnosis. Differences in clinical characteristics and survival was assessed between patient groups defined by race, gender, age, smoking status, body habitus and treatment received. Survival was computed using the Kaplan–Meier method while univariate and multivariate models were employed to assess for significant association between patient survival and variables of interest.

Results:
A total of 27 patients were included in this analysis. The majority of patients were females (89%) and predominantly of Caucasian (66.7%) or Black (14.8%) race. The median age at diagnoses was 63 years (range: 20-77) and 61.5% of patients were non-smoker. Approximately 52% underwent surgical resection. The median overall survival (OS) was 151 months (95%CI: 39-165) while 1-year and 5-year survival rates were 95.2% and 73.2% respectively. Nineteen patients (71%) remain alive at the time of this analysis. Male patients (HR: 4.58, 95%CI: 0.76-27.67, p=0.098) and smokers (HR: 23.79; 95%CI: 0.98-579.54; p<0.052) appeared to have an inferior survival. No statistically significant difference in survival was recorded in patient subgroups defined by age, race, surgical intervention or body weight.

Conclusion:
DIPNECH is a rare condition with increasing rate of diagnosis. The overall prognosis is good in comparison to other lung neoplasms but up to a quarter of the patients do not survive beyond five years post diagnosis. Male gender and associated use of tobacco products may be associated with poor outcome. References: 1. Chassagnon, G., et al., DIPNECH: when to suggest this diagnosis on CT. Clin Radiol, 2015. 70(3): p. 317-25. 2. Ruffini, E., et al., The significance of associated pre-invasive lesions in patients resected for primary lung neoplasms. Eur J Cardiothorac Surg, 2004. 26(1): p. 165-72.

Abstract not provided