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BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial.METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 μg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438.FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths.INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination.FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development.

OBJECTIVES: Cancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness.MATERIALS AND METHODS: GLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity.RESULTS: Increasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody.CONCLUSION: The progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.

Previous studies have shown interference between epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and chemotherapy in the cell cycle, thus reducing efficacy. In this randomised controlled trial we investigated whether intercalated erlotinib with chemotherapy was superior compared to erlotinib alone in untreated advanced EGFR-mutated nonsmall cell lung cancer (NSCLC).Treatment-naïve patients with an activatingBetween April 2014 and September 2016, 22 patients were randomised equally into both arms; the study was stopped due to slow accrual. Median follow-up was 64 months. Median PFS was 13.7 months (95% CI 5.2-18.8) for CPE and 10.3 months (95% CI 7.1-15.5; hazard ratio (HR) 0.62, 95% CI 0.25-1.57) for erlotinib monotherapy; when compensating for number of days receiving erlotinib, PFS of the CPE arm was superior (HR 0.24, 95% CI 0.07-0.83; p=0.02). ORR was 64% for CPEIntercalating erlotinib with cisplatin-pemetrexed provides a longer PFS compared to erlotinib alone in

Immunotherapy for metastasized non‐small‐cell lung cancer (NSCLC) can show long‐lasting clinical responses. Selection of patients based on programmed death‐ligand 1 (PD‐L1) expression shows limited predictive value for durable clinical benefit (DCB). We investigated whether early treatment effects as measured by a change in circulating tumor DNA (ctDNA) level is a proxy of early tumor response to immunotherapy according to response evaluation criteria in solid tumors v1.1 criteria, progression‐free survival (PFS), DCB, and overall survival (OS). To this aim, blood tubes were collected from advanced‐stage lung adenocarcinoma patients (n = 100) receiving immune checkpoint inhibitors (ICI) at baseline (t0) and prior to first treatment evaluation (4–6 weeks; t1). Nontargetable (driver) mutations detected in the pretreatment tumor biopsy were used to quantify tumor‐specific ctDNA levels using droplet digital PCR. We found that changes in ctDNA levels were strongly associated with tumor response. A > 30% decrease in ctDNA at t1 correlated with a longer PFS and OS. In total, 80% of patients with a DCB of ≥ 26 weeks displayed a > 30% decrease in ctDNA levels. For patients with a PD‐L1 tumor proportion score of ≥ 1%, decreasing ctDNA levels were associated with a higher frequency a DCB (80%) and a prolonged median PFS (85 weeks) and OS (101 weeks) compared with patients with no decrease in ctDNA (34%; 11 and 39 weeks, respectively). This study shows that monitoring of ctDNA dynamics is an easy‐to‐use and promising tool for assessing PFS, DCB, and OS for ICI‐treated NSCLC patients., Predictive markers to reliably monitor response in patients with lung adenocarcinoma treated with immune checkpoint inhibitors are currently lacking. Here, we show that tumor‐informed monitoring of a single bloodborne mutation using droplet digital PCR enabled the identification of early disease progression and durable treatment responses. The association between patient survival and changes in mutant circulating tumor DNA (ctDNA) levels was unrelated to programmed death‐ligand 1 expression. Altogether, our data show that ctDNA dynamics could offer a promising cost‐effective approach to monitor patients with metastasized lung adenocarcinoma.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors.PATIENTS AND METHODS: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors.RESULTS: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 64% to 87%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented.CONCLUSION: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.

Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of >99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

We read with great interest the article ‘Addressing immune-related adverse events of cancer immunotherapy: how prepared are rheumatologists?’ by Kostine et al .1 The introduction of immune checkpoint inhibitor (ICI) therapy has been a major breakthrough in the management of metastatic cancer. On the downside, ICI therapy may induce unwanted autoimmune effects, the so-called immune-related adverse effects (irAEs). Various irAEs have been described that resemble a regular rheumatic disease, including polymyalgia rheumatica (ICI-PMR).2 3 The authors report that rheumatologists may lack confidence in diagnosing irAEs. Therefore, recommendations for the diagnosis of rheumatic irAEs are needed. Based on our experience with ICI-PMR, we propose that imaging could be an important part of such recommendations. We investigated six consecutive patients with ICI-PMR by ultrasonography, and five of these patients also by [18F]-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) scan. Five patients fulfilled the provisional American College of Rheumatology/European League Against Rheumatism classification criteria for PMR.4 A normal C-reactive protein level in the absence of an erythrocyte sedimentation rate (ESR) test precluded PMR classification in one patient. However, this patient fulfilled both the clinical and ultrasound criteria for PMR,4 and showed findings suggestive of PMR on the FDG-PET/CT scan.5 The median age was 73 years (range 59–83; online supplementary table 1). Patients received anti-programmed cell death protein 1 (PD-1) treatment, that …

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.

The prognostic value of markers of cancer stem cells and epithelial to mesenchymal transition in small cell lung cancer is not known. We retrospectively studied these markers in the biopsy tissue of patients with small cell lung cancer and correlated them with overall survival and the strongest known prognostic marker circulating tumor cells.Background: Small cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and epithelial to mesenchymal transition have been associated with increased chemoresistance and metastatic spread in SCLC. Patients and Methods: The biopsy specimens of 38 SCLC patients were used for marker evaluation by immunohistochemistry. The markers for CSCs were CD44 and SOX2. The markers for epithelial to mesenchymal transition were E-cadherin, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, vimentin, and c-MET. Staining was scored as low (weak) or high (strong) intensity for SOX2, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, and c-MET and using the immunoreactive score for CD44, E-cadherin, and vimentin, expressed as low or high expression. Results: High expression of c-MET (c-METH) and low expression of E-cadherin (E-cad(L)) showed a trend toward a better prognosis (P = .07 and P = .09, respectively). The combination of c-METH and E-cad(L) resulted in significantly better survival (P = .007). The tested markers were not associated with CTCs, although a trend was seen for c-(METE)-E-H-cad(L) (P = .09) with low CTCs. The CSC markers SOX2 and CD44 were not associated with overall survival in this patient cohort. Conclusion: SCLC with a mesenchymal-like phenotype (c-METHE-cad(L)) is associated with longer survival and showed a trend toward lower CTCs. (C) 2016 Elsevier Inc. All rights reserved.

Objective The relevance of EGFR gene amplification in patients with EGFR-mutated advanced non-small cell lung cancer (NSCLC) has not been fully elucidated. Moreover, a limited number of studies have been published on methods to estimate gene amplifications based on routinely performed targeted next generation sequencing (NGS). In this study we aimed to determine whether PCR-based targeted NGS data on tumor tissue can be used to estimate presence of gene amplifications and explored the prognostic value of EGFR gene amplification in EGFR mutated NSCLC patients. Materials and methods A total of 3,194 good quality targeted NGS data files were retrieved from 2014 to 2017. Among those, 1,729 NSCLC samples originated from 1,586 NSCLC patients of whom 134 had an EGFR mutation (8.2%). Clinical data were available for 66 of the patients. Raw sequencing data were re-analyzed using a custom designed pipeline. The presence of an amplification was based on the read depth of a given amplicon relative to a set of reference amplicons from the sample or relative to a set of normal control samples. Reference amplicons were selected based on low degree of variation in read depth amongst all tested samples. Amplifications were regarded significant when the ratio was ≥3 and the z score was ≥3.5. Technical validation was done by FISH and MLPA. Cox regression analysis on overall survival was done with each of the amplification parameters using SPSS. Results No amplifications were detected for the ALK, KIT, NRAS, PDGFRA, GNAQ and MAP2K1 gene loci, whereas amplifications for BRAF, ESR1, GNA11, HRAS, KRAS, MET and PIK3CA were observed at a low frequency. Depending on the amplification analysis strategy (within sample or relative to normal controls), 19% and 13% of the EGFR mutated group had an EGFR amplification, respectively. In EGFR wild type patients, amplifications were detected in 5% and 4% of the patients using the two methods, respectively. The sensitivity and specificity of the NGS based estimation of amplifications for EGFR was 94% (14/15) and 97% (34/35) respectively for both data analysis approaches. Patients with concurrent EGFR mutations and amplifications (estimated within sample) treated with EGFR-TKI had a significantly worse overall survival compared with those without concurrent EGFR amplifications (ratio, p=0.047 and z score, p=0.015). Cox regression analysis indicated a borderline significant interaction between ratio and z score (p=0.052). Conclusion Routinely obtained amplicon-based NGS data can be used to identify gene amplifications. The presence of EGFR amplifications in EGFR mutant patients is predictive of a worse overall survival. Keywords: Lung adenocarcinoma, EGFR, survival, tyrosine kinase inhibitor Citation Format: Pei Meng, Jiacong Wei, Miente Martijn Terpstra, Anke van Rijk, Menno Tamminga, Frank Scherpen, Arja ter Elst, Mohamed Z. Alimohamed, Lennart F. Johansson, T. Jeroen N. Hiltermann, Harry J. Groen, Klaas Kok, Anthonie J. van der Wekken, Anke van den Berg. Clinical value of EGFR gene amplifications detected using amplicon based targeted next generation sequencing data in lung adenocarcinoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4335.

Cytokine interleukin-1β (IL-1β) has multiple pro-tumorogenic effects on tumor microenvironment, thereby promoting carcinogenesis, tumor invasiveness, and immunosuppression. CAN is a selective IL-1β inhibitor that aims to target tumor-promoting inflammation to reduce immune suppression, thereby potentiating effects of immunotherapy with PD-1 inhibitors such as PEM. Ph 3 CANTOS study has shown IL-1β inhibition with CAN was associated with reduced incidence of lung cancer (LC) and LC mortality in pts with atherosclerosis, providing a rationale to investigate therapeutic role of CAN in LC. CANOPY-1 (NCT03631199) is a PBO-controlled, double-blind, randomized, ph 3 trial designed to evaluate efficacy and safety of PEM + Ctx ± CAN in tx naive pts with stage IIIB/IIIC (not eligible for definitive chemo-radiation curative tx) or stage IV squamous and nonsquamous NSCLC. The study was divided into 2 parts: part 1 is open labelled, safety run-in part where pts received CAN 200 mg s.c Q3W + PEM 200 mg i.v Q3W + platinum-based Ctx (as induction during first 4 cycles only); Cohort A (A, non-squamous), carboplatin + pemetrexed; Cohort B (B, non-squamous), cisplatin + pemetrexed; Cohort C (C, squamous or non-squamous), carboplatin + paclitaxel. Part 2 is randomized and evaluates efficacy and safety of CAN comb regimen vs PBO comb regimen. Primary objective of safety run-in part: recommended ph 3 dose regimen (RP3R) of CAN comb. Secondary objectives: ORR, DCR, DOR, safety, PK, and immunogenicity. As of 14 May 2019 (follow-up of ≥42 days from C1D1 unless pt discontinued earlier), 10 pts in A, 11 pts in B, and 9 pts in C were treated, of which 73% were male, median age was 63 yrs. In total, 24/30 (80%) pts enrolled were still receiving tx; primary reason for tx discontinuation was progressive disease (3 pts in A and 1 pt each in B and C) and 1 pt died due to study indication. 1 pt reported DLT during first 42 days of study tx (C: grade 3 hepatitis, not related to CAN). RP3R of CAN in comb with standard dose PEM + Ctx was 200 mg SC Q3W based on Bayesian logistic regression model (BLRM). Serious AEs regardless of causality were reported in 8 (27%) pts (2 pts in A and 3 pts each in B and C), none of which were considered to be related to CAN. Most common AEs (≥20%, any grade) across all cohorts (n=30) were nausea (37%), vomiting (30%), constipation and fatigue (each 23%), and neutrophil count decrease (20%). 14 pts (47%) experienced grade 3 AEs and 1 pt experienced grade 4 AE (cardiac tamponade [unrelated]). No fatal serious AEs were reported. AEs leading to discontinuation of one of the study drugs were reported in 3 (10%) pts (hepatitis, peripheral neuropathy, and polyneuropathy) but none were CAN related. AEs leading to dose reduction and dose interruption of one of study drugs were reported in 3 (10%) pts and 5 (17%) pts, respectively. Only 1 DLT was reported with CAN + PEM + Ctx. Based on BLRM and all relevant clinical data, the RP3R of CAN as 200 mg SC Q3W comb was considered safe and well tolerated. Enrollment for the randomized part is completed. Citation Format: Bruce E. Johnson, Tae Min Kim, T. Jeroen N. Hiltermann, Fabrice Barlesi, Christian Grohe, Yasushi Goto, Orvar Gunnarsson, Tobias Overbeck, Noemi Reguart, Martin Wermke, Gilberto Castro Castro, Enriqueta Felip, Alastair Greystoke, Benjamin J. Solomon, Stephanie Deudon, Anne-Laure Louveau, Vanessa Passos, Daniel SW Tan. CANOPY-1: Safety run-in results from phase (ph) 3 study of canakinumab (CAN) or placebo (PBO) in combination (comb) with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT214.

Cytokine interleukin-1β (IL-1β) has multiple pro-tumorogenic effects on tumor microenvironment, thereby promoting carcinogenesis, tumor invasiveness, and immunosuppression. CAN is a selective IL-1β inhibitor that aims to target tumor-promoting inflammation to reduce immune suppression, thereby potentiating effects of immunotherapy with PD-1 inhibitors such as PEM. Results of phase 3 CANTOS study have shown that IL-1β inhibition with CAN was associated with reduced incidence of lung cancer and lung cancer mortality, thus providing a rationale to investigate therapeutic role of CAN in lung cancer. CANOPY-1 (NCT03631199) is a placebo-controlled, double-blind, randomized, phase 3 trial designed to evaluate efficacy and safety of PEM + Ctx ± CAN in previously untreated pts with stage IIIB/IIIC (not eligible for definitive chemo-radiation curative tx) or stage IV squamous and nonsquamous NSCLC. The study was divided into 2 parts: part 1 is non-randomized, safety run-in part where pts received CAN 200 mg s.c Q3W + PEM 200 mg i.v Q3W + platinum-based Ctx [Cohort A (non-squamous), carboplatin + pemetrexed; Cohort B (non-squamous), cisplatin + pemetrexed; Cohort C (squamous or non-squamous), carboplatin + paclitaxel]. Part 2 of the study randomizes pts to evaluate efficacy and safety of CAN combination regimen vs placebo combination regimen. Primary objective of safety run-in part: RP3R for CAN in combination with PEM + Ctx. Secondary objectives: ORR, DCR, DOR, safety, PK, and immunogenicity. As of 14 May 2019 (follow-up of ≥42 days from C1D1 unless pt discontinued earlier), 10 pts in cohort A (A), 11 pts in cohort B (B), and 9 pts in cohort C (C) were treated, of which 73% were male, median age was 63 yrs. In total, 24/30 (80%) pts enrolled were still receiving tx; primary reason for tx discontinuation was progressive disease (5 pts; 3 pts in A and 1 pt each in B and C) and 1 patient died due to study indication. Dose-limiting toxicity (DLT) occurring during first 42 days of study tx was reported only in 1 pt (cohort C: grade 3 hepatitis, not related to CAN). Recommended phase 3 regimen (RP3R) of CAN in combination with standard dose of PEM + Ctx was confirmed as 200 mg SC Q3W based on Bayesian logistic regression model (BLRM). Serious AEs regardless of study drug relationship were reported in 8 (27%) pts (2 pts in A and 3 pts each in B and C), none of which considered to be related to CAN. Most common AEs (≥20%, any grade) across all cohorts (n=30) were nausea (37%), vomiting (30%), constipation and fatigue (each 23%), and neutrophil count decrease (20%). Overall, 14 pts (47%) experienced grade 3 AEs and 1 pt experienced grade 4 AE (cardiac tamponade unrelated to study drugs). No fatal serious AEs were reported. AEs leading to discontinuation of one of the study drugs were reported in 3 (10%) pts (hepatitis, peripheral neuropathy, and polyneuropathy) but none were CAN related. AEs leading to dose reduction and dose interruption of one of study drugs were reported in 3 (10%) pts and 5 (17%) pts, respectively. Only 1 DLT was reported with this triplet combination of CAN + PEM + Ctx. Based on BLRM and all relevant data, the RP3R of CAN as 200 mg SC Q3W combination was considered safe and well tolerated. Enrolment is ongoing in randomized phase 3 part of study to evaluate efficacy and safety. Citation Format: Bruce E. Johnson, Tae Min Kim, T. Jeroen N. Hiltermann, Fabrice Barlesi, Christian Grohe, Yasushi Goto, Orvar Gunnarsson, Tobias Overbeck, Noemi Reguart, Martin Wermke, Gilberto Castro, Enriqueta Felip, Alastair Greystoke, Benjamin J. Solomon, Noelia Nebot, Stephanie Deudon, Anne-Laure Louveau, Vanessa Q. Passos, Daniel SW Tan. Safety run-in results from phase 3 study of canakinumab (CAN) or placebo in combination with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC (CANOPY-1) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C100. doi:10.1158/1535-7163.TARG-19-C100

Introduction: Immunotherapy is a breakthrough in non-small cell lung cancer (NSCLC), showing long lasting responses in metastasized disease. However, only a limited number of patients responds to treatment. Selection of patients by known biomarkers (eg PD-L1, TMB) and combining PD-(L)1 inhibitors with other treatments (eg. chemotherapy or CTLA4 inhibitors) enriches the population for responders, however still around 60% do not respond with a durable response. We hypothesized that in KRAS mutant positive advanced NSCLC the detection of changes of ctDNA levels will be a relevant predictive marker in immunotherapy ( and stronger than eg PD-L1 expression in tumor tissue).. Purpose: To test ctDNA as an early predictive marker for durable response in KRAS mutated lung cancer, in patients treated with PD-1 inhibitors after progression on platinum containing chemotherapy. Methods: Patients with advanced NSCLC treated with nivolumab with a known KRAS or BRAF mutation from primary biopsy, with disease progression after platinum based chemotherapy are eligible. ctDNA obtained from EDTA plasma at baseline, 1, 2, 4, 6 weeks and thereafter every 12 weeks compared to durable response as primary outcome (ie. benefit from treatment> 6month). Secondary outcomes were tumor response according to RECISTv1.1, progression-free and overall survival. ctDNA was determined and analyzed using a digital droplet PCR assay (Biorad) that was confirmed by a second technique (Idylla). ctDNA was categorized according to its response pattern in time: increasing, decreasing and undetectable KRAS mutant allele frequencies over time. Results: In this ongoing study 31 patients with a known KRAS and 2 patients with a BRAF mutation participated. 24 (72%) had detectable levels of KRAS (n=22) or BRAF (n=2) in plasma. Concordance between Idylla and ddPCR was good and correlation was high (R2 =0,52). Of first 27 patients ctDNA increasing n=11, decreasing n=10, no change n=6. Data on all included patients will be presented. Conclusion: Early detection of decreasing KRAS/BRAF mutant alleles in ctDNA predicted durable tumor responses, and increasing levels were associated with early disease progression. Citation Format: T.Jeroen.N. Hiltermann, Lucie B.M. Hijmering-Kappelle, Maria L. Aguirre Azpurua, Anthonie.J. vd Wekken, Birgitta.I. Hiddinga, Wim Timens, Harry.J.M. Groen, Ed Schuuring. ctDNA a promising predictive marker for treatment with PD-1 inhibitors in KRAS mutated NSCLC after platinum based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 399.

In the CellSearch&reg, system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4&prime, 6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 &plusmn, 74,948, as compared to 359 control samples with an average and standard deviation of 4191 &plusmn, 4463 (p &lt, 0.001). In patients, only 18% &plusmn, 21% and in controls 23% &plusmn, 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% &plusmn, 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells.

Background: Lung cancer remains the number one cause of cancer related death worldwide. Immune modulating therapy have shown long lasting survival, however, they are only effective in about 20% of patients. Although PD-L1 expression predicts tumor response, it is not a robust predictor. Circulating tumor cells (CTCs) or tumor derived extracellular vesicles (tdEV) may be another tumor response predictor. CTCs have been associated with survival in NSCLC. Our hypothesis is that CTC or tdEV changes predict early tumor response and it is an early marker of treatment efficacy. Methods: Patients with proven advanced NSCLC referred to our center for immunotherapy by PD-L1 or PD-1 antagonists were eligible for inclusion. CellSave tubes were drawn at start of therapy (T0) and after 4 weeks of treatment (T1). EpCAM expressing CTCs were detected by the CellSearch system. EpCAM expressing tdEV which are present in the blood during CTC enumeration, were counted in the images from the CellTracks Analyzer, which were analyzed with the open source program ACCEPT. Response to therapy was measured by the response evaluation criteria in solid tumors (RECIST) and according to clinical evaluation of the treating physician. Patients were divided in groups based on response to therapy and presence of CTCs. Cox regression analyses were used to study the association between CTCs and tdEVS with both OS and PFS. Results: 114 patients have been included (104 T0 samples, 71 T1 samples, 52 both a T0 and T1 sample). 53 patients have progressive disease (PD), 24 stable disease (SD), 23 partial response (PR) and 2 a complete response; 12 could not be evaluated yet. CTCs were present in 33 T0 samples (32%), and 20 T1 samples (28%). When CTCs were present at T0, patients had no response to therapy in 83% of cases, while patients without CTCs had no response in 68% of cases. Patients with increased CTC counts at T1 responded in 9% of the cases, versus 33% in patients with decreased CTC counts. CTCs were significantly correlated to survival at both time points independently (T0, HR=1.06, p=0.042; T1, HR=1.04, p=0.009). Comparing patients with a decrease of CTCs with those with an increase, Cox regressions showed a HR of 8.9 (p=0.041), while using the difference of CTCs between T1 and T0 as a continuous variable in the cox regression gave a HR of 1.41 (p=0.02).tdEVS were present in 90% of cases (median 7, range 0-1752)at T0 and in 94% at T1 (median 5, range 0-1975). Cox regression showed a significant correlation with survival of tdEVs at T0 (HR=1.0, p=0.004) and T1 (HR=1.0, p=0.009) and the change in tdEVs counts between T0 and T1 (HR=1.01, p=0.04) Conclusion: CTC and tdEV decrease in the first weeks of immunotherapy was associated with tumor response, whereas an increase in CTC was associated with no response to immunotherapy, indicating a possible use as an early marker. Citation Format: Menno Tamminga, Sanne de Wit, Joost F. Swennenhuis, T. Jeroen N. Hiltermann, Ed M. Schuuring, Leon W.M. Terstappen, Harry J. Groen. Circulating tumor cells and tumor-derived extracellular vesicles as a possible marker for tumor response and survival in patients with non-small cell lung cancer treated with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3606.

Introduction. Composition of cell suspensions enriched for Circulating Tumor Cells (CTC) by the CellSearch system has not yet been explored as the system only presents DAPI+/Cytokeratin+ objects to the operator to identify CTC. We used ACCEPT (http://github.com/LeonieZ/ACCEPT) to identify and characterize all DAPI+ nuclei in EpCAM enriched samples and explored avenues to increase the number of nuclei that can be assigned to a cell lineage. Addition of CD16, expressed on granulocytes and NK-cells, the detection of a cellular plasma membrane and the use of LED light, as opposed to the mercury arc lamp, to improve the excitation of CD45-APC were tested. Methods. In 127 controls and 300 samples from 192 metastatic lung cancer patients, CD16-PerCP was added to the CellSearch CTC identification antibody cocktail. A CellTracks Analyzer equipped with six filter cubes was used to acquire the images. For exploration, after the CellTracks image acquisition 5 cartridges were scanned again on a microscope that contains a LED light source to determine if this would improve the detection of CD45-APC. Also, wheat germ agglutinin (wga) conjugated to AlexaFluor488 stains the cellular plasma membrane and was added to the CellSearch antibody cocktail to discriminate between bare nuclei and unstained cells in 4 samples. The stack of fluorescence images was analyzed with the open source imaging program ACCEPT to define and identify various cell populations. Results. In 427 samples 26,285 ±26,590 [603-156,519] (mean, SD, [range]) nuclei were identified with ACCEPT in the CellSearch images of which only 14% ±15 were identified as leukocytes or CTC, remaining 86% ±15 cells unidentified. Adding CD16 increased the leukocyte population by 47% ±17, thereby reducing the unidentified population to 39% ±19 of the total number of nuclei. CD16-staining was found on 13% of the CTC. Using a LED-light source improved the identification of APC+ cells, and reduced the amount of unidentified nuclei in these samples from 29% (±5) to 14% (±10). Exploration with wga indicated that 43% (±11) (mean 987 cells, range 331-2,638 cells) of the unstained cells contained a cellular membrane. Conclusion. In 427 blood samples run on CellSearch, only 14% of the nuclei obtained after EpCAM enrichment could be assigned as a leukocyte or a CTC. By adding CD16 to the antibody cocktail, the unidentified population is reduced from 86% to 39%. Also, 13% of nuclei scored as CTC express CD16, suggesting these are granulocytes that are incorrectly designated as tumor cells. Using a LED light source improves the identification of dimly stained CD45+ cells. Exploration with wga indicates that 57% of the unidentified nuclei are simply bare nuclei and the origin of 43% still remains unknown. Further studies are needed to determine whether EpCAM+/Cytokeratin- CTC are present among the nucleated cells of which the origin is not yet known. Citation Format: Sanne De Wit, Leonie L. Zeune, T. Jeroen N. Hiltermann, Harry J. Groen, Leon W.M. Terstappen. Quantifying cell populations in CTC enriched samples with the open-source imaging program ACCEPT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3645.

Background: The number of circulating tumor cells (CTCs) detected by the CellSearch system is usually low or absent in NSCLC, possibly by elimination of CTCs in the capillary system. Our hypothesis is that the number of CTCs will be higher in the pulmonary vein draining the primary tumor compared to peripheral blood vessels and is influenced by the manipulation of the tumor during surgery. Methods: In an ongoing study, patients with NSCLC undergoing open thoracotomy or video assisted surgery (VATS) lobectomy are included. During surgery, blood was drawn from the draining pulmonary vein (dv) of the lobe containing the tumor (T1,T2) and from the radial artery (ra) (T0,T1,T2). T0 was at start of surgery, T1 upon identification of the draining pulmonary vein and T2 right before the pulmonary vein was dissected. Dissection of the pulmonary artery occurred in open surgeries before, and in VATS after T2. EpCAM+ CTCs were detected by CellSearch and EpCAM- CTCs after filtration of the EpCAM depleted samples. EpCAM+ CTCs were isolated by FACS and analyzed for copy number variations (CNV) by single-cell whole genome sequencing and genomic alterations of EpCAM- CTC by FISH on the microsieves. Results: So far, 17 patients have been included, one with a benign inflammatory tumor (pt 8). CTC counts were higher in the pulmonary vein than in the distal artery, with CTC clumps observed in some patients (* in table). No consistent effect of manipulation could be determined. In patient 4 and 5, EpCAM+ CTCs from the vein showed CNVs aberrations, whereas in patient 6 CTCs in the radial artery were of normal epithelial origin. EpCAM- CTCs of patient 1 and 2 were of both tumor and normal origin. Conclusion: In NSCLC patients high amounts of single and clumps of EpCAM+ and EpCAM-CTCs were observed in the draining pulmonary vein during surgery. In contrast, the amount of CTCs measured in the radial artery is low (even after correction of blood flow from non-tumor vessels), suggesting loss of CTCs in the cardiac system. EpCAM+ and EpCAM- CTCs in the draining vein and the radial artery during surgeryDraining vein T1 (EpCAM+)Draining vein T1 (EpCAM-)Draining vein T2 (EpCAM+)Draining vein T2 (EpCAM-)radial artery T0 (EpCAM+)radial artery T0 (EpCAM-)radial artery T1 (EpCAM+)radial artery T1 (EpCAM-)Radial artery T2 (EpCAM+)Patient 185*3515NDND122Patient 210*41911NDND050Patient 350*130101*103*2000Patient 42117110000Patient 5127312231Patient 600050027210Patient 7>3000*>26000132000000Patient 8#017596*1000050Patient 9000001000Patient 10 Lower lobe73*255204*173NDND000Patient 10 Middle lobe1426016NDND000Patient 11NDND05NDNDNDND0Patient 121013NDND01NDPatient 13440400000Patient 10121102010NDPatient 102321000000Patient 10305>1000*>100001NDND0Patient 1042820*7NDND100 Citation Format: Menno Tamminga, S de Wit, Joost F. Swennenhuis, Ellen Heitzer, Michael Speicher, T. Jeroen N. Hiltermann, Ed M. Schuuring, Leon W.M. Terstappen, Harry J. Groen. Circulating tumor cells measured in the pulmonary vein and radial artery during surgery of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5594.

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.

Introduction: The immune system plays an important role in tumor progression and treatment response such as with new immune modulating therapies. It is unclear in lung cancer what the effect of smoking is upon intratumoral immune cell composition and their function. In our study publicly available expression data were used to find evidence for the influence of smoking on the composition of intratumoral immune cell fractions and their prognostic impact. Methods: We searched the Gene Expression Omnibus (GEO) for human non-small cell lung cancer (NSCLC) samples. Analysis was confined to samples hybridized to the Affymetrix HG-U133 Plus 2.0 (GEO accession number GPL570) platforms. Expression profiles were downloaded and curated for duplicates and corrupt files. Different immune cell fractions were estimated using CIBERSORT and the LM22 leukocyte signature matrix. We combined this information with available clinical data. We used Cox regression analyses to evaluate the prognostic impact of the different immune fractions and Mann-Whitney U tests to test significant differences. Results: Smokers with NSCLC had a significantly (p Non-smokers significantly benefit from a higher percentage of intratumoral memory B-cells in the immune infiltrate as compared to normal tissue (HR 0.92, 95% CI 0.85-1.00, p=0,049), while the smokers significantly benefit from higher intratumoral immune factions of resting CD4+ T-cells (HR 0.93, 95% CI 0.89-0.98, p=0.001). Higher percentages of regulatory T-cells (HR 1.23, 95% CI 1.03-1.47, p=0.025), neutrophils (HR 1.11, 95% CI 1.02-1.21, p=0.014) and follicular helper cells (HR 1.12, 95% CI 1.01-1.24, p=0.03) had a significant detrimental effect on survival of smokers. There was a borderline beneficial effect for smokers of the intratumoral NK cell faction (HR 0.61, 95% CI 0.36-1.02, p=0.057). For non-smokers, a detrimental effect was observed for the intratumoral plasma cell fraction (HR 1.04, 95% CI 1.00-1.09, p=0.073) and the naïve B-cell fraction (HR 1.17, 95%CI 0.99-1.39, p=0.075). Conclusion: In NSCLC significant different composition of intratumoral immune cells were observed between a smoking and non-smoking population. These differences were associated with differences in overall survival. Citation Format: Menno Tamminga, T. Jeroen N. Hiltermann, Ed Schuuring, Rudolf S. Fehrmann, Harry J. Groen. Effect of smoking on tumor-infiltrating immune cell composition and prognosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2017-4569

Introduction: Breakthroughs in cancer research have resulted in mutation-specific targeted therapies (precision medicine). Most of these new drugs are only effective in patients with an actionable molecular profile. Thus, predictive molecular testing enables oncologists to select individual patients for the most appropriate (targeted) therapy and to reduce the burden of overtreatment. The number of clinically relevant predictive markers that are routinely analyzed is growing rapidly, resulting in the identification of rare mutations, mutations with unknown relevance and coexistence of two or more mutations in the same sample. Incorporating these into the optimal treatment for the individual patient can be complex. Methods: A total of 2461 sequential tumor biopsies were analyzed at our institute using targeted next generation sequencing (Ion Torrent platform). 230 of these patients were discussed at a weekly Molecular Tumor Board (MTB) meeting. Cases involved 170 lung and 21 colorectal carcinomas, 24 melanomas, 1 GIST and a range of other malignancies with uncommon and rare mutations. The board is composed of pulmonologists, medical oncologists, pathologists and clinical scientists in molecular pathology. The goal of the MTB is to discuss the biological and clinical relevance of rare mutations or uncommon profiles and to suggest treatment options based on registered, off-label or trial-based drugs presently available in the Netherlands. Results: In this abstract we report on four patients with an ERBB2 exon 20 mutation and 1 patient with ERBB2 amplification received anti-HER2 treatment after an MTB consensus decision. Two patients with an insertion in exon 20 of ERBB2: (c.2313_2324dup; p.(Y772_A775dup)) received first line therapy with afatinib and showed a partial response and stable disease respectively. One patient with a c.2524G>A; p.(V842I) mutation received afatinib and showed stable disease for 3 months. A patient with another ERBB2 exon 20 insertion (c.2326_2327insTAT:p.(G776delinsVC)) received afatinib but had progressive disease within two months. One patient with an ERBB2 amplification by FISH and high (3+) HER2(ERBB2) expression, showed a partial response to trastuzumab. All patients had stage IV and would without genomic knowledge been treated with chemotherapy. Conclusion: Lung cancer patients with sporadic ERBB2 mutations might benefit from targeted ERBB2 therapy. For an optimal treatment decision, patients with rare mutations in general, may benefit from discussion in a multidisciplinary molecular tumor board. In the future, both the considerations for targeted therapy as well as treatment response and toxicity should be registered in an open-access database and shared with other national and international Molecular Tumor Board initiatives to allow comparison with traditional treatments. Citation Format: Arja ter Elst, Nils A. 't Hart, Anthonie J. van der Wekken, Wim Timens, Lucie B. Hijmering-Kappelle, Geke A. Hospers, Hilde Jalving, Elise M. van der Logt, Leon C. van Kempen, Sjoukje F. Oosting, Matthew R. Groves, T Jeroen Hiltermann, Anke van den Berg, Harry J. Groen, Ed Schuuring. Treatment decision-making of rare ERBB2 (HER2) mutations in lung cancer; a role for multidisciplinary molecular tumor boards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 754. doi:10.1158/1538-7445.AM2017-754

Introduction: The immune system plays an important role in tumor progression and treatment response, for example with the new immune modulating therapies. In lung cancer the effect of the individual immune cell types is unclear. In our study, we used publicly available expression data to find evidence whether there are differences in the composition of immune cell fractions within the microenvironment between squamous cell carcinoma and adenocarcinoma of the lung. Methods: We searched the Gene Expression Omnibus (GEO) for human NSCLC samples. Analysis was confined to samples hybridized to the Affymetrix HG-U133 Plus 2.0 (GEO accession number GPL570) platforms. Expression profiles were downloaded and curated for duplicates and corrupt files. Different immune cell fractions were estimated using CIBERSORT and the LM22 leukocyte signature matrix. We combined this information with available clinical data. We used Cox regression analyses to evaluate the prognostic impact of the different immune fractions and Mann-Whitney U tests to test significant differences. Results: Comparing the immune cell composition of adenocarcinoma (n=587) and squamous cell carcinoma (n=254), we observed in adenocarcinoma increased percentages of naïve and memory B-cells, resting and active CD4+ T-cells, regulatory T-cells, active NK cells, monocytes, M2 macrophages, resting dendritic cells and resting mast cells, and decreased percentages of plasma cells , CD8+ T-cells, follicular helper cells, resting NK cells, gamma delta T-cells, M0 and M1 macrophages and active mast cells. In adenocarcinoma, a higher percentage of memory B-cells in the immune infiltrate was associated with increased survival (HR 0.97, 95% CI 0.95-0.99, p=0.02), while higher percentages of neutrophils (HR of 1.07, 95% CI 1.01-1.13, p=0.02), M0 macrophages (1.02, 95% CI 1.01-1.04, p=0.01) and follicular helper cells (1.07, 95% CI 1.02-1.13, p=0.01) were associated with a worse survival. In both histologies, the resting mast cell immune fraction showed a positive association with survival (adenocarcinoma HR 0.96, 95% CI 0.93-0.99, squamous cell carcinoma HR 0.93, 95% CI 0.89-0.98, p Conclusion: In NSCLC significant differences in intratumoral immune cell composition were observed between adenocarcinoma and squamous cell carcinoma. Citation Format: Menno Tamminga, T. Jeroen Hiltermann, Ed Schuuring, Rudolf S. Fehrmann, Harry J. Groen. Prognostic effect of tumor-infiltrating immune cell composition in non-small cell lung cancer by histology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2946. doi:10.1158/1538-7445.AM2017-2946

Background: The number of circulating tumor cells (CTC) detected by the CellSearch system is low or absent in 7.5 mL of venous peripheral blood of metastatic and non-metastatic non-small cell lung cancer (NSCLC) patients. Our hypothesis is that CTC can be detected in the pulmonary vein draining the lobe containing the primary tumor before its removal and would be influenced by manipulation of the lobe during surgery. Methods: Patients with early stage NSCLC undergoing open lobectomy were included. During surgery, blood was drawn centrally (pulmonary vein draining the lobe containing the tumor) and peripherally (radial artery) at two time points: directly after thoracotomy and identification of the draining pulmonary vein (T1) and just before closure of the draining pulmonary vein after manipulation, dissection and closure of the pulmonary artery (T2). EpCAM+ CTC were detected by CellSearch. The EpCAM+ CTC depleted sample was subsequently filtered through 5µm micosieves and fluorescently labeled for identification of EpCAM- CTC (DAPI+, Cytokeratin+ and CD45-). Results: So far, three patients have been included. Remarkably, cell clumps were detected among EpCAM+ CTC at T1 in the pulmonary vein in all patients, and as well at T2 in patient #3. CTC in the pulmonary vein at T1 were morphologically different than those detected at T2 and in the arteries. Experiments to confirm malignancy of both subtypes of CTC are pending, using chromosome aneuploidy status and copy number variation analysis to compare them with the resected tumor. Conclusion: In early stage NSCLC high amounts single and clumps of CTC are observed in the draining pulmonary vein during surgery. The amount measured in the radial artery is lower, indicating loss of CTC in the circulatory system before peripheral measurement. If the cells found really are CTC that are shed from the primary tumor during surgery, it will have clinical consequences for the surgery techniques used. EpCAM+ and EpCAM- Circulating tumor cells found during surgery in non-small cell lung cancerPatient #1Patient #2Patient $3#CTC/7.5 mLEpCAM+EpCAM-EpCAM+EpCAM-EpCAM+EpCAM-Baseline (T0)NANANANA32Draining vein T1*8535*104*50130Draining Vein T2151911*10110Radial artery T1120500Radial artery T22101012Radial Artery T1+3NANANANA02Radial artery T1+5NANANANA05Radial artery T2+5NANANANA21*Presence of CTC clusters Citation Format: Menno Tamminga, Sanne de Wit, Joost F. Swennenhuis, Caroline van de Wauwer, Theo J. Klinkenberg, T. Jeroen Hiltermann, Harry J. Groen, Leon W. M. Terstappen. Circulating tumor cells measured in the pulmonary vein and the radial artery during surgery of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2017-3783

Introduction The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing as more and more targeted therapies become available. Presence in blood of circulating tumor cells (CTC), tumor derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA) measured with different approaches are being explored for their potential to represent a liquid biopsy in the European and Dutch CANCER-ID projects (https://www.cancer-id.eu/ & https://www.utwente.nl/tnw/cancer-id/). Here, we determine in just one 7.5 mL tube of blood the presence of CTC, tdEV and ctDNA and investigate the relation with survival of metastatic NSCLC patients. Methods In total 106 advanced NSCLC patients were enrolled in the study. In 86 patients EpCAM+ CTC, EpCAM- CTC & tdEV were enumerated and in 50 patients EpCAM+ CTC, EpCAM- CTC, tdEV & ctDNA, all from one CellSave blood tube. ctDNA from a separate plasma tube is available for all patients but not yet analyzed. Before placing the sample in the CellSearch system, plasma was aspirated and stored at -80°C. EpCAM+ CTC were enumerated by CellSearch and EpCAM- CTC after filtration of the EpCAM+ CTC depleted blood through 5µm pore filters, as described by de Wit et al. (Sci. Rep. doi: 10.1038/srep12270, 2015). tdEV were defined by a multidimensional gate as cytokeratin+/DAPI-/CD45- vesicles and identified in the CellSearch images, using the open source image analysis program ACCEPT. The stored plasma was used for ctDNA quantification with the FAST-SeqS approach, described by Belic et al. (ClinChem 61, 838, 2015). In several patients with EpCAM- CTC, fluorescent in situ hybridization was performed on the filter to establish the cancerous origin of the EpCAM- CTC. Results In 24% of the patients ≥1 EpCAM+ CTC as well as ≥1 EpCAM- CTC were detected in 7.5 mL of blood. In 30% of the patients tdEV were present at a frequency >45 per 7.5 mL. This frequency is based on the mean + 2SD from 42 healthy controls. In 20% of the patients >10% ctDNA load was found. No significant correlation was found between the presence of these biomarkers. Presence of all four biomarkers was detected in 6% of patients and at least one of four was found in 52% of patients. One or more EpCAM+ CTC were associated with poor overall survival (p=0.010 for 86 patients and p=0.019 for 50 patients), whereas EpCAM- CTC were not (p=0.495 n=86; p=0.571 n=50). The latter is surprising since some CTC were shown to be cytogenetically aberrant, conform the primary tumor. The presence of >45 tdEV (p=0.271 n=50) and >10% ctDNA (p=0.082 n=50) did not reach significance. Conclusions In this study EpCAM+ CTC, EpCAM- CTC, tdEV or ctDNA was detected in one tube of blood in 52% of the NSCLC patients. Only the presence of EpCAM+ CTC was associated with poor overall survival, raising the question whether or not the extraction of molecular information from these other biomarkers can be used to predict response to treatment in NSCLC. To increase the percentage of patients from which a liquid biopsy can be obtained, the analyzed blood volume will need to be increased. Citation Format: Sanne de Wit, Menno Tamminga, Joost F. Swennenhuis, Leonie L. Zeune, Ellen Heitzer, Michael Speicher, T Jeroen N. Hiltermann, Leon WMM Terstappen, Harry JM Groen. Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-250. doi:10.1158/1538-7445.AM2017-LB-250

Several studies have shown heterogeneity in lung cancer, with parallel existence of multiple subclones characterized by their own specific mutational landscape. The extent to which minor clones become dominant in distinct metastasis is not clear. The aim of our study was to gain insight in the evolution pattern of lung cancer by investigating genomic heterogeneity between primary tumor and its distant metastases. Whole exome sequencing (WES) was performed on 24 tumor and five normal samples of two small cell lung carcinoma (SCLC) and three non-SCLC (NSCLC) patients. Validation of somatic variants in these 24 and screening of 33 additional samples was done by single primer enrichment technology. For each of the three NSCLC patients, about half of the mutations were shared between all tumor samples, whereas for SCLC patients, this percentage was around 95. Independent validation of the non-ubiquitous mutations confirmed the WES data for the vast majority of the variants. Phylogenetic trees indicated more distance between the tumor samples of the NSCLC patients as compared to the SCLC patients. Analysis of 30 independent DNA samples of 16 biopsies used for WES revealed a low degree of intra-tumor heterogeneity of the selected sets of mutations. In the primary tumors of all five patients, variable percentages (19-67%) of the seemingly metastases-specific mutations were present albeit at low read frequencies. Patients with advanced NSCLC have a high percentage of non-ubiquitous mutations indicative of branched evolution. In contrast, the low degree of heterogeneity in SCLC suggests a parallel and linear model of evolution.

Lung cancer is a devastating disease with a poor prognosis. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) represent different forms of lung cancer that are associated with distinct genetic causes and display different responses to therapy in the clinic. Whereas SCLC is often sensitive to chemotherapy at start of treatment, NSCLC are less chemo-sensitive. In NSCLC different histological subtypes are distinguished and increasing efforts are made to identify subtypes that respond to specific therapies, such as those harbouring epidermal growth factor receptor (EGFR) mutations that have benefit from treatment with EGFR inhibitors. Targeting of the apoptotic machinery represents another approach that aims to selectively kill cancer cells while sparing normal ones. Here we describe different ways that are currently explored to induce apoptosis in lung cancer cells, specifically pathways controlled by TNF-related apoptosis-inducing ligand (TRAIL), BCL-2 family members and apoptosis inhibitory proteins (IAPs). Preclinical studies are discussed and for some agents results from early clinical studies and future perspectives are considered. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Purpose: Lung cancer is divided into two main groups, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter subdivided in squamous, large cell and adenocarcinoma. Many studies have shown intra-tumor heterogeneity in lung cancer with multiple minor clones characterized by their own specific mutational landscape. These minor clones can become predominant under selective pressure caused by therapy. Aim of our study is to investigate the relation between primary lung cancer and multiple distant metastases using whole exome sequencing. Materials and methods: Normal tissue, primary lung tumor, and multiple metastases were obtained from 2 SCLC and 3 NSCLC patients resulting in a total of 29 samples (Table 1). DNA was isolated from total, macro-dissected, or laser micro-dissected tissue to obtain a purity of at least 80% tumor cells. Library preparation was based on the SureSelect All Exon V5 bait (Agilent) and paired-end sequencing was done using Illumina® HiSeq2000. Genome analysis toolkit (GATK) was used to analyze data and the 1000-Genome database was used to remove single nucleotide polymorphisms. Patients' characteristics and overview of sequenced metastases samplesSubtypeSexAge at diagnosisCOPDSmokingPack yearTime interval between diagnosis and death (days)No. of somatic mutationsMetastasesLarge cell carcinomaMale65YesYes302308446Lymph node (1x)Adrenal gland (1x)Squamous cell carcinomaMale68YesYes>100111306Liver (1x)Spleen (1x)Heart (1x)AdenocarcinomaFemale51NoNo02579Liver (1x)Lymph nodes (2x)Ovary (1x)Vertebra (1x)Jejunum (1x)Small cell carcinoma-1Female79YesYes>508347Liver (1x)Adrenal gland (1x)Small cell carcinoma-2Female58YesYes406260Liver (1x)Lymph nodes (4x) Results: In four patients more than 70% of the observed variants were true somatic mutations. In the adenocarcinoma patient, only 32% of the variants were called as true somatic mutations. After excluding personal variants, 79 to 446 true somatic mutations were observed in any of the tumor samples of the five lung cancer patients (Table 1). Comparison of the mutations observed in the primary and metastatic samples revealed that around 50% of the mutations were shared between all tumor samples of the three NSCLC patients, whereas more than 95% of mutations were shared in all samples of the two SCLC patients. Conclusion: We found a high percentage of metastasis specific mutations in NSCLC patients. In contrast, SCLC patients show a limited number of metastasis specific mutations, consistent with the aggressiveness of these tumor and the very poor prognosis. Citation Format: Ali Saber, Klaas Kok, Martijn M. Terpstra, Wim Timens, Sijmen Aukema, T. Jeroen Hiltermann, Harry J. M. Groen, Anke van den Berg. Whole exome sequencing reveals a distinct mutation pattern in metastatic small cell lung cancer compared to non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2015-4757

Purpose: A small proportion of lung cancer patients have an inversion involving the ALK gene on chromosome 2. These ALK+ patients initially respond to crizotinib, but later develop progressive disease. This resistance can be due to gatekeeper mutations in the kinase domain of the ALK gene like L1196M and G1269A, copy number gain of the EML4-ALK fusion gene, and mutations in EGFR and KRAS. We aimed at identifying fusion genes that may explain the resistance mechanism in patients progressing on crizotinib. Materials and Methods: Frozen tissue of the resistant tumor samples was available for four EML4-ALK positive patients (Table 1). Paired end RNA sequencing was performed using the Illumina TruSeq RNA platform. DeFuse (v.0.6.1) was used to identify fusion products. Candidate fusion genes were validated by RT-PCR. In addition, we used the IGV browser to identify mutations in resistance-associated genes in the RNA-seq data. Results: Thirteen fusions genes were identified with high confidence (Table 1). In three of the four patients we detected an EML4-ALK fusion product. Four novel fusion products with a functional ORF were confirmed by RT-PCR. Three of these fusion products were present in patient 1 (Table 1). The fourth fusion involving CLIP4-VSNL1 was detected in patient 3. Interestingly, three of the 4 fusion products observed in patient 3 involved 5 genes that are all close to the ALK locus, i.e. EML4, ALK, CLIP4, VSNL1 and MCFD2 (Table 1). In addition, we found two resistance-related mutations in the tyrosine kinase domain of the ALK gene, i.e. p.C1156Y and p.G1269A, in patients 2 and 3, respectively. Fusion products in four crizotinib resistant lung cancer patientsPatientAgeSmokingALK immunohistochemistryTumor response (PFS in months)Fusion genesORF147CurrentNot availableNo (1.4)RAB35-ATP2A2YesSLC10A7-TTC29YesSTAB2-NUAK1YesKMT2B-AC002115.9NoPIAS1-SLC24A6NoZNF827-ARHGAP10No227None+Yes (9.2)EML4-ALKYesNRG1-RBPMSNo342None+Yes (10.4)EML4-ALKYesCLIP4-VSNL1YesMCFD2-CLIP4NoCPSF6-RBMS2No456Current+Yes (15.7)EML4-ALKYes Conclusion: We found four new fusion products with a functional ORF using RNA sequencing of crizotinib resistant tumor samples. Further analysis of the primary tumor samples is ongoing and will reveal if these fusion genes might have been induced by the crizotinib treatment. Two of the four patients have a known resistance-inducing mutation in the ALK gene. Citation Format: Ali Saber, Anthonie van der Wekken, Klaas Kok, Martijn M. Terpstra, Mirjam F. Mastik, Wim Timens, Ed M.D. Schuuring, T. Jeroen Hiltermann, Harry J.M. Groen, Anke van den Berg. Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2015-4245