T. Jeroen N. Hiltermann

The detection of EpCAM + and EpCAM - circulating tumor cells

EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM+ CTC detected by CellSearch and EpCAM– CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM– CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM+ cells could be detected with CellSearch, whereas most spiked cells with EpCAMlow or EpCAM– expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM– CTC. In this pilot study, the presence of EpCAM+ CTC was associated with poor outcome, whereas the EpCAM– CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM– and EpCAM+ CTC.

Randomized, Placebo-Controlled Phase III Study of Docetaxel Plus Carboplatin With Celecoxib and Cyclooxygenase-2 Expression As a Biomarker for Patients With Advanced Non–Small-Cell Lung Cancer: The NVALT-4 Study

PURPOSE Cyclooxygenase-2 (COX-2) protein expression in patients with non-small-cell lung cancer (NSCLC) may be not only a prognostic marker but also predictive for COX-2 inhibition. We hypothesized that COX-2 expression is associated with shorter survival and that celecoxib, being a potent COX-2 inhibitor, increases tumor response and survival. PATIENTS AND METHODS A phase III study was performed in patients with stage IIIb/IV NSCLC who had pathologic confirmation, no prior chemotherapy, Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate organ function. Treatment consisted of docetaxel and carboplatin every 3 weeks for five cycles. Patients were randomly assigned to receive celecoxib 400 mg or placebo twice daily. COX-2 expression on tumor cells was detected by immunohistochemistry. Primary end point was overall survival (OS). RESULTS From July 2003 to December 2007, 561 patients were randomly assigned. Toxicity was mild, and no increase in cardiovascular events was observed. Tumor response was 38% in the celecoxib arm and 30% in the placebo arm (P = .08). Median progression-free survival was 4.5 months (95% CI, 4.0 to 4.8) for the celecoxib arm and 4.0 months (95% CI, 3.6 to 4.9) for the placebo arm (hazard ratio [HR], 0.8; 95% CI, 0.6 to 1.1; P = .25). Median OS was 8.2 months (95% CI, 7.5 to 8.8) for both treatment arms (HR, 0.9; 95% CI, 0.6 to 1.2; P = .32). COX-2 expression did not independently predict survival. Benefit from celecoxib, restricted to patients with low COX-2 expression, was not significant when adjusted for prognostic factors. CONCLUSION In advanced NSCLC, celecoxib does not improve survival. In this study, COX-2 expression was not a prognostic biomarker and had no predictive value when celecoxib was added to chemotherapy.

Targeting apoptosis pathways in lung cancer

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.

Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer.

Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56–4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14–14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04–0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

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.

Moving forward with circulating tumor cells and lung cancer

Lung cancer is still the deadliest tumor around the world. Annually over 1 million people dye due to lung cancer (1). The most common cause of death in (lung) cancer patients is due to metastases. Tumor load increases exponentially by dissemination and growth of neoplastic cells into organs distinct from that in which they originated (2). Non-small cell lung carcinoma (NSCLC) accounts for around 85% of all lung cancer cases and is the leading cause of cancer-related mortality worldwide. At presentation approximately 70% of patients present with advanced disease, for whom curative therapy will not be available (3). During the last decade new drugs have been developed for specific subtypes of lung cancer. Emphasis is now on targeted therapy, with drug aiming at a mutated protein that has become an oncogen (4).

Mutations in EMT-Related Genes in ALK Positive Crizotinib Resistant Non-Small Cell Lung Cancers

Crizotinib is an effective drug for patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC), but upon treatment, the tumors inevitably become crizotinib resistant in time. The resistance mechanisms are only partly understood. In this study, we aim to identify gene mutations associated with resistance in ALKpositive advanced non-squamous NSCLC treated with crizotinib. Four ALK positive patients with progressive disease following crizotinib treatment were identified with paired pre- and post-crizotinib tumor tissue from our previously published cohort. Somatic variants in these samples were detected by whole exome sequencing. In one of the four patients, an ALK-resistance associated mutation was identified. In the other three patients, no ALK-resistance associated mutations were present. In these patients we identified 89 relevant somatic mutations in 74 genes that were specific to the resistant tumors. These genes were enriched in 15 pathways. Four pathways, were related to epithelial-mesenchymal transition (EMT): proteoglycans in cancer, HIF-1 signaling, FoxO signaling pathway, and ECM-receptor interaction. Analysis of other EMT-related pathways revealed three additional genes with mutations specific to the crizotinib-resistant tumor samples. The enrichment of mutations in genes associated with EMT-related pathways indicates that loss of epithelial differentiation may represent a relevant resistance mechanism for crizotinib.

Mutation patterns in small cell and non-small cell lung cancer patients suggest a different level of heterogeneity between primary and metastatic tumors.

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.

Abstract 754: Treatment decision-making of rare ERBB2 (HER2) mutations in lung cancer; a role for multidisciplinary molecular tumor boards

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. 9t 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

Abstract LB-250: Liquid biopsy in NSCLC: EpCAM+ and EpCAM- circulating tumor cells, tumor derived extracellular vesicles and cell-free circulating tumor DNA

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