Ali Saber

The mutational landscape of Hodgkin lymphoma cell lines determined by whole-exome sequencing

The mutational landscape of Hodgkin lymphoma cell lines determined by whole-exome sequencing

Resistance mechanisms after tyrosine kinase inhibitors afatinib and crizotinib in non-small cell lung cancer, a review of the literature

Targeted treatment of advanced non-small cell lung cancer patients with afatinib in EGFR mutation or crizotinib in ALK break positive patients results in profound tumor responses but inevitably induces resistance. In this review we present currently known resistance mechanisms for afatinib and crizotinib two recently approved drugs. Resistance mechanisms identified for afatinib include c-MET amplification and the V843I EGFR mutation. Expression of FGFR1, increased IL6R/JAK/STAT signaling, enhanced interference with aerobic glycolysis and autophagy are associated with resistance to afatinib. Most common resistance mechanisms for ALK break positive cases are gatekeeper mutations in the ALK gene. Also activation of the EGFR pathway, KRAS mutations, the autophagy pathway and epithelial mesenchymal transition (EMT), have been associated with resistance. Many of the proposed resistance mechanisms need to be functionally studied to proof a causative relationship with resistance.

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.

Overall survival in EGFR mutated non-small-cell lung cancer patients treated with afatinib after EGFR TKI and resistant mechanisms upon disease progression

Purpose To determine survival in afatinib-treated patients after treatment with first-generation EGFR tyrosine kinase inhibitors (TKIs) and to study resistance mechanisms in afatinib-resistant tumors. Methods Characteristics and survival of patients treated with afatinib after resistance to erlotinib or gefitinib in two large Dutch centers were collected. Whole exome sequencing (WES) and pathway analysis was performed on available pre- and post-afatinib tumor biopsies and normal tissue. Results A total of 38 patients were treated with afatinib. T790M mutations were identified in 22/29 (76%) pre-afatinib treatment tumor samples. No difference in median progression-free-survival (2.8 months (95% CI 2.3–3.3) and 2.7 months (95% CI 0.9–4.6), p = 0.55) and median overall-survival (8.8 months (95% CI 4.2–13.4) and 3.6 months (95% CI 2.3–5.0), p = 0.14) were observed in T790M+ patients compared to T790M- mutations. Somatic mutations in TP53, ADAMTS2, CNN2 and multiple genes in the Wnt and PI3K-AKT pathway were observed in post-afatinib tumors of six afatinib-responding and in one non-responding patient. No new EGFR mutations were found in the post-afatinib samples of the six responding patients. Further analyses of post-afatinib progressive tumors revealed 28 resistant specific mutations in six genes (HLA-DRB1, AQP7, FAM198A, SEC31A, CNTLN, and ESX1) in three afatinib responding patients. No known EGFR-TKI resistant-associated copy number gains were acquired in the post-afatinib samples. Conclusion No differences in survival were observed in patients with EGFR-T790M treated with afatinib compared to those without T790M. Tumors from patients who had progressive disease during afatinib treatment were enriched for mutations in genes involved in Wnt and PI3K-AKT pathways.

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.

Whole exome sequencing reveals a distinct mutation pattern in metastatic small cell lung cancer compared to non-small cell lung cancer

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. 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

Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients

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. 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