Heterogeneity In Lung Cancer Research Articles

Abstract A1-25: Intratumor heterogeneity in never-smoker Asian EGFR mutant lung adenocarcinoma

Abstract Lung cancer has the highest cancer associated mortality rate in many countries across the world. In contrast to western populations, approximately half of lung adenocarcinoma cases in Singapore harbour activating epidermal growth factor receptor (EGFR) mutations, with preponderance for never smokers and female gender. Although EGFR tyrosine kinase inhibitors (TKIs) confer high response rates of up to 70%, drug resistance invariably ensues - most commonly through the “acquisition” of EGFR T790M mutation. While the extent and pattern of intratumoral heterogeneity (ITH) in non-small cell lung cancer (NSCLC) were recently described, these studies examined histologically and molecularly diverse cohort of patients, majority being current or ex-smokers. Here we report ITH in eight never-smoker EGFR mutant lung adenocarcinoma cases of Asian ethnicity. All eight patients had no prior treatment history and harboured an activating EGFR mutation (5 L858R, 2 exon 19 deletion, 1 exon 20 insertion). They underwent lobectomy for Stage IA, IB NSCLC. Tumors were harvested using a systematic sectoring protocol according to standard operation procedures, with tissue banked for exome sequencing, RNA-sequencing and SNP array. A total of 46 tumor sectors (at least 4 regions from each of the 8 tumors) were subject to whole exome sequencing, with matched normal samples. With an average sequencing depth of 100x, we identified 860 somatic exonic SNVs (601 being non-synonymous) and 49 indels across all samples. The median number of SNVs per patient was 112 and per sector was 49. Notably, activating EGFR mutations were identified across all tumor sectors of all but two patients (for whom it was identified in 3 of 5 and 5 of 7 sectors respectively). In addition, we did not identify the EGFR T790M mutation in any of the sequenced tumor sectors, suggesting that, this resistance mutation is not present at detectable frequencies even as a branch or subclonal event in a treatment naïve scenario. Of 20 genes that were significantly mutated across 46 individual tumor sectors, only two overlapped with published recurrently mutated genes in NSCLC. In conclusion, we show that activating EGFR mutations are ubiquitous truncal events in 6 of 8 Asian never-smoker lung adenocarcinoma – consistent with its role as a therapeutically tractable driver gene. The T790M mutation commonly associated with TKI resistance was not detected as a subclonal event in treatment naïve patients. Further, our study reveals the unique mutation spectra of Asian EGFR mutant lung adenocarcinoma, highlighting the value of multi-region sequencing in characterising the genomic architecture of defined molecular subsets of NSCLC from different ethnic backgrounds. Citation Format: Rahul Nahar, Weiwei Zhai, Angela Takano, Alexis Jiaying Khng, Xingliang Liu, Chong Hee Lim, Audrey S.M. Teo, Cheryl Xueli Chan, Apoorva Gogna, Kiat-Hon Lim, Tina Koh, Huay Mei Poh, Yin Yeng Lee, Liang He Chen, Tong Zhang, Vidhya Gomathi Krishnan, N Gopalakrishna Iyer, Pauline Ng, Wan Teck Lim, Bing Lim, Eng-Huat Tan, Daniel S.W. Tan, Axel M. Hillmer. Intratumor heterogeneity in never-smoker Asian EGFR mutant lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-25.

Abstract 4815: Tumor heterogeneity of advanced primary lung cancer evaluated by multiregion sequencing

Abstract Background: Cancers are composed of cells with distinct genetic and phenotypic characteristics. Tumor heterogeneity in each patient may influence on personalized medicine strategies that depend on results from single tumor-biopsy samples. However, tumor heterogeneity in advanced primary lung cancer has not been well studied. The purpose of this study was to investigate the tumor heterogeneity in advanced primary lung cancer. Methods: We sequenced fourteen regions from four patients who were suspicious for primary lung cancer with multiple lymph node metastasis. Tumor samples were obtained from primary tumor and metastatic lymph node(s) or distant metastasis using bronchoscopic forcep biopsy, endobronchial ultrasound-guided transbronchial needle aspiration, and percutaneous needle biopsy. The profiles of somatic mutations, copy number alterations and gene expression were compared among primary tumor and metastatic sites in each patient. Clonal evolution was evaluated using the concept of cancer cell fraction by SciClone analysis. Results: We performed multiregion whole exome sequencing and RNA sequencing on three non-small cell lung cancers (2 adenocarcinomas [TH1 and TH4] and 1 squamous cell carcinoma [TH2]) and one small cell lung cancer (TH3). EML4-ALK fusion was noticed in TH1 and TH4. There was profound tumor heterogeneity in terms of somatic mutation, copy number alteration, or gene expression in each patient. The frequency of private somatic mutation from spatially distinct regions were 59% in TH1, 44% in TH2, 13% in TH3, and 30% in TH4. Known driver mutation was also heterogeneous in TH1 and TH2. Analysis of the clonal architecture confirmed the presence of clonal heterogeneity in each patient. Phylogenetic reconstruction revealed the evidence of branched clonal evolution. Conclusions: Tumor heterogeneity was noticed from spatially distinct regions from advanced primary lung cancers. The single biopsy specimens may not represent the whole landscape of genetic alteration in advanced primary lung cancers. Citation Format: Sang-Won Um, Je-Gun Joung, Su Yeon Lee, Joon Seol Bae, Jinha Park, Woong-Yang Park. Tumor heterogeneity of advanced primary lung cancer evaluated by multiregion sequencing. [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 4815. doi:10.1158/1538-7445.AM2015-4815

Abstract 1310: Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC)

Abstract Early phase I trials with monoclonal antibodies targeting PD-1/PD-L1 have demonstrated durable clinical responses in patients with NSCLC. However, the prognostic/predictive role of tumor PD-L1 expression has not been determined. Current assays for evaluation of PD-L1 protein expression are not standardized. Here, we demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF), and compare results obtained using three PD-L1 antibodies targeting the intracellular (IC) and extracellular (EC) domains. We measured PD-L1 protein expression using two antibodies against the IC domain (E1L3N [Cell Signaling Technology] and SP142 [Spring Biosciences Inc.]) and one antibody binding the EC domain (E1J2J [Cell Signaling Technology]) in 49 NSCLC whole tissue sections and a TMA with the same 49 cases. Mel624 cells stably transfected with PD-L1, as well as Mel624 parental cells and human term placenta were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA® method of QIF. For IHC, PD-L1 positivity was determined using previously reported cut-points for tumor (1%, 5%, and 50%) and stroma (5%). IHC inter-assay concordance was evaluated using kappa coefficient. AQUA scores were measured in an average of 28.6 fields of view per case or by TMA at two-fold redundancy. Linear regression coefficients (R2) were used to compare antibody QIF scores. Tumor-infiltrating lymphocytes were scored in hematoxylin/eosin stained slides using current consensus guidelines. Chromogenic IHC assays showed fair to poor concordance (kappa 0.124 - 0.552) for all cut-points. QIF showed PD-L1 immunopositivity was heterogeneous for all three PD-L1 antibodies. R2 values were lower when E1J2J (EC) was compared to E1L3N and SP142 (IC) (R2 = 0.090 and 0.079), while coefficients were higher when the IC antibodies were compared (R2 = 0.658). E1L3N and SP142 significantly correlated with high TILs (p = 0.007 and p = 0.021) but E1J2J did not (p = 0.281). E1J2J significantly correlated with older age (p = 0.038), but no other clinicopathological feature. SP142 significantly correlated with lymph node positive status (p = 0.030) Objective determination of PD-L1 protein levels in NSCLC reveals significant heterogeneity within tumors. There is significant inter-assay variability, even between assays detecting the same protein domain. This could be due to different antibody affinities, cross reactivity, or distinct target epitopes. Efforts to determine the clinical value of these observations are underway. Citation Format: Joseph McLaughlin, Kurt A. Schalper, Daniel E. Carvajal-Hausdorf, Vasiliki Pelekanou, Vamsidhar Velcheti, Herbert Haack, Matthew R. Silver, Roy Herbst, Patricia LoRusso, David L. Rimm. Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC). [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 1310. doi:10.1158/1538-7445.AM2015-1310

Abstract 3761: Distinct transcriptional programs drive phenotypic heterogeneity in small cell lung cancer

Abstract Small-cell lung cancer (SCLC), a lethal neuroendocrine cancer, lacks targetable oncogenes and is monolithically treated with standard combination chemotherapy. Interestingly, distinct subpopulations of neuroendocrine and non-neuroendocrine cells have been identified in mouse models of SCLC, but their existence in human SCLC remains unknown. Phenotypic heterogeneity, an important phenomenon in cellular reprogramming and therapeutic resistance, can arise from cell state transitions (i.e. network-level changes dynamically controlled by transcriptional regulators). The motivation for this study is to determine whether phenotypically distinct cell states exist in human SCLC, and investigate transcription factor (TF) dynamics that maintain these states. We implemented a mixed bioinformatics and experimental approach that defines inter-tumor heterogeneity in SCLC patients and cell lines as a spectrum of neuroendocrine (NE) and mesenchymal (MC) differentiation delineated by two anti-correlated gene co-expression networks. Features such as adhesion, surface markers and kinases effectively summarize heterogeneity in SCLC cell lines and patients as three distinct phenotypic states: NE, MC and intermediate. To characterize the transcriptional influence that governs these distinct phenotypic states, we constructed a TF regulatory network using the mutual information based method ARACNE. Boolean network model dynamics of the top 19 predicted TFs of the NE and MC networks gives rise to 3 distinct clusters of stable phenotypic states or ‘attractors’, each given by a unique TF network configuration. These TF network states were validated at both gene and protein level in SCLC cell lines and patients, identifying specific network states governing NE, MC and intermediate attractors. In silico TF perturbation experiments (single or combination TF activation/knockdown) performed to explore the possibility of state transitions, indicated that the NE state is more easily reprogrammable via single TF manipulations than the MC state, which required a combination of 3-4 simultaneous TF manipulations. SOX2, FOXA2 and OVOL2 were identified as master regulators of MC → NE state transitions while NOTCH1, MYC, SMAD3, and NFKB1 were master regulators of the NE → MC state transitions. Experimentally, phenotypic state transitions such as NE → Intermediate and MC → Intermediate were successfully induced using HDAC inhibitors, but not with demethylating agents or cisplatin. Etoposide treatment could also induce NE → Intermediate state transitions in NE cell lines but MC cell lines were resistant. Drug rebound experiments reveal that the transitioned cells fall back into NE/MC differentiated states upon removal of the drug. Thus, classification of human SCLC into 3 distinct phenotypic states - NE, MC and intermediate serves as a useful mapping tool for defining heterogeneity that could lead into personalized treatment strategies. Citation Format: Akshata R. Udyavar, Megan Hoeksema, David J. Wooten, Mukesh Bansal, Andrea Califano, Lourdes Estrada, Jonathan Irish, Pierre Massion, Vito Quaranta. Distinct transcriptional programs drive phenotypic heterogeneity in 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 3761. doi:10.1158/1538-7445.AM2015-3761

Abstract 4757: Whole exome sequencing reveals a distinct mutation pattern in metastatic small cell lung cancer compared to non-small cell lung cancer

Abstract 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

Abstract LB-295: Analysis of miR-21 isomiRs in lung cancer

Abstract microRNAs (miRNA) are small non-coding RNAs that target mRNA transcripts. Primarily, they bind to the 3′UTR of mRNA sequences and repress protein translation. They are involved in the regulation of over 60% of human genes. miRNAs are deregulated in cancer and expression signatures have been correlated with cancer diagnosis, progression and prognosis. Thus, they are being pursued for clinical application. isomiRs, or miRNA variants that are generated post-transcriptionally, were originally classified as experimental error in cloning and next generation sequencing studies. However, over the past five years it has become increasingly clear that isomiRs are evolutionarily conserved, prevalent and functional. miR-21 is overexpressed and associated with poor prognosis in multiple malignancies and as such it is currently under development as a therapeutic target. Thus, we examined the isomiR landscape of miR-21 in lung cancer using data from The Cancer Genome Atlas (lung adenocarcinoma n = 420 and squamous cell carcinoma n = 296). Raw sequence files were extracted from TCGA, cleaned and analyzed using STATA 13. We observed that there are significantly more isomiRs of miR-21 in both lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) compared to non-involved adjacent lung tissue. Specifically, LUAD had total of 244 5p variants and LUSC had 232 5p variants, compared with 74 and 105 in non-involved tissue, respectively. The most prevalent isomiR of miR-21 was not the canonical ‘miRBase’ isomiR and the most common edit was a 5′ deletion with 3′ addition. There were 15 miR-21-5p isomiRs in LUAD and 12 in LUSC that were prevalent (defined as detected in more than 50% of patients) and were expressed at a significantly higher level defined as detected with a FPKM value greater than 50) in tumor versus non-involved tissue samples. The average fold change was 5.7 in LUAD (range 2.8-21.1) and 2.7 in LUSC (range 1.7-5.6). Of the prevalent miR-21-5p isomiRs, 6 in LUAD and 4 in LUSC had a change in the canonical miR-21 seed sequence. An analysis of the targets of these isomiRs using the target prediction algorithms Targetscan and Diana revealed limited overlap in gene targets (0% to 54%) compared with canonical miR-21-5p targets. Our work highlights a high complexity of miRNA sequence heterogeneity in lung cancer. Combining the characterization of miR-21 isomiRs and pursuing translational laboratory studies will allow us to determine the functionality of these isomiRs and their relevance for human cancer diagnosis and treatment. Citation Format: Jennie D. Leikin, Adriana Zingone, Derek Brown, Khadijah Mitchell, Mohammed Khan, Oscar Vidal, Brid Ryan. Analysis of miR-21 isomiRs in 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 LB-295. doi:10.1158/1538-7445.AM2015-LB-295

Abstract 2982: Genome sequencing reveals the multicentric nature of multiple synchronous lung adenocarcinomas

Abstract Background: Genomic heterogeneity of lung cancers from different patients has been well documented, reflecting distinct genetic background and different carcinogen exposures. On the other hand, our recent study has demonstrated limited heterogeneity between different regions within the same tumor (Zhang, et al. Science, 2014: 346:256-9). Multiple synchronous lung cancer (MSLC) is a unique set of lung cancers with multiple tumors arising in the context of identical constitutional genetic background and shared carcinogen exposures. MSLC presents a clinical dilemma as to whether they are intrapulmonary metastasis or true multicentric primary tumors. Methods: We applied whole-genome/exome sequencing to 15 tumors plus one metastatic regional lymph node from six patients with multiple synchronous lung adenocarcinomas. All nonsynonymous mutations were subjected to mass spectrometry or Sanger sequencing for validation. Results: A total of 876 nonsynonymous mutations were detected and validated. With the exception of a metastatic lymph node that shared 52 mutations with the primary tumor suspected as its origin by clinicopathological assessment, all tumors, including one highly suspicious for intrapulmonary metastasis, showed distinct genomic profiles, indicating that all MSLC in this study are consistent with true multicentric primary lung adenocarcinomas. When compared to TCGA study, these synchronous multifocal tumors are no more similar than similarly staged lung adenocarcinomas from unrelated patients. Although similar mutational spectra were observed between different tumors from some patients, at least three patients showed significantly different mutational spectra in different tumors indicating multiple mutational processes in play during the development of individual, independent tumors within the same lung subjected to common exposures on the same constitutional genetic background. Interestingly, the same known cancer genes including EGFR, KRAS and STK11/LKB1 demonstrated distinct mutational patterns in different tumors from the same patients suggesting that even on the identical genetic background and common exposures, development of multicentric lung adenocarcinomas can be driven by distinct molecular events in different tumors, but there may be constraint around certain genes/pathways critical for carcinogenesis in specific patients. Conclusions: The dramatic genomic heterogeneity in multiple synchronous lung adenocarcinomas within the same patients with identical constitutional genetic background and environmental exposures indicates the complexity of lung adenocarcinoma carcinogenesis. These data suggest that detailed molecular profiling up to genome-scale analyses of all lesions may be necessary for MSLC to distinguishing multiple primary lung cancers from true intrapulmonary metastasis and accurately exploit biomarker-driven management. Citation Format: Jianjun Zhang, Yu Liu, Lin Li, Jianhua Zhang, Guangliang Yin, Dongmei Lin, Xiangyang Liu, Hannah Cheung, Sahil Seth, Xingzhi Song, Xizeng Mao, Jiexin Zhang, Shujun Cheng, Andrew Futreal, Yanning Gao. Genome sequencing reveals the multicentric nature of multiple synchronous lung adenocarcinomas. [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 2982. doi:10.1158/1538-7445.AM2015-2982

Abstract 4602: Exome sequencing of UKLS lung cancer CT screened early stage cancers

Abstract INTRODUCTION Developments in both low-dose CT-screening within populations at high risk of lung cancer and sequencing analysis of lung tumours have improved the prospects for early detection and clinical management of lung cancer. Such approaches potentially could provide biological insight into the early stages of lung cancer development, which until now have been refractory to genomic analysis, since lung tumours are rarely identified at a presymptomatic timepoint. BRIEF DESCRIPTION OF PERTINENT EXPERIMENTAL PROCEDURES Individuals with at least a 5% risk of developing lung cancer within 5 years (according to the LLPv2 lung cancer risk score) were recruited to the UK Lung low dose CT Screening (UKLS) trial. Ten of the early stage NSCLC UKLS tumours were prepared for exome sequencing. Tumour DNA was extracted from FFPE material and DNA was also extracted from matched blood samples. Tumour and blood libraries were prepared using an Agilent SureSelect exome-capture kit and sequenced to 100x and 50x, depth respectively, on the Illumina HiSeq platform (Oxford Gene Technology, UK). Sequence data were aligned against the GRCh37 human reference and matched samples were subject to local realignment in pairs. Somatic variants were identified using a combination of EBCall, MuTect and VarScan2 and were post-filtered to remove false positive variants (alignment artifacts and FFPE-hypersensitive sites) by comparison between samples. TCGA (The Cancer Genome Atlas) lung adenocarcinoma and squamous cell carcinoma samples from smokers and former smokers (who had complete data for a minimal set of clinical and epidemiological variables) were compared to the UKLS samples described above. To ensure comparable genomic regions were studied, both the UKLS and TCGA somatic variants were restricted to protein-coding regions covered by the Agilent SureSelect array but disjoint from simple-repetitive regions. SUMMARY OF THE NEW, UNPUBLISHED DATA Despite detection at an early stage, somatic mutations were identified in all CT-detected tumours, including non-synonymous variants in known driver genes. Between 71 and 471 variants were detected within non-repetitive protein-coding regions, which is comparable to, if somewhat lower than, the number of variants in the same regions in the TCGA ever-smoker samples (p = 0.17; Kruskal-Wallis). The results suggest that the majority of somatic variants exist in lung tumours prior to the evolution of symptoms and are consistent with recent studies of lung cancer heterogeneity wherein the majority of mutations occur prior to subclonal branching. STATEMENT OF CONCLUSIONS Somatic variations in the host genome are readily detected in FFPE material from pre-symptomatic lung tumours. Mutational burden is comparable to lung adenocarcinoma and squamous carcinoma from mature symptomatic individuals providing further evidence for the long latency of lung tumour development. Citation Format: Russell Hyde, Michael Davies, Martin Ledson, John A. Holemans, Richard D. Page, John Gosney, David R. Baldwin, Anand Devaraj, David M. Hansell, Stephen W. Duffy, John K. Field. Exome sequencing of UKLS lung cancer CT screened early stage cancers. [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 4602. doi:10.1158/1538-7445.AM2015-4602

Genomic heterogeneity of lung cancers and its potential clinical implications.

7530 Background: Substantial genomic heterogeneity of lung cancers between different patients, reflecting distinct genetic background and potentially different carcinogen exposures in different pat...

MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC).

We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation. Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation. Median MET H-score was 140 (range 0-400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25-50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively). MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation.

Non-Small Cell Lung Cancers in Women and Men: Differences in Biology, Behavior, and Outcomes.

Lung cancer is the leading cause of cancer deaths in men and women, both because of its high incidence rates and poor prognosis without effective therapies. Environmental carcinogens, most predominantly tobacco smoke, play a significant role. There are continuously emerging data to suggest the biological process differs between lung cancers in men and women. Differences are seen in a variety of cellular pathways and responses to carcinogens and therapies. Particular note in this article is made of carcinogen processing by cytochrome P450s, estrogen receptor pathways, epidermal growth factor receptor mutations, and how these are not necessarily independent cellular processes. These topics are only very briefly summarized here and it was not possible to include many important references. The heterogeneity of lung cancers in men and women, as well as smokers and nonsmokers, are likely to become more apparent with further studies. Work previously done in our laboratory (EDRN, PIs David Chia & Lee Goodglick) served to further emphasize these differences. This report is dedicated to the memory of Lee Goodglick with whom I had the privilege to work for many years prior to his untimely death.

Non-small cell lung cancer: HER2 oncogene status

to study HER2 protein and HER2 gene, their heterogeneity in non-small cell lung cancer. 218 intraoperative non-small cell lung samples were examined using tissue matrix methods. HER2 protein was determined by immunohistochemistry (clone 4B5, <<Ventana>> and HER2 gene and CEP1 7 were evaluated by in situ hybridization (SISH, <<Ventana>>). Positive and indefinite statuses were found in 59 (27%) and 47 (22%) cases, respectively; intratumor heterogeneity was detected in 32 (30%) cases. Amplification of the HER-2 gene was found in 12 (6%) cases; that of the HER2 gene along with an increase in CEPI 7 was observed in 7 (3%) cases; elevated CEP1 7 levels were seen in 19 (9%) cases. Intratumor heterogeneity of HER2 gene amplification was not found; however, one case of adenocarcinoma showed high-level HER2 gene amplification in the gland-like areas and low-level HER2 gene amplification in the solid areas. HER2-positive status and amplification were more common in adenocarcinoma than in squamous cell carcinoma (p<0.001). There was a moderate correlation between HER2 immunohistochemical status and amplification (r=0.38; p<0.001). Thus, in non-small cell lung cancer, there is an elevated HER2 protein level and, well less frequently, altered activity in the HER2 gene (amplification) as a cause of enhanced protein synthesis.

Abstract 983: Intratumor heterogeneity in non-small cell lung cancer inferred by multi-region exome sequencing

Abstract Lung cancer is the most common cancer worldwide, and a leading cause of cancer-related death. Despite improvements in molecular diagnosis and targeted therapies, the 5-years overall survival remains poor. To obtain a better insight into the genetic architecture of the most common type of lung cancer, non-small lung cancer (NSCLC), we performed multi-region DNA sequencing on 7 resected NSCLC samples. Ultra-deep sequencing of a comprehensive cancer gene panel and whole-exome sequencing revealed intratumor heterogeneity in all samples, with several putative tumor driver mutations present in some but not all regions of a tumor. Phylogenetic tree analyses based on non-synonymous mutations revealed a branched evolution pattern in all tumors. An adenosquamous tumor showed striking intratumor heterogeneity, with only a third of all non-synonymous mutations shared across all tumor regions, and clear separation of adenocarcinoma and squamous cell carcinoma tumor regions in the remaining two-third of the mutations. Our multi-region exome sequencing data also revealed regional differences in DNA copy number alterations. Some tumors, relatively homogeneous in terms of mutations, displayed high levels of intratumor heterogeneity in terms of DNA copy number changes, indicating the presence of distinct patterns of intratumor heterogeneity that might contribute to disease progression in different tumours. Overall, our multi-region deep exome sequencing data revealed intratumor heterogeneity in NSCLC, demonstrating branched evolution, both in terms of non-synoymous mutations and DNA copy number alterations, which has important implications for our understanding of the clonal evolution of NSCLC. Citation Format: Elza De Bruin, Nicholas McGranahan, Lucy Yates, Mariam Jamal-Hanjani, Max Salm, Richard Mitter, Seema Shafi, Nirupa Murugaesu, Andrew Rowan, Marco Gerlinger, David Wedge, Stuart Horswell, Ignacio Varela, Warren Tom, Chaitali Parikh, Timothy Harkins, Clarence Lee, Nik Matthews, Aengus Stewart, Peter Campbell, Charles Swanton. Intratumor heterogeneity in non-small cell lung cancer inferred by multi-region exome sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 983. doi:10.1158/1538-7445.AM2014-983

The Effect Of Respiratory Motion On Pet Imaging Of Tumor Heterogeneity In Non-small Cell Lung Cancer (NSCLC)

The Effect Of Respiratory Motion On Pet Imaging Of Tumor Heterogeneity In Non-small Cell Lung Cancer (NSCLC)

10: Intra-tumour heterogeneity in early-stage lung cancer inferred by multi-region sequencing

10: Intra-tumour heterogeneity in early-stage lung cancer inferred by multi-region sequencing

Tumor cell heterogeneity in Small Cell Lung Cancer (SCLC): phenotypical and functional differences associated with Epithelial-Mesenchymal Transition (EMT) and DNA methylation changes.

Small Cell Lung Cancer (SCLC) is a specific subtype of lung cancer presenting as highly metastatic disease with extremely poor prognosis. Despite responding initially well to chemo- or radiotherapy, SCLC almost invariably relapses and develops resistance to chemotherapy. This is suspected to be related to tumor cell subpopulations with different characteristics resembling stem cells. Epithelial-Mesenchymal Transition (EMT) is known to play a key role in metastatic processes and in developing drug resistance. This is also true for NSCLC, but there is very little information on EMT processes in SCLC so far. SCLC, in contrast to NSCLC cell lines, grow mainly in floating cell clusters and a minor part as adherent cells. We compared these morphologically different subpopulations of SCLC cell lines for EMT and epigenetic features, detecting significant differences in the adherent subpopulations with high levels of mesenchymal markers such as Vimentin and Fibronectin and very low levels of epithelial markers like E-cadherin and Zona Occludens 1. In addition, expression of EMT-related transcription factors such as Snail/Snai1, Slug/Snai2, and Zeb1, DNA methylation patterns of the EMT hallmark genes, functional responses like migration, invasion, matrix metalloproteases secretion, and resistance to chemotherapeutic drug treatment all differed significantly between the sublines. This phenotypic variability might reflect tumor cell heterogeneity and EMT during metastasis in vivo, accompanied by the development of refractory disease in relapse. We propose that epigenetic regulation plays a key role during phenotypical and functional changes in tumor cells and might therefore provide new treatment options for SCLC patients.

Abstract B27: Phenotypic plasticity and heterogeneity in small cell lung cancer (SCLC): Novel molecular subtypes and potential for targeted therapy.

Abstract Background: SCLC (15% of lung cancers) exhibits: 1) rapid growth and early fatal metastasis; 2) neuroendocrine features; 3) high initial responsiveness to chemotherapy and radiation; 4) aggressive recurrence with 5% 5-year patient survival. Gene expression and mutation profiling efforts to identify oncogenic mutations, gene amplifications or signatures with clinical utility in SCLC have thus far been unfruitful. In addition, prognostic or diagnostic markers for SCLC are scarce. Hence, there is a dire need for investigating molecular subtypes and oncogenic drivers in SCLC. We hypothesize that deregulated networks, rather than single genes, drive SCLC phenotype. Results: We previously identified a SCLC-specific gene co-expression network (Blue module, by Weighted Gene Co-expression Network Analysis - WGCNA) from a lung cancer patient dataset, and derived a SCLC-specific hub network (SSHN) signature that: 1) separated SCLC from other lung cancer types and normal lung in both genomic and proteomic independent datasets; 2) identified 2 SCLC subtypes with high and low SSHN expression in both patient specimens and cultured cell lines. Spleen tyrosine kinase (SYK) was validated as a candidate oncogenic driver of one subtype, as SYK targeted small-interfering RNA significantly decreased viability via increased death in high SYK-expressing SCLC cell lines. Due to the lack of larger SCLC patient datasets, we have now applied the SSHN classifier to the 53 SCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE) and validated the SSHN-high and low subtypes. From this larger dataset, it is evident that the SSHN-defined subtypes are not totally separate. Rather, they are connected by gradual intermediate shades. This gradation became clearer by applying WGCNA to SCLC cell lines from CCLE, which identified 2 gene co-expression modules – Blue and Turquoise, that overlap with modules from patient datasets described above. The Blue module is enriched in neuroendocrine signaling, the Turquoise in mesenchymal adhesion-related pathways. Eigengene expression of the 2 modules (MEblue, MEturquoise) is anti-correlated, and all 53 SCLC cell lines are distributed along this anti-correlation diagonal. Expression of the neuroendocrine marker CD56 is highest in cells at one end of this diagonal (MEblue-high cell lines), and decreases towards the other end (MEturquoise-high cell lines), whereas the mesenchymal marker CD44 has an opposite trend. Multi-dimensional flow cytometry data, visualized with viSNE, indicated that SCLC cell lines are heterogeneous with respect to several additional cell surface and cytoplasmic markers and that, in general, there is a gradient of expression of these markers that tends to correlate with the neuroendocrine (e.g., SYK) to mesenchymal (e.g., TGFbeta receptor II) phenotype gradient. Finally, at the neuroendocrine end of the phenotypic spectrum (MEblue-high) cells grow in suspension, whereas they become increasingly adherent towards the mesenchymal end (MEturquoise-high). Conclusion: Our data provide strong evidence for a heterogeneous phenotypic space in SCLC that may define distinct subtypes. This heterogeneity was previously unsuspected in human SCLC, although evidence for it was reported in genetic mouse models of SCLC {Calbo J, et.al, Cancer Cell, 2011}. Classification of human SCLC cell lines along a neuroendocrine to mesenchymal differentiation gradient should apply to human tumors as well, since the WGCNA network classifiers overlap. However, further studies in patients are warranted to prove the existence of distinct SCLC subtypes, as well as to probe their translational value for biomarkers and targeted treatment. Citation Format: Akshata Ramrao Udyavar, Megan Hoeksema, Kirsten Diggins, Jonathan Irish, Pierre P. Massion, Vito Quaranta. Phenotypic plasticity and heterogeneity in small cell lung cancer (SCLC): Novel molecular subtypes and potential for targeted therapy. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B27.

Genes and Pathology of Non-Small Cell Lung Carcinoma

While histopathology has traditionally been the cornerstone of treatment decisions in the management of lung cancer patients, the complexity and heterogeneity of histological classification has had a limited impact in the routine practice of oncology. This has changed dramatically in the last few years, owing to discoveries of genomic aberrations and results of clinical trials of novel and targeted therapies. These discoveries have resulted in a new way of classifying non-small cell lung cancer (NSCLC), based on the occurrence of putative or proven driver and targetable genomic changes. The rapidity by which the landscape of mutation and genomic changes is being identified also has led to a new paradigm and approaches to pathological diagnosis of NSCLC. In this context, international consortia have proposed new classifications of lung adenocarcinoma and guidelines for molecular testing in lung cancer and have provided concrete recommendations on new ways to practice lung cancer pathology.

EMT markers in lung adenocarcinoma pleural effusion spheroid cells

Epithelial-to-mesenchymal transition (EMT) is a process in which cells undergo a developmental switch from epithelial to mesenchymal phenotype. This process has been related to embryologic morphogenesis but also to cancer progression and metastasis. The aim of the current study was to investigate the expression of EMT-related markers in adherent and spheroid cell cultures derived from malignant pleural effusions (MPEs) of patients affected by lung adenocarcinoma. On the basis of efficient in vitro propagation, six cases of MPEs were selected and analyzed by immunocytochemistry staining for EMT markers and by RT-PCR for transcription factors known to orchestrate EMT. EMT markers immunostaining showed in spheroids a statistically significant correlation between the loss of E-cadherin immunoreactivity and overexpression of N-cadherin (P < 0.001). Likewise loss of EpCAM epithelial marker was coincident with Vimentin overexpression (P < 0.001). RT-PCR analysis of transcription factors Snail, Slug, and Twist showed a highly variable expression, although a general trend to increase was observed. Importantly, in some selected cases it was possible to establish a precise relationship between spheroid formation, EMT switch and increased upregulation of the marker related to cancer stemness such as ALDH positivity. Therefore, MPE-derived cell cultures, while recapitulating the heterogeneity of lung cancer, are a suitable system to study the mechanisms at the basis of EMT and to understand its relationship with the generation of cancer stem cells.

Update on Recent Prognostic Markers in Adenocarcinoma

SUMMARY Biomarkers have evolved from individual markers to gene signatures validated in multi-institutional studies and independent datasets. Multiple gene signatures have been associated with survival, as well as defining outcome following specific adjuvant chemotherapy. The heterogeneity of lung cancer has necessitated the incorporation of multiple genes in mRNA signatures. The use of plasma miRNA profiles in the diagnosis or prognosis of early-stage lung carcinoma also appears promising. However, challenges remain, with questions regarding reproducibility, standardized techniques, cost and differences between gene signatures. Before they can be widely adopted, molecular profiles must be validated in large, prospective trials evaluating the specific patient population for which the profiles are to be utilized.