Multi-region Exome Sequencing Research Articles

Multiregion exome sequencing indicates a monoclonal origin of esophageal spindle-cell squamous cell carcinoma.

Esophageal spindle-cell squamous cell carcinoma (ESS) is a rare biphasic neoplasm composed of a carcinomatous component (CaC) and a sarcomatous component (SaC). However, the genomic origin and gene signature of ESS remain unclear. Using whole-exome sequencing of laser-capture microdissection (LCM) tumor samples, we determined that CaC and SaC showed high mutational commonality, with the same top high-frequency mutant genes, mutation signatures, and tumor mutation burden; paired samples shared a median of 25.5% mutation sites. Focal gains were found on chromosomes 3q29, 5p15.33, and 11q13.3. Altered genes were mainly enriched in the RTK-RAS signaling pathway. Phylogenetic trees showed a monoclonal origin of ESS. The most frequently mutated oncogene in the trunk was TP53, followed by NFE2L2, KMT2D, and MUC16. Prognostic associations were found for CDC27, LRP2, APC, and SNAPC4. Our data highlight the monoclonal origin of ESS with TP53 as a potent driver oncogene, suggesting new targeted therapies and immunotherapies as treatment options. © 2024 The Pathological Society of Great Britain and Ireland.

Mapping single-cell transcriptomes in the intra-tumoral and associated territories of kidney cancer.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8+ Tcell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target.

Genomic landscapes of canine splenic angiosarcoma (hemangiosarcoma) contain extensive heterogeneity within and between patients.

Cancer genomic heterogeneity presents significant challenges for understanding oncogenic processes and for cancer’s clinical management. Variation in driver mutation frequency between patients with the same tumor type as well as within an individual patients’ cancer can shape the use of mutations as diagnostic, prognostic, and predictive biomarkers. We have characterized genomic heterogeneity between and within canine splenic hemangiosarcoma (HSA), a common naturally occurring cancer in pet dogs that is similar to human angiosarcoma (AS). HSA is a clinically, physiologically, and genomically complex canine cancer that may serve as a valuable model for understanding the origin and clinical impact of cancer heterogeneity. We conducted a prospective collection of 52 splenic masses from 43 dogs (27 HSA, 15 benign masses, and 1 stromal sarcoma) presenting for emergency care with hemoperitoneum secondary to a ruptured splenic mass. Multi-platform genomic analysis included matched tumor/normal targeted sequencing panel and exome sequencing. We found candidate somatic cancer driver mutations in 14/27 (52%) HSAs. Among recurrent candidate driver mutations, TP53 was most commonly mutated (30%) followed by PIK3CA (15%), AKT1 (11%), and CDKN2AIP (11%). We also identified significant intratumoral genomic heterogeneity, consistent with a branched evolution model, through multi-region exome sequencing of three distinct tumor regions from selected primary splenic tumors. These data provide new perspectives on the genomic landscape of this veterinary cancer and suggest a cross-species value for using HSA in pet dogs as a naturally occurring model of intratumoral heterogeneity.

Abstract 2144: Holistic sampling of clonal dynamics using cfDNA in lung TRACERx

Abstract Background: Minimal residual disease (MRD) detection using liquid biopsy has the potential to improve patient outcomes in non-small cell lung cancer (NSCLC). Liquid biopsy may also provide representative clonal sampling through the disease course, but current clonal deconvolution methods are ineffective in plasma samples with <1% tumor content, common in the localised or MRD setting. Methods: We analysed 1071 plasma samples from 198 TNM I-III NSCLC patients in TRACERx who underwent multiregion exome sequencing of primary tumor and relapse tissue, with 416 standard of care surveillance scans. Seventy-four patients suffered a relapse. We targeted a median of 200 tumor-specific mutations per patient consisting of clonal and subclonal variants and sequenced cell-free DNA (cfDNA) using anchored multiplex PCR to a median unique depth of 2230X with 5 supporting duplicates. We used library-specific trinucleotide background models to call MRD. We developed ECLIPSE (Extraction of CLonality from LIquid bioPSiEs), to perform formal clonal deconvolution in <1% purity plasma samples by leveraging copy number and mutation clone identities from tumor tissue. Results: Median MRD lead time was 119 days (range 0-1137) in patients with pre-operative circulating tumor DNA (ctDNA) detection. In the 13% of relapse patients lacking pre-operative ctDNA detection the median MRD lead time was 0 days (range 0-589). MRD lead times positively correlated with clonal ctDNA fraction doubling times (DTs). Cancer cell fractions (CCFs) of subclones estimated in plasma with ECLIPSE and tissue collected concurrently were proportional (P < 0.001, R2 = 0.6). Subclonal mutations that would appear clonal in single biopsies could be separated from true clonal mutations using their CCF in plasma (P < 0.001, OR = 0.44), distinguishing clonal from subclonal mutations, where the latter are unlikely to represent effective therapeutic targets. Where cfDNA and tissue was available at relapse we detected 28/29 metastatic tissue subclones in cfDNA with an additional 8 cfDNA-unique subclones. These subclones were more frequently estimated as present in only a subset of metastatic cells using cfDNA (P = 0.008, OR = 5.5) consistent with localisation to unsampled metastatic sites. Metastatic competent subclones had higher CCFs in pre-operative plasma (P < 0.001, OR = 4.5). Shifts in clonal dynamics were concurrent with treatment. Finally, patients with cfDNA-detected polyphyletic metastatic seeding had shorter disease-free survival than those with monophyletic seeding (HR = 2.89, 95% CIs 1.46-5.73). Conclusions: Tumor-informed anchored multiplex PCR most commonly detected MRD before clinical relapse and allowed determination of clonal ctDNA DT. Using ECLIPSE, plasma samples of <1% purity allow formal measurements of clonal dynamics from diagnosis to relapse, which impacts patient outcome and has the potential to guide personalised medicine. Citation Format: Alexander Mark Frankell, Christopher Abbosh, Aaron Garnett, Judit Kisistok, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Aamir Shahpurwalla, Aaron Odell, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel E. Cook, Clare Puttick, Maise Al-Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Paula Roberts, Ariana Huebner, Nicolas Carey, Joan Riley, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Josh Stahl, Nicolai Birkbak, the Lung TRACERx consortium, Charles Swanton. Holistic sampling of clonal dynamics using cfDNA in lung TRACERx [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 2144.

Abstract PR008: Immunosuppressive niche engineering at the onset of human colorectal cancer

Abstract The evolutionary dynamics of tumor initiation remain undetermined, and the interplay between neoplastic cells and the immune system is hypothesized to be critical in transformation. Colorectal cancer (CRC) presents a unique opportunity to study the transition to malignancy as pre-cancers (adenomas) and early-stage cancers are frequently detected and surgically removed. Here, we demonstrate a key role for the immune response in tumor initiation by studying tumor-immune eco-evolutionary dynamics from pre-cancer to carcinoma using a computational model, ecological analysis of digital pathology data, and multi-region exome sequencing and neoantigen prediction in a total of 62 patient samples. Modelling indicates there are several potential routes to malignancy, each of which uniquely sculpts tumor ecology and intra-tumor antigenic heterogeneity (aITH). In patient samples, the immune microenvironment was characterized using the spatial distribution of 17 markers across registered whole-slide images, as well as patterns of intra-lesion aITH measured using multi-region exome sequencing and neoantigen prediction. The patient data were best described by a model where adenomas that become immunogenic early on do not progress to CRC because they are under immune control; progression therefore proceeds in adenomas with low immunogenicity. In these tumors, immune suppression is initially low, but gradually an immunosuppressive niche that is depleted in CD8+ cytotoxic T cells expands. There was little evidence for immune blockade (PD-L1 expression) in tumor initiation or progression. These results suggest that re-engineering the immunosuppressive niche may prove to be an effective immunotherapy in CRC. Citation Format: Chandler D. Gatenbee, Ann-Marie Baker, Ryan O. Schenck, Maximilian Strobl, Jeffrey West, Mark Robertson-Tessi, Trevor A. Graham, Alexander R.A. Anderson. Immunosuppressive niche engineering at the onset of human colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR008.

Multi-region exome sequencing reveals the intratumoral heterogeneity of surgically resected small cell lung cancer

Small cell lung cancer (SCLC) is a highly malignant tumor which is eventually refractory to any treatment. Intratumoral heterogeneity (ITH) may contribute to treatment failure. However, the extent of ITH in SCLC is still largely unknown. Here, we subject 120 tumor samples from 40 stage I-III SCLC patients to multi-regional whole-exome sequencing. The most common mutant genes are TP53 (88%) and RB1 (72%). We observe a medium level of mutational heterogeneity (0.30, range 0.0~0.98) and tumor mutational burden (TMB, 10.2 mutations/Mb, range 1.1~51.7). Our SCLC samples also exhibit somatic copy number variation (CNV) across all patients, with an average CNV ITH of 0.49 (range 0.02~0.99). In terms of mutation distribution, ITH, TMB, mutation clusters, and gene signatures, patients with combined SCLC behave roughly the same way as patients with pure SCLC. This condition also exists in smoking patients and patients with EGFR mutations. A higher TMB per cluster is associated with better disease-free survival while single-nucleotide variant ITH is linked to worse overall survival, and therefore these features may be used as prognostic biomarkers for SCLC. Together, these findings demonstrate the intratumoral genetic heterogeneity of surgically resected SCLC and provide insights into resistance to treatment.

FP10.03 Multi-Region Exome Sequencing Reveals the Intratumoral Heterogeneity of Surgically Resected Small Cell Lung Cancer

FP10.03 Multi-Region Exome Sequencing Reveals the Intratumoral Heterogeneity of Surgically Resected Small Cell Lung Cancer

Abstract CT023: Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study

Abstract Introduction Minimal residual disease (MRD) detection in solid tumors describes isolation of circulating tumor DNA (ctDNA) molecules in plasma following definitive treatment of a cancer. Detection of MRD following surgical tumor excision categorizes patients as high risk for disease recurrence. Establishing an MRD approach to treating early-stage NSCLC will facilitate escalation of standard of care (SoC) treatment only in patients destined to relapse from their cancer and overcome challenges associated with conventional adjuvant drug-trial design. Here, we present data from the lung TRACERx study where patients with early-stage NSCLC underwent phylogenetic ctDNA profiling following resection. Methods Patient specific anchored-multiplex PCR (AMP) enrichment panels were generated for 78 lung TRACERx patients who underwent surgery for stage I-III NSCLC; 608 plasma samples were analyzed. Extensive patient-specific cfDNA enrichment panels targeted a median of 196 (range 72 to 482) clonal and subclonal variants detected in primary tumor tissue by multi-region exome sequencing. A novel MRD-caller controlled and estimated background sequencing error to maximize ctDNA detection at low mutant allele frequencies (MAFs). Analytical validation experiments benchmarked assay performance. Results Analytical validation of a 50-variant AMP-MRD assay demonstrated a sensitivity of 89% for mutant DNA at a MAF of 0.008% (with 25ng of DNA input into the assay), specificity was 100% experimentally and 99.9% (95% CI: 99.67 to 99.99%) modelled in-silico. 45 patients suffered relapse of their primary NSCLC; ctDNA was detected at or before clinical relapse in 37 of 45 patients. In these 37 patients the median ctDNA lead-time (time from ctDNA detection to clinical relapse) was 151 days (range 0 to 984 days) and the median time to relapse from surgery was 413 days (range 41 to 1242 days). In 10 of 10 patients who developed second primary cancers during follow-up no ctDNA was detected, reflecting specificity of the MRD assay toward the primary tumor. In 23 patients who remained relapse-free during a median of 1184 days of study follow-up, ctDNA was detected in 1 of 199 time-points analyzed. Analysis of SoC adjuvant surveillance imaging (CT, PET-CT or MRI, 220 encounters) revealed examples of MRD positive patients where SoC radiological surveillance was negative for impending relapse. Through application of large cfDNA enrichment panels targeting up to 483 variants per patient we observed dynamic changes in clonal composition and copy-number status prior to NSCLC relapse, categorized relapse as monoclonal or polyclonal and identified distinct subclonal dynamics during systemic intervention for disease recurrence. Conclusions ctDNA is an adjuvant biomarker capable of both detecting MRD following surgery and defining the clonality of relapsing disease. These data pave the way for clinical trials predicated on escalation of adjuvant standard of care in NSCLC patients who exhibit MRD positive status following surgery. Citation Format: Chris Abbosh, Alexander Frankell, Aaron Garnett, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel Cooke, Clare Puttick, Maise Al Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Ariana Huebner, Nicolas Carey, Joan Riley, Paula Roberts, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Nicolai Birkbak, Josh Stahl, Charles Swanton, Lung TRACERx consortium. Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study [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 CT023.

Extreme intratumour heterogeneity and driver evolution in mismatch repair deficient gastro-oesophageal cancer

Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.

Multiregion exome sequencing of ovarian immature teratomas reveals 2N near-diploid genomes, paucity of somatic mutations, and extensive allelic imbalances shared across mature, immature, and disseminated components.

Immature teratoma is a subtype of malignant germ cell tumor of the ovary that occurs most commonly in the first three decades of life, frequently with bilateral ovarian disease. Despite being the second most common malignant germ cell tumor of the ovary, little is known about its genetic underpinnings. Here we performed multi-region whole exome sequencing to interrogate the genetic zygosity, clonal relationship, DNA copy number, and mutational status of 52 pathologically distinct tumor components from 10 females with ovarian immature teratomas, with bilateral tumors present in 5 cases and peritoneal dissemination in 7 cases. We found that ovarian immature teratomas are genetically characterized by 2N near-diploid genomes with extensive loss of heterozygosity and an absence of genes harboring recurrent somatic mutations or known oncogenic variants. All components within a single ovarian tumor (immature teratoma, mature teratoma with different histologic patterns of differentiation, and yolk sac tumor) were found to harbor an identical pattern of loss of heterozygosity across the genome, indicating a shared clonal origin. In contrast, the 4 analyzed bilateral teratomas showed distinct patterns of zygosity changes in the right versus left sided tumors, indicating independent clonal origins. All disseminated teratoma components within the peritoneum (including gliomatosis peritonei) shared a clonal pattern of loss of heterozygosity with either the right or left primary ovarian tumor. The observed genomic loss of heterozygosity patterns indicate that diverse meiotic errors contribute to the formation of ovarian immature teratomas, with 11 out of the 15 genetically distinct clones determined to result from nondisjunction errors during meiosis I or II. Overall, these findings suggest that copy-neutral loss of heterozygosity resulting from meiotic abnormalities may be sufficient to generate ovarian immature teratomas from germ cells.

Multi-region exome sequencing reveals genomic evolution from preneoplasia to lung adenocarcinoma

There has been a dramatic increase in the detection of lung nodules, many of which are preneoplasia atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or invasive adenocarcinoma (ADC). The molecular landscape and the evolutionary trajectory of lung preneoplasia have not been well defined. Here, we perform multi-region exome sequencing of 116 resected lung nodules including AAH (n = 22), AIS (n = 27), MIA (n = 54) and synchronous ADC (n = 13). Comparing AAH to AIS, MIA and ADC, we observe progressive genomic evolution at the single nucleotide level and demarcated evolution at the chromosomal level supporting the early lung carcinogenesis model from AAH to AIS, MIA and ADC. Subclonal analyses reveal a higher proportion of clonal mutations in AIS/MIA/ADC than AAH suggesting neoplastic transformation of lung preneoplasia is predominantly associated with a selective sweep of unfit subclones. Analysis of multifocal pulmonary nodules from the same patients reveal evidence of convergent evolution.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

P2.02-013 Investigation of Genomic and TCR Repertoire Evolution of AAH, AIS, MIA to Invasive Lung Adenocarcinoma by Multiregion Exome and TCR Sequencing

P2.02-013 Investigation of Genomic and TCR Repertoire Evolution of AAH, AIS, MIA to Invasive Lung Adenocarcinoma by Multiregion Exome and TCR Sequencing

Tu1833 - Multi-Region Exome Sequencing Reveals the Clonal Evolution of Colitis-Associated Colorectal Cancer

Tu1833 - Multi-Region Exome Sequencing Reveals the Clonal Evolution of Colitis-Associated Colorectal Cancer

In the literature: March 2016

The way clones resistant to anti-EGFR (Epidermal Growth Factor Receptor) inhibition evolve in non-small cell lung cancer is far from being determined. In an international cooperative effort led by investigators at Johns Hopkins Hospital in Baltimore, the origin of acquired resistance mediated by the EGFRT790M mutation has been further elucidated. That mutation, recognised as gatekeeper and present in about 60% of pretreated cases with anti-EGFRs, could occur either from the selection of previously existing EGFR790M cell clones or by the evolution of originally EGFR790M-negative drug-tolerant cells. In a series of elegant experiments, the authors show that these drug-tolerant cells have a diminished apoptotic response to third generation EFFR inhibitors specifically tackling EGFR790M. Moreover, the use of potent inhibitors of the antiapoptotic factors BCL2 and BCL-xL, such as navitoclax, was able to restore sensitivity. Thus, drug-tolerant cells that are capable of surviving initial drug therapy may provide a reservoir of cells from which genetic mechanisms of acquired resistance can evolve. These …

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

Intratumoral heterogeneity in kidney cancer

Clear cell renal cell carcinoma (CCRCC) is characterized by mutation of the VHL gene and loss of a segment of chromosome 3. A new study using multi-region exome sequencing has identified substantial intratumoral heterogeneity within large primary CCRCCs, which has profound implications for understanding tumor evolution and for developing effective therapies.

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73-75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development.

Abstract 4603: Intratumor heterogeneity in clear cell renal cell carcinoma (ccRCC): Multi-region sequencing redefines the mutational landscape of ccRCCs.

Abstract Background: Current efforts to define the genomic landscapes of solid tumours are based on the analysis of single tumour biopsies. We recently demonstrated through multi-region exome sequencing (M-seq) of 2 ccRCC that these cancers are genetically heterogeneous (Gerlinger et al. NEJM, 2012). Thus, single biopsy approaches are likely to underestimate the genetic complexity and the incidence of driver gene mutations. Methods: We applied M-seq and copy number profiling to an average of 7 regions from each of 7 advanced ccRCCs to reveal the genomic landscapes and to reconstructed the life histories of these tumours through phylogenetic analysis. Results: M-seq identified approximately 100 non-synonymous somatic mutations per case on average; twice as many mutations than revealed on average in a single biopsy or the 54 mutation found on average in single biopsies of metastatic ccRCCs analysed by The Cancer Genome Atlas. M-seq revealed that all tumours were heterogeneous with more than 50% of non-synonymous somatic mutations and copy number aberrations not present across all analysed tumour regions. The mutational spectrum and chromosomal instability scores differed between regions of individual tumours. Phylogenetic reconstruction showed branched evolution in all cases. Loss of chromosome 3p and mutations in the VHL and PBRM1 genes were the only ccRCC drivers which were altered ubiquitously throughout all regions in all tumours where they occurred. These were the only known ccRCC driver mutations which were recurrently located on the trunk of the phylogenetic trees, indicating that these truncal genetic events are sufficient for tumour initiation. All other known ccRCC driver mutations and the vast majority of chromosomal driver aberrations were confined to spatially separated subclones, represented as branches on the phylogenetic trees. Thus, genetic alterations of VHL, PBRM1 and chromosome 3p appear to initiate the generation of genetic diversity and the acquisition of additional driver events promoting expansion of spatially separated subclones. The truncal driver mutations were similar across all tumours but clinical outcomes differed significantly between patients, indicating that subclonal drivers may determine clinical outcomes. Conclusions: M-seq revealed intra-tumour heterogeneity and parallel evolution of multiple subclones in each of 7 metastatic tumors, indicating that these are common characteristic of ccRCCs. VHL, PBRM1 and chromosome 3p were the only recurrent truncal driver events identified. All other driver mutations were heterogeneous and these are likely to influence clinical outcome. Thus, M-seq reveals complex heterogeneous genomic landscapes in ccRCCs and demonstrates that the detection of subclonal driver mutations may be highly relevant for personalized cancer medicine approaches. Citation Format: Marco Gerlinger, Stuart Horswell, James Larkin, Andrew J. Rowan, Pierre Martinez, Ignacio Varela, Claudio R. Santos, Rosalie Fisher, Max P. Salm, Zoltan Szallasi, Nicholas Matthews, Aengus Stewart, P Andrew Futreal, Charles Swanton. Intratumor heterogeneity in clear cell renal cell carcinoma (ccRCC): Multi-region sequencing redefines the mutational landscape of ccRCCs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4603. doi:10.1158/1538-7445.AM2013-4603

Abstract 964: Intra-tumor heterogeneity and Darwinian selection revealed by multi-region exome sequencing of renal cell carcinomas

Abstract Background: Genetic intra-tumor heterogeneity (ITH) may foster tumor adaptation by providing selectable phenotypes for Darwinian evolution. Extensive genetic heterogeneity may also hinder personalized medicine strategies that rely on the portrayal of the mutational landscape from single tumor biopsies. Methods: To examine ITH, we have subjected multiple biopsies from primary renal cell carcinomas and associated metastatic sites to exome capture sequencing (n=2), SNP-array based chromosomal aberration and ploidy profiling analysis (n=4). Phylogenetic relationships of tumor regions were reconstructed by clonal ordering of non-synonymous somatic mutations. The phenotypic consequences of genetic ITH were characterised by immunohistochemistry, mutation functional analysis and mRNA expression profiling. Results: Phylogenetic reconstruction revealed branched evolutionary tumor growth with 63-69% of somatic mutations identified from single biopsies not detectable across all sequenced tumor regions. Independent tumor suppressor gene loss of function mutations with distinct regional distributions were detected within individual tumors: SETD2 harbored 5 different mutations in 2 tumors and PTEN and KDM5C two different mutations in one tumor, each. Thus, despite genetic divergence during tumor progression, phenotypic convergent evolution occurred, indicating a high degree of early mutational diversity. ITH was observed for a mutation in the kinase domain of mTOR, correlating with S6 and 4EBP phosphorylation specifically in cancer regions carrying the mutation and constitutive activation of mTOR kinase activity in vitro. Expression of a renal cancer-specific prognostic signature differed between tumor regions. Chromosomal aberration analysis revealed extensive ITH with 26/30 tumor biopsies from four tumors harboring divergent allelic imbalance profiles. Ploidy profiling revealed heterogeneity in two out of four tumors and identified an aneuploid tumor cell population in a metastasis that probably evolved from a tetraploid intermediate detectable in the primary tumor. Conclusions: Genetic ITH was present in all tumors and occurs through spatially separated heterogeneous somatic mutations and chromosomal and ploidy aberrations leading to both, phenotypic intra-tumor diversity (mTOR activating mutation) and convergent loss of function(SETD2, PTEN and KDM5C). ITH can lead to underestimation of the tumor genomic landscape portrayed from single biopsies and may present significant challenges to personalized medicine and biomarker development. ITH, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. Targeting mutations commonly located on the trunks, rather than the branches of phylogenetic trees, may improve therapeutic outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 964. doi:1538-7445.AM2012-964