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Supplemental Figure S1. Number of predicted neoantigens. Supplemental Figure S2. Association between expressed branch predicted neoantigens and relapse. Supplemental Figure S3. Substantial ITH in the number of unique T cell rearrangements. Supplemental Figure S4. Substantial ITH in tumor-enriched T cell repertoire in 8 patients whose blood samples were available. Supplemental Figure S5. A minority of T cell clones are shared between different regions within a tumor. Supplemental Figure S6. The top 5 clones within each tumor are conserved across all regions of that given tumor. Supplemental Figure S7. Tumor-enriched MOI in patients with available paired blood. Supplemental Figure S8. CD4-dominant T cell infiltrate in localized lung adenocarcinomas by immunohistochemical (IHC) staining. Supplemental Figure S9. Correlation between predicted neoantigen burden and TCR metrics. Supplemental Figure S10. Association between intratumor TCR heterogeneity and patient relapse. Supplemental Figure S11. Follow up time (months) in patients who recurred and those who have not. n.s., not significant.

Table S1. Patient demographics and characteristics.

Supplemental Figure Legends

BackgroundLung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy.MethodsIn order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a young female never-smoker with late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T-cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors.ResultsWe observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases including a set of prevalent T cell clonotypes which were completely excluded from left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression.ConclusionsOur study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.

Background Lung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy. Methods In order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a 32-year-old female never-smoker with left lung primary late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors. Results We observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases. Only TP53 mutation was detected in all tumors suggesting an early event while other cancer gene mutations were later events which may have followed subclonal diversification. A set of prevalent T cell clonotypes were completely excluded from left-side thoracic tumors indicating distinct T cell repertoire profiles between left-side and non left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression. Conclusions Our study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.

Recent studies have revealed that cancer patients had a higher risk of having coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), compared to patients without cancer. The expression of angiotensin-converting enzyme 2 (ACE2), the receptor of SARS-CoV-2, was aberrantly expressed in many tumors. In this study, by exploring the TCGA and GTEx public databases, we investigated ACE2 expression and its association with prognosis in non-small cell lung cancer (NSCLC), the most susceptible caner type. We found that lung was one of the major organs with highly expressed ACE2. Furthermore, ACE2 expression was significantly elevated in lung adenocarcioma (LUAD) and lung squamous cell carcinoma (LUSC) compared to normal tissues. DNA methylation might be one possible mechanism leading to ACE2 upregulation. Despite that, the AEC2 expression was not statistically associated with disease-free survival (DFS) and overall survival (OS) for LUAD patients, and higher ACE2 expression was associated with prolonged DFS in LUSC patients. Taken together, we observed ACE2 was highly expressed in LUAD and LUSC despite the controversial role of ACE2 expression in predicting prognosis in these two common lung cancer types.

Background & Aims Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

Recurrent oncogenic fusion genes play a critical role in the development of various cancers and diseases and provide, in some cases, excellent therapeutic targets. To date, analysis tools that can identify and compare recurrent fusion genes across multiple samples have not been available to researchers. To address this deficiency, we developed Co-occurrence Fusion (Co-fuse), a new and easy to use software tool that enables biologists to merge RNA-seq information, allowing them to identify recurrent fusion genes, without the need for exhaustive data processing. Notably, Co-fuse is based on pattern mining and statistical analysis which enables the identification of hidden patterns of recurrent fusion genes. In this report, we show that Co-fuse can be used to identify 2 distinct groups within a set of 49 leukemic cell lines based on their recurrent fusion genes: a multiple myeloma (MM) samples-enriched cluster and an acute myeloid leukemia (AML) samples-enriched cluster. Our experimental results further demonstrate that Co-fuse can identify known driver fusion genes (e.g., IGH-MYC, IGH-WHSC1) in MM, when compared to AML samples, indicating the potential of Co-fuse to aid the discovery of yet unknown driver fusion genes through cohort comparisons. Additionally, using a 272 primary glioma sample RNA-seq dataset, Co-fuse was able to validate recurrent fusion genes, further demonstrating the power of this analysis tool to identify recurrent fusion genes. Taken together, Co-fuse is a powerful new analysis tool that can be readily applied to large RNA-seq datasets, and may lead to the discovery of new disease subgroups and potentially new driver genes, for which, targeted therapies could be developed. The Co-fuse R source code is publicly available at https://github.com/sakrapee/co-fuse .

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity. Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088–97. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

EGFR-mutant non-small-cell lung cancer (NSCLC) patients inevitably develop drug resistance when treated with EGFR tyrosine kinase inhibitors (TKIs). Systematic genetic analysis is important to understand drug-resistant mechanisms; however, the clinical significance of co-occurring genetic alterations at baseline, co-acquired mutations at progressive disease (PD), and the clonal evolution remain underinvestigated. We performed targeted sequencing of pre-treatment and PD tumor samples from 54 EGFR-mutant NSCLC patients. Ten additional patients were sequenced using whole-exome sequencing to infer the clonal evolution patterns. We observed a domain-dependent effect of PIK3CA mutation at baseline on patient progression-free survival (PFS). In addition, at baseline, 9q34.3/19p13.3 (NOTCH1/STK11/GNA11) showed a co-deletion pattern, which was associated with a significantly worse PFS (p = 0.00079). T790M-postive patients with other concurrent acquired oncogenic mutations had a significantly shorter PFS (p = 0.005). Besides acquired T790M mutation, chromosomal instability (CIN) related genes, including AURKA and TP53 alterations, were the most frequently acquired events. CIN significantly increased during TKI treatment in T790M-negative patients and is a candidate resistance mechanism to the first-generation TKIs. Clonal evolution analyses suggest that the composition and relationship among resistant subclones, particularly relationship with T790M subclone, affect patients’ outcomes. Overall, our findings of novel co-occurring alterations and clonal evolution patterns can be served as predictive biomarkers to stratify patients and help to better understand the drug-resistant mechanism to TKIs.

Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy., Relevant features of T cell repertoire in human cancer remain to be delineated. Here the authors show, by TCR sequencing in a large cohort of lung cancer patients, that while a majority of T cell clones are shared between tumor and adjacent lung tissue, less frequent tumor-unique T cell clones correlate with worse prognosis.

// Kelly Quek 1, 2, * , Jun Li 2, * , Marcos Estecio 3 , Jiexin Zhang 4 , Junya Fujimoto 5 , Emily Roarty 2 , Latasha Little 2 , Chi-Wan Chow 5 , Xingzhi Song 2 , Carmen Behrens 5 , Taiping Chen 4 , William N. William 1 , Stephen Swisher 6 , John Heymach 1, 7 , Ignacio Wistuba 5 , Jianhua Zhang 2 , Andrew Futreal 2 , Jianjun Zhang 1, 2 1 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Genomic Medicine, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 3 Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 4 Department of Bioinformatics and Computational Biology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 5 Department of Translational Molecular Pathology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 6 Department of Thoracic Surgery, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 7 Department of Cancer Biology, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA * Co-first authors Correspondence to: Jianjun Zhang, email: JZhang20@mdanderson.org Andrew Futreal, email: AFutreal@mdanderson.org Jianhua Zhang, email: JZhang22@mdanderson.org Keywords: intra-tumor heterogeneity, non-small cell lung cancer, DNA methylation Received: November 30, 2016 Accepted: January 27, 2017 Published: February 28, 2017 ABSTRACT Cancers are composed of cells with distinct molecular and phenotypic features within a given tumor, a phenomenon termed intratumor heterogeneity (ITH). Previously, we have demonstrated genomic ITH in localized lung adenocarcinomas; however, the nature of methylation ITH in lung cancers has not been well investigated. In this study, we generated methylation profiles of 48 spatially separated tumor regions from 11 localized lung adenocarcinomas and their matched normal lung tissues using Illumina Infinium Human Methylation 450K BeadChip array. We observed methylation ITH within the same tumors, but to a much less extent compared to inter-individual heterogeneity. On average, 25% of all differentially methylated probes compared to matched normal lung tissues were shared by all regions from the same tumors. This is in contrast to somatic mutations, of which approximately 77% were shared events amongst all regions of individual tumors, suggesting that while the majority of somatic mutations were early clonal events, the tumor-specific DNA methylation might be associated with later branched evolution of these 11 tumors. Furthermore, our data showed that a higher extent of DNA methylation ITH was associated with larger tumor size (average Euclidean distance of 35.64 (> 3cm, median size) versus 27.24 (

SUMMARY Chromatin interactions play important roles in transcription regulation. To better understand the underlying evolutionary and functional constraints of these interactions, we implemented a systems approach to examine RNA polymerase-II-associated chromatin interactions in human cells. We found that 40% of the total genomic elements involved in chromatin interactions converged to a giant, scale-free-like, hierarchical network organized into chromatin communities. The communities were enriched in specific functions and were syntenic through evolution. Disease-associated SNPs from genome-wide association studies were enriched among the nodes with fewer interactions, implying their selection against deleterious interactions by limiting the total number of interactions, a model that we further reconciled using somatic and germline cancer mutation data. The hubs lacked disease-associated SNPs, constituted a nonrandomly interconnected core of key cellular functions, and exhibited lethality in mouse mutants, supporting an evolutionary selection that favored the nonrandom spatial clustering of the least-evolving key genomic domains against random genetic or transcriptional errors in the genome. Altogether, our analyses reveal a systems-level evolutionary framework that shapes functionally compartmentalized and error-tolerant transcriptional regulation of human genome in three dimensions.

Introduction: Although small-cell lung cancer (SCLC) is sensitive to first-line radiation and chemotherapy, nearly all patients recur often with treatment-resistant disease. Therefore, delineating subclonal architecture and molecular evolution of SCLC after treatment by comparing genomic profiles of recurrent and paired treatment-naïve tumors may provide new insights into mechanisms underlying recurrence and susceptibility to further treatment. Methods: Treatment-naïve and paired recurrent tumor samples from 9 patients with limited-stage SCLC treated with concurrent chemoradiation (CCRT) underwent whole exome sequencing (WES) and data is currently available for 6 pairs. All 6 patients had disease recurrence more than 3 months after finishing treatment. Genomic landscape including somatic mutations, somatic copy number aberrations (SCNA), subclonal architecture and transversion rate (percentage of base changes from pyrimidine to purine and vice-versa) were compared between treatment-naïve and paired recurrent tumor samples. Circulating cell-free DNA (cfDNA) collected at the time of recurrence was also subject to deep sequencing of 461 cancer genes. Results: SCNA analyses revealed significantly more complex chromosomal gains in relapsed SCLCs compared to pre-treatment primary tumors (7.7% vs 13.2% genes with copy number gain in primary and relapsed SCLC respectively, p=0.034). As expected, TP53 and RB1 were the most commonly mutated genes (83% and 67% respectively) and tobacco exposure related signatures were predominant in all samples. Primary and relapsed tumor pairs showed very similar genomic landscape including tumor mutational burden (11.8/MB in primary SCLC vs 9.7/MB in relapsed tumors), intra-tumor heterogeneity complexity (MATH score 38.2 in primary vs 39.2 in relapse) and transversion rates, that have been reported to be associated with alkylating-agent treatment (62% in primary vs 65% in relapse). An average of 72.25% (range: 53-86%) somatic mutations, including all canonical cancer gene mutations, were shared between primary SCLC and relapsed tumors. In addition, the subclonal architecture was also very similar between treatment-naïve primary SCLC and paired relapsed tumors. Of note, 5 out of 6 patients achieved disease control on retreatment with chemotherapy. Conclusion: Copy number gains may play a role in post-CCRT relapse of SCLC. Paired pre-treatment and relapse samples show genomic similarities which could account for benefit to subsequent treatment. Additional sample analysis and ctDNA assessment is ongoing to better address these findings, especially in patients with chemoresistant relapse. These findings also suggest that other molecular changes involving gene expression (eg. methylation) may play a role in SCLC relapse and resistance. Citation Format: Marcelo V. Negrao, Ming Tang, Ying Jin, Yamei Chen, Xiao Hu, Huarong Tang, Haimiao Xu, Kelly Quek, Jianhua Zhang, Xizeng Mao, Xingzhi Song, John V. Heymach, Ignacio Wistuba, Lauren A. Byers, Bonnie S. Glisson, Andrew Futreal, Ming Chen, Jianjun Zhang. Exome sequencing of paired primary and relapsed small cell lung cancers reveals increased copy number aberration complexity to be associated with disease relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 213.

Cancer is the result of the accumulation of genetic and epigenetic alterations in key genes, which ultimately lead to uncontrolled growth of mutated cells. Genetic alterations range from small base substitutions to large chromosomal structural rearrangements. Many cancer genomes carry tens to hundreds of somatic structural rearrangements, which may have functional consequences. However, the patterns and characterization of somatic rearrangements in human cancers are still in early stages. In this thesis, I describe the genomic landscape of somatic structural rearrangements in human pancreatic cancers. I then investigate the potential mechanisms involved in somatic rearrangement formation and test whether such rearrangements can be used as biomarkers to monitor patient therapy. Chapter 1 - Introduction: This chapter is a broad overview of somatic structural rearrangements in human cancers, motivation for studying them, and their potential use for clinical applications. Chapter 2 – A workflow to increase verification rate of somatic chromosomal rearrangements using high throughput next generation sequencing: I established a high throughput workflow to rapidly verify somatic structural variants and identify the exact location of breakpoints using benchtop next generation sequencing and computational tools. The workflow examined more than 300 predicted breakpoints and identified breakpoint location in more than 80% of somatic events at base level. The results demonstrated that next generation sequencing is comparable to the conventional Sanger sequencing and can complete the verification workflow in a shorter timeframe, enabling rapid validation of events. Chapter 3 – Patterns of somatic breakpoints may indicate repair mechanisms that were active or absent during the generation of genomic rearrangements: This chapter describes the spectrum of somatic rearrangements and breakpoints detected in 120 primary pancreatic ductal adenocarcinomas genomes. The analysis includes characterization of the breakpoint junctions to infer which potential DNA repair processes are occurring. The results revealed that the majority of tumours exhibited repair of chromosome structural breaks using microhomology suggesting that NHEJ is the main mechanism of DNA repair in pancreatic cancer. Tumours with BRCA1 or BRCA2 gene mutations or with a high contribution of a BRCA-like mutational signature showed a higher frequency of somatic breakpoints with microhomology length 1 to 5 bp and lower frequency of breakpoints with a blunt end (0 bp) when compared to tumours harbouring BRCA wild type or low BRCA mutational signature. The similarity in breakpoint characteristics between tumours with BRCA mutation and BRCA mutational signature reinforce previous findings that a subtype of pancreatic tumour might have deficiency in the HR pathway and could respond to PARP1 inhibitors. Furthermore, the analysis of the DNA sequence surrounding the breakpoints revealed strong signal of A+Ts rich regions suggesting that the formation of somatic rearrangements could also be mediated by either retrotransposition activity or chromosomal fragile sites. Taken together, the analyses of breakpoint junctions highlighted that the formation of somatic rearrangements in pancreatic carcinogenesis is complex – potentially with more than one mechanism active within a cancer genome. Chapter 4 – Identification of personalized DNA-based biomarkers for pancreatic cancer and the assessment of whether they can be used to monitor tumour burden and response to chemotherapy: In this chapter, I first evaluated the performance of sequencing and PCR based methods to detect ctDNA. Subsequently, I quantified ctDNA in the serum or plasma of the three pancreatic cancer patients. Little or no detection of ctDNA was observed in the analysed serum samples. For one patient, a tumour specific rearrangement was detected in the serum, and this patient had already presented with metastatic disease. Based on the results, it was hypothesized that ctDNA released from pancreatic cancer might be limited by: i) nature of pancreatic cancer; ii) the physiological location of the pancreas which could influence the amount of ctDNA released in the circulation, as the fragmented tumour DNA might be cleared by the liver and therefore reduced the opportunity to detect ctDNA in the circulation; iii) volume of plasma or serum used to isolate cfDNA; iv) the status of disease progression. In this analysis, the ctDNA was detected only in the sample collected at late stage pancreatic cancer (after the diagnosed of liver metastasis). Thus, in the context of pancreatic cancer disease, the quantification of ctDNA might be more suitable for recurrent disease, which has metastasized from the pancreas. Chapter 5: I concluded my findings throughout my studies in Chapter 2, 3 and 4 with a summary and future directions.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

140 Background: Non-small cell lung cancer (NSCLC) is characterized by a high mutational load. Accordingly, it is also among the tumor types which respond best to immune checkpoint blockade, likely through its ability to enhance the anti-tumor T cell response. However, the lung is constantly exposed to the outside environment, which may result in a continuous state of inflammation targeting pathogens rather than tumor cells. Therefore, a greater understanding of the T cell receptor (TCR) landscape and phenotypes across normal lung and tumor is warranted. Methods: Here, we performed sequencing of the CDR3 variable region of the beta chain of the TCR as well as whole exome sequencing on peripheral blood, normal lung and tumor in 235 NSCLC patients. We further analyzed the immune microenvironment by Cytometry by Time-of-Flight (CyTOF) in 10 NSCLC patients with paired normal lung and tumor. Results: Comparison of the TCR repertoire showed 9% (up to 15%) of T cells were shared between normal lung and tumor, though the most dominant were generally shared (up to 95%). Interestingly, T cell clonality was higher in the normal lung than tumor in almost all patients (89%, p < 0.0001) suggesting potential differences in the ongoing immune response in different regions of the lung. A substantial number of non-synonymous exonic mutations (NSEM) were detected in tumors (average = 566 NSEM) but also in the normal lung (average = 156 NSEM), with many shared (up to 45.6%). CyTOF confirmed marked differences in the immune microenvironment, including higher frequency of VISTA+ antigen-presenting cells in the tumor (p = 0.04). Finally, analysis of clinicopathological attributes revealed a greater T cell diversity in the periphery in patients with increased overall survival (OS, p = 0.001), while patients with a more similar normal lung/tumor T cell repertoire showed decreased OS (p = 0.028). Conclusions: These results suggest that a substantial proportion of infiltrating T cells in NSCLC tumors may be lung-resident T cells associated with response to environmental factors. However, normal lung and NSCLC tumors carry T cells of distinct phenotypes, which highlights differences in the ongoing antigenic response within the lung.

8545 Background: Our previous study has suggested that complex genomic intratumor heterogeneity (gITH) was associated with an increased risk of relapse in patients with localized lung adenocarcinomas (LUAD). We have launched a study to investigate molecular and immune profile ITH of Stage IA NSCLC (a patient population with no optimal biomarker to guide postsurgical therapy) to understand the molecular evolution during early carcinogenesis and to identify biomarkers for early detection and intervention. Here, we report the preliminary analysis on gITH. Methods: We performed multiregion whole exome sequencing on 30 Stage IA LUAD and matched normal lung tissue to a median sequencing depth of 494x. 15 patients have relapsed within 3 years post-surgery (cases) and 15 patients have not relapsed with a minimum of 5-year postsurgical follow up (controls). Cases and controls are 1:1 matched for the key prognostic factors including tumor size, smoking status, age, gender, ethnicity and lobectomy or wedge resection. Shannon diversity index (SDI) was used to quantify ITH in each individual tumor. Kaplan-Meier method was used to evaluate the relationship between ITH and disease-free survival (DFS) as well as overall survival (OS). Results: Consistent with our previous study, 108 of 110 (98.2%) canonical cancer gene mutations were shared events by all regions of individual tumor. Compared to non-relapsed controls, tumors from relapsed cases demonstrated significantly higher degree of ITH (SDI of 1.78 in cases vs 1.58 for controls, p = 0.016). Higher degree of gITH was associated with shorter DFS (p = 0.008) and shorter OS (p = 0.0153). Significantly higher mutation burden was observed in tumors from relapsed patients (median of 10.86 mutations per MB in cases vs 7.45 mutations per MB in controls, p = 0.03). Analysis of gITH on a larger cohort and on predicted neoantigen, methylation, gene expression and immune profiles are in progress. Conclusions: Majority of cancer gene mutations are clonal events during early carcinogenesis of LUAD. Complex gITH may be associated with more aggressive biology and inferior clinical outcome in patients with Stage IA LUAD, therefore, may be evaluated as a potential biomarker.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.

Introduction: While remission rates for childhood acute lymphoblastic leukemia (ALL) now exceed 80%, relapsed ALL remains the leading cause of non-traumatic death in children. Recently, a high-risk group of B-progenitor ALL patients has been identified. Such cases exhibit a gene expression profile similar to that of BCR-ABL1 positive (Ph+) ALL but are BCR-ABL1 negative, and also experience poor treatment outcomes. This subset, termed Ph-like ALL, is characterised by a range of genetic alterations that activate cytokine receptor and kinase signalling, allowing potential targeting by available tyrosine kinase inhibitors (TKI). The frequency of Ph-like ALL in the Australian community and the prognosis in the setting of the first MRD (minimal residual disease) intervention trial by the Australian and New Zealand Children's Haematology/Oncology Group (ANZCHOG ALL8) is unknown. Method: We retrospectively screened 250 unselected samples that were available from children diagnosed with B-ALL, for Ph-like ALL. The children, aged between 1 and 18 years, were enrolled on the ANZCHOG ALL8 trial and recruited from 2002-2011. The criteria for stratification to the high-risk group, based upon Berlin-Frankfurt-Munster (BFM) protocols, were BCR-ABL1 or MLL t(4;11) translocation; poor prednisolone response at day 8; failure to achieve remission by day 33 or high MRD (>5 x10-4) at day 79. MRD was measured by RQ-PCR for patient-specific immunoglobulin and T-cell receptor rearrangements. All patients received a standard BFM four drug induction chemotherapy regimen including a prednisolone pre-phase and intrathecal methotrexate. High-risk patients received a further three novel intensive blocks of chemotherapy followed by transplant in most cases. Patients were screened for Ph-like ALL using a custom Taqman Low Density Array (TLDA) based upon previous reports. Fusions were then confirmed by RT-PCR for 30 known fusions, Sanger sequencing, mRNA sequencing and/or FISH. Results: Ten percent (25/250) of children in this cohort were identified as having Ph-like ALL, with most fusions converging on kinase activating pathways (Table 1). Three Ph-like ALL patients were considered high-risk, the remaining 22 (88%) were medium risk. Five children with Ph-like ALL, that did not have a fusion identified by RT-PCR, are currently under further investigation. Furthermore, 15 of the 20 (75%) of rearrangements involved CRLF2 with 10 (66%) of these children relapsing. Strikingly, 56% (14/25) of children in the ALL8 cohort who were identified as Ph-like subsequently relapsed compared to 16% (36/225) who were not, with significantly worse event free survival (p Conclusion: Here we demonstrate a significantly higher frequency of relapse amongst Australian children with Ph-like ALL compared to non Ph-like disease despite a MRD-adjusted intensification regimen. In this cohort, these children should be classified as high-risk due to high treatment failure rates with standard/medium risk regimens. Importantly, rapid identification of these patients may guide future intervention with targeted therapies, such as TKI, matched to the causative genetic lesion in this high-risk group. Figure 1. Fusions identified in Ph-like ALL from ANZCHOG ALL8 cohort. Figure 1. Fusions identified in Ph-like ALL from ANZCHOG ALL8 cohort. Figure 2. Kaplan-Meier estimates of event free survival for patients with Ph-like ALL and non Ph-like ALL (all risk groups). Figure 2. Kaplan-Meier estimates of event free survival for patients with Ph-like ALL and non Ph-like ALL (all risk groups). Disclosures Hughes: ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mullighan:Incyte: Consultancy, Honoraria; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.