Kelly Quek

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

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.

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.

Abstract 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.

Acute lymphoblastic leukemia (ALL) remains a leading cause of cancer-related death in children and young adults. Since the 1960s, improvements in the treatment of children with ALL have led to 10-year survival rates now exceeding 85%.[1][1] Philadelphia-like (Ph-like) ALL is characterized by a gene

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 (<= 3cm), p = 0.014), advanced age (average Euclidean distance of 34.95 (above 65) verse 28.06 (below 65), p = 0.046) and increased risk of postsurgical recurrence (average Euclidean distance of 35.65 (relapsed patients) versus 29.03 (patients without relapsed), p = 0.039).

Recent studies have revealed that cancer patients had a higher risk of having coronavirus disease 2019 , 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.

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non-small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

<ns4:p><ns4:italic>Helicobacter pylori</ns4:italic>is a gastric pathogen that causes several gastroduodenal disorders such as peptic ulcer disease and gastric cancer. Eradication efforts of<ns4:italic>H. pylori</ns4:italic>are often hampered by antimicrobial resistance in many countries, including Vietnam. Here, the study aimed to investigate the occurrence of antimicrobial resistance among<ns4:italic>H. pylori</ns4:italic>clinical isolates across 13 hospitals in Vietnam. The study further evaluated the clarithromycin resistance patterns of<ns4:italic>H. pylori</ns4:italic>strains. In order to address the study interests, antimicrobial susceptibility testing, epsilometer test and PCR-based sequencing were performed on a total of 193 strains isolated from patients, including 136 children (3–15 years of age) and 57 adults (19–69 years of age). Antimicrobial susceptibility testing showed that the overall resistance to amoxicillin, clarithromycin, levofloxacin, metronidazole, and tetracycline was 10.4%, 85.5%, 24.4%, 37.8%, and 23.8% respectively. The distribution of minimum inhibitory concentrations (MICs) of clarithromycin-resistant strains was 85.5% with MIC &gt;0.5 μg/mL. The majority of the clarithromycin resistant isolates (135 of 165 subjects) have MICs ranging from 2 μg/mL to 16 μg/mL. Furthermore, sequencing detection of mutations in 23S rRNA gene revealed that strains resistant and susceptible to clarithromycin contained both A2143G and T2182C mutations. Of all isolates, eight clarithromycin-resistant isolates (MIC &gt;0.5 μg/mL) had no mutations in the 23S rRNA gene. Collectively, these results demonstrated that a proportion of clarithromycin-resistant<ns4:italic>H. pylori</ns4:italic>strains, which are not related to the 23S rRNA gene mutations, could be potentially related to other mechanisms such as the presence of an efflux pump or polymorphisms in the CYP2C19 gene. Therefore, the present study suggests that providing susceptibility testing prior to treatment or alternative screening strategies for antimicrobial resistance is important for future clinical practice. Further studies on clinical guidelines and treatment efficacy are pivotal for successful eradication of<ns4:italic>H. pylori</ns4:italic>infection.</ns4:p>

Nature 521, 489–494 (2015); doi: 10.1038/nature14410 In this Article, the affiliations of authors Michael Quinn and Orla McNally should read “22Department of Obstetrics and Gynaecology, The University of Melbourne, and The Royal Women’s Hospital, Parkville, Victoria 3052, Australia”. Their affiliations have been corrected in the HTML and PDF versions online.

Central MessageDelineating genomic evolution and intratumor heterogeneity of non–small cell lung cancer by multiregion deep sequencing provides novel insights for diagnosis, prevention, and treatment.See Editorial Commentary page 1203.Feature Editor's Note—“When the views entertained in this volume on the origin of species, or when analogous views are generally admitted, we can dimly forsee that there will be a considerable revolution in natural history.”—Charles DarwinRecent landmark studies on intratumoral heterogeneity require us to revise our current conceptual framework for understanding the evolution of cancer. Whole-exome sequencing on multiple regions of single human tumors has demonstrated striking intraclonal diversity in cancer: whereas some somatic mutations are clonal in nature (present in all tumor cells), many are subclonal (present in only some tumor cells). Critical to our understanding of cancer as a complex adaptive and evolving biologic ecosystem, these discoveries have demonstrated that cancer evolves not on a linear trajectory but through parallel evolution of subclones. Thus, intratumoral heterogeneity underlies, and fuels, tumor evolution. This concept has vital clinical implications for patients suffering from cancer, particularly in areas of targeted therapy and drug resistance. For example, an “actionable” driver mutation may be a suboptimal target for therapy if confined to a subclonal branch of the tumor's evolutionary tree. In the accompanying Feature Editorial, the authors discuss the major findings of the TRACERx (Tracking Non-Small Cell Lung Cancer Evolution through Therapy) study recently published in The New England Journal of Medicine, in which the nature of intratumoral heterogeneity is defined for 100 patients with non–small cell lung cancer treated surgically within a multicenter cohort. The findings, reported herein, are striking, and will drive innovation in the prevention, diagnosis, and treatment of lung, and all, human cancers. We can expect to see our own rapid evolution of this field, from which we ultimately can hope to provide therapeutic interventions that reduce tumor adaptiveness.Bryan Burt, MD Delineating genomic evolution and intratumor heterogeneity of non–small cell lung cancer by multiregion deep sequencing provides novel insights for diagnosis, prevention, and treatment. See Editorial Commentary page 1203. Non–small cell lung cancer (NSCLC) continues to be a prevalent and deadly cancer, and the variability of recurrences argues for other tumor, patient, or treatment-related factors beyond current staging, which influence prognosis. One significant aspect of lung cancer biology that has received increased attention relates to the heterogeneity and evolution of the tumor itself. Tumor heterogeneity results from tumor evolution and in the meanwhile serves as a substrate for tumor evolution and natural selection and is influenced by the host system (such as antitumor immune surveillance) as well as anticancer therapies. Delineating cancer evolutionary history may provide pivotal insight to our understanding of cancer development, progression, and therapeutic resistance. Pioneering studies by multiregion profiling1Zhang J. Fujimoto J. Zhang J. Wedge D.C. Song X. Zhang J. et al.Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing.Science. 2014; 346: 256-259Crossref PubMed Scopus (696) Google Scholar, 2McGranahan N. Furness A.J.S. Rosenthal R. Ramskov S. Lyngaa R. Saini S.K. et al.Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.Science. 2016; 351: 1463-1469Crossref PubMed Scopus (1944) Google Scholar, 3Gerlinger M. Rowan A.J. Horswell S. Math M. Larkin J. Endesfelder D. et al.Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.N Engl J Med. 2012; 366: 883-892Crossref PubMed Scopus (5729) Google Scholar, 4Liu Y. Zhang J. Li L. Yin G. Zhang J. Zheng S. et al.Genomic heterogeneity of multiple synchronous lung cancer.Nat Commun. 2016; 7: 13200Crossref PubMed Scopus (111) Google Scholar and by comparing paired primary and relapsed tumors5Paik P.K. Shen R. Won H. Rekhtman N. Wang L. Sima C.S. et al.Next-generation sequencing of stage IV squamous cell lung cancers reveals an association of PI3K aberrations and evidence of clonal heterogeneity in patients with brain metastases.Cancer Discov. 2015; 5: 610-621Crossref PubMed Scopus (107) Google Scholar, 6Brastianos P.K. Carter S.L. Santagata S. Cahill D.P. Taylor-Weiner A. Jones R.T. et al.Genomic characterization of brain metastases reveals branched evolution and potential therapeutic targets.Cancer Discov. 2015; 5: 1164-1177Crossref PubMed Scopus (617) Google Scholar have shed light on cancer genomic evolution and suggested the potential impact of genomic heterogeneity on cancer biology and patient outcome. However, these studies have been limited to small retrospective cohorts. The Tracking Non-Small Cell Lung Cancer Evolution through Therapy (TRACERx) study7Jamal-Hanjani M. Wilson G.A. McGranahan N. Birkbak N.J. Watkins T.B.K. Veeriah S. et al.Tracking the evolution of non–small-cell lung cancer.N Engl J Med. 2017; 376: 2109-2121Crossref PubMed Scopus (1314) Google Scholar is an ambitious study targeting the evolution of NSCLC. It is a multicenter cohort study aiming to prospectively evaluate tumor evolution of 842 patients with NSCLC treated initially with surgical therapy. The objective is to examine the tumor heterogeneity at the time of surgical resection, at relapse, and after sequential lines of other treatments through high-depth, multiregion whole-exome sequencing of surgically resected specimen and sequentially collected tumor biopsy samples at the time of relapse. Results are also correlated with plasma circulating tumor DNA (ctDNA), which is analyzed from the blood samples collected throughout the patient's clinical course. The results from the first 100 patients (stage IA-IIIB) enrolled in TRACERx study were recently published in The New England Journal of Medicine.7Jamal-Hanjani M. Wilson G.A. McGranahan N. Birkbak N.J. Watkins T.B.K. Veeriah S. et al.Tracking the evolution of non–small-cell lung cancer.N Engl J Med. 2017; 376: 2109-2121Crossref PubMed Scopus (1314) Google Scholar This report was directed at investigating the intratumoral heterogeneity (ITH) architecture of surgically excised tumors and evaluating whether ITH is associated with clinical outcome. In this elegant analysis, 327 tumor regions from 100 tumors were subjected to high-depth whole-exome sequencing (median sequencing depth of 426×) and sophisticated bioinformatics analyses. The authors demonstrated extensive genomic ITH at the nucleotide and chromosomal level; the median of 30% of somatic mutations and the median of 48% of copy number alterations (CNAs) were subclonal. Lung squamous cell carcinoma demonstrated greater clonal mutational burden than lung adenocarcinoma, probably reflecting heavier smoking history in patients with lung squamous cell carcinoma; however, subclonal mutation burden or ratio did not seem to differ between the histologies. As expected, early clonal mutations were associated with smoking (COSMIC signature 4) in the majority of tumors. In contrast, subclonal mutations were significantly enriched for genomic signatures related to spontaneous deamination of methylated cytosines (COSMIC signature 1A) and APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) (COSMIC signatures 2 and 13),8Alexandrov L.B. Nik-Zainal S. Wedge D.C. Aparicio S.A. Behjati S. Biankin A.V. et al.Signatures of mutational processes in human cancer.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6215) Google Scholar suggesting continuous genomic evolution with different mutational mechanisms in play, which occurred at different times during cancer development and progression. The authors also tested their central hypothesis whether ITH is associated with clinical outcome. Their preliminary analysis demonstrated that a high proportion (≥48%, the cohort median) of subclonal CNAs was associated with worse disease-free survival (DFS) as compared with a low proportion of subclonal CNAs. This finding remained significant after adjustment for age, smoking, histology, adjuvant therapy, and tumor stage (hazard ratio, 3.70; 95% confidence interval, 1.29-10.65; P = .01). However, no significant association with DFS was observed between the groups when stratified by percentage of subclonal mutations. Although the published work is only the preliminary analysis of the first 100 of proposed 842 patients, the rich information and novel findings are quite provocative for the future translational and clinical research. Herein, we outline some of the concepts. First, this work provided another piece of evidence that genomic heterogeneity is associated with clinical outcome, even in early stages of NSCLC. It is somewhat surprising that more commonly regarded ITH in point mutations was not found to be associated with survival in this patient cohort; this finding is in contrast with previous reports in lung cancer1Zhang J. Fujimoto J. Zhang J. Wedge D.C. Song X. Zhang J. et al.Intratumor heterogeneity in localized lung adenocarcinomas delineated by multiregion sequencing.Science. 2014; 346: 256-259Crossref PubMed Scopus (696) Google Scholar, 2McGranahan N. Furness A.J.S. Rosenthal R. Ramskov S. Lyngaa R. Saini S.K. et al.Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.Science. 2016; 351: 1463-1469Crossref PubMed Scopus (1944) Google Scholar and other malignancies.9Landau D.A. Carter S.L. Stojanov P. McKenna A. Stevenson K. Lawrence M.S. et al.Evolution and impact of subclonal mutations in chronic lymphocytic leukemia.Cell. 2013; 152: 714-726Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar One possible explanation is that the imbalance in many major prognostic factors, such as stage, age, smoking, and adjuvant therapy, in this relative small patient cohort may have masked an actual association between mutational ITH and survival. In addition, there have been only 20 relapses at the time of data analyses, and the postsurgical follow-up of this study has been rather short. The median follow-up duration for the cohort has been approximately 18 months and less than 2 years in 80 patients. Therefore, it is premature to conclude that point-mutation ITH is not associated with survival in locoregional NSCLC. Hopefully, this question will be more definitively answered when additional patients from TRACERx or similar studies are analyzed after prolonged follow-up. Nevertheless, the association between the high level of CNA ITH and worse DFS, which remained significant on a multivariate analysis after adjusting for known prognostic factors, suggests that chromosomal instability may have greater impact on outcome than somatic mutations. Chromosomal alterations involving the gain or loss of chromosomal segments or even whole chromosomes could affect hundreds or thousands of genes and may thus disrupt multiple key molecular processes. Second, by the use of multiregion sequencing, subclonal driver mutations were identified in 75% of patients in this study. This introduces a challenge to our current personalized oncology approach, which is based on sequencing driver genes from single biopsies acquired either by bronchoscopy or guided transcutaneous biopsy. Multiregion sequencing is not practical for patients with metastatic diseases or unresectable tumors. However, ctDNA is not spatially limited to certain tumor regions and may have the advantage in detecting subclonal mutations compared with single biopsies. Indeed, in an accompanying article published in Nature,10Abbosh C. Birkbak N.J. Wilson G.A. Jamal-Hanjani M. Constantin T. Salari R. et al.Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.Nature. 2017; 545: 446-451Crossref PubMed Scopus (937) Google Scholar the TRACERx team has investigated the role of ctDNA in molecular diagnosis and disease monitoring and was able to identify clonal and subclonal mutations present in matched tumor samples. With the rapid progress being made in liquid biopsy sequencing technologies, sequencing ctDNA could become a practical alternative approach to multiregion tumor sequencing. Third, in this study all tumors were primarily resected. How neoadjuvant therapies such as chemotherapy, targeted therapy, radiation, or immune therapy would impact ITH architectures of NSCLC remains unknown. One can hypothesize that residual tumor cells that survive neoadjuvant therapies could be the subclones resistant to neoadjuvant therapies. Therefore, investigating the postneoadjuvant therapy residual tumors may provide valuable information on mechanisms of drug resistance. For example, because targetable driver mutations such as EGFR, MET, and BRAF are all clonal, the residual cancer cells in the resected tumors and lymph nodes after appropriate targeted treatment would provide valuable material to investigate the mechanisms underlying resistance to these agents. Therefore, well-designed window-of-opportunity neoadjuvant clinical trials would be valuable for studying drug resistance. Fourth, in addition to serving as a potential prognostic biomarker, ITH itself could become a potential therapeutic target. Given the important role of genomic instability in tumor evolution, modulating genomic stability such as targeting APOBEC family, a common cause of subclonal diversification of NSCLC, or inhibiting DNA repair pathways could become a novel therapeutic strategy. This strategy has been recently tested. For example, poly (ADP-ribose) polymerase inhibitors have proven to be active in homologous recombination–deficient tumors.11Mirza M.R. Monk B.J. Herrstedt J. Oza A.M. Mahner S. Redondo A. et al.Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer.N Engl J Med. 2016; 375: 2154-2164Crossref PubMed Scopus (1471) Google Scholar Fifth, the majority of studies on ITH, including the current study, have mainly focused on the genomic ITH. However, ITH can be present at different molecular levels (genetic, epigenetic, gene, and protein expression, etc) of cancer cells and also in tumor microenvironment constituting of epithelial cells, blood and lymphatic vessels, cytokines, or infiltrating immune cells. ITH of any of the aforementioned components may impact tumor evolution and patient outcome. Our pilot study has demonstrated that a greater level of DNA methylation ITH was associated with larger tumor size, advanced patient age, and increased risk of postsurgical recurrence in NSCLC.12Quek K. Li J. Estecio M. Zhang J. Fujimoto J. Roarty E. et al.DNA methylation intratumor heterogeneity in localized lung adenocarcinomas.Oncotarget. 2017; 8: 21994-22002Crossref PubMed Scopus (27) Google Scholar Most recently, we reported substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions from localized lung cancers and that a greater degree of TCR ITH was associated with an increased risk of postsurgical recurrence and shorter DFS.13Reuben A. Gittelman R.M. Gao J. Zhang J. Yusko E.C. Wu C.J. et al.TCR repertoire intratumor heterogeneity in localized lung adenocarcinomas: an association with predicted neoantigen heterogeneity and postsurgical recurrence.Cancer Discov. 2017; 7: 1088-1097Crossref PubMed Scopus (123) Google Scholar Tumor evolution is a complex process, during which cancer cells accumulate molecular alterations that change their phenotypic features by interacting with the tumor microenvironment. To systematically understand tumor evolution, future studies are required to depict the overall molecular (genetic, epigenetic, gene, and protein expression) ITH of cancer cells as well as the tumor microenvironment, particularly the immune contexture. Again, well-designed trials/projects that include surgical locoregional disease control of lung cancer will be invaluable in sorting out this process. Last but not least, the TRACERx data show a possible molecular poor prognostic marker for lung cancer, in this case high-level CNA ITH, which could be prospectively validated in the future. In the past, clinical, pathologic, and radiologic prognostic markers for NSCLC have not translated into strategies for treatment intensification in high-risk patients, and we continue to use a “one-size fits all” approach. One of the reasons for this was probably the limited number of effective agents, but the therapeutic arsenal for cancer treatment has drastically changed over the last years, especially for NSCLC, where targeted and immune therapies are now approved. Therefore, validated molecular markers and mutational signatures from studies such as TRACERx could play a major role in patient selection in future trials aiming to intensify neoadjuvant and adjuvant treatment for localized or locally advanced disease. The TRACERx study is an important step to advance our understanding of NSCLC tumor evolution and its impact on cancer biology and patient outcome. We are looking forward to the updated analyses of the study and more such efforts to dissect the evolutionary history of lung cancer and other malignancies, leading to novel diagnostic, preventive, and therapeutic strategies and eventually cure. Authors have nothing to disclose with regard to commercial support. Tracing the origin, tracking the evolution, and the treatment of the futureThe Journal of Thoracic and Cardiovascular SurgeryVol. 155Issue 3PreviewThe classic approach of using hematoxylin and eosin staining for tumor slides is a cornerstone in the management of non–small cell lung cancer (NSCLC). Although this modality provides valuable histologic information, it does not delve into the biologic determinants of NSCLC. With the advent of gene mutation analysis, driver alterations, such EGFR, ALK, and KRAS, can predict response to select targeted therapies.1 As this mutational information had been garnered from patients with advanced metastatic disease, there is little clarity regarding the origins of these mutations in early-stage malignancy as well as the effects that early gene aberrancies have on the course of the disease. Full-Text PDF Open Archive

Hepatocellular carcinoma (HCC) accounts for approximately 85%-90% of all liver cancer cases and has poor relapse-free survival. There are many gene expression studies that have been performed to elucidate the genetic landscape and driver pathways leading to HCC. However, existing studies have been limited by the sample size and thus the pathogenesis of HCC is still unclear. In this study, we performed an integrated characterization using four independent datasets including 320 HCC samples and 270 normal liver tissues to identify the candidate genes and pathways in the progression of HCC. A total of 89 consistent differentially expression genes (DEGs) were identified. Gene-set enrichment analysis revealed that these genes were significantly enriched for cellular response to zinc ion in biological progress group, collagen trimer in the cellular component group, extracellular matrix structural (ECM) constituent conferring tensile strength in the molecular function group, protein digestion and absorption, mineral absorption and ECM-receptor interaction. Network system biology

Multiple synchronous lung tumors (MSLT), particularly within a single lobe, represent a diagnostic and treatment challenge. While histologic assessment was once the only method to possibly distinguish multiple primary lung cancers, there is a growing interest in identifying unique genomic features or mutations to best characterize these processes.In order to differentiate multiple primary lung malignancies from intrapulmonary metastases in patients with MSLT, we performed whole exome sequencing (WES) on 10 tumor samples from 4 patients with MSLT.Shared mutations between tumors from the same patient varied from 0-91%. Patient 3 shared no common mutations; however, in Patients 2 and 4, identical mutations were identified among all tumors from each patient, suggesting that the three tumors identified in Patient 3 represent separate primary lung cancers, while those of Patients 1, 2 and 4 signify hematogenous and lymphatic spread.A high proportion of shared mutations between different lung tumors is likely indicative of intrapulmonary metastatic disease, while tumors with distinct genomic profiles likely represent multiple primary malignancies driven by distinct molecular events. Application of genomic profiling in the clinical setting may prove to be important to precise management of patients with MSLT.

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 &lt; 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.

Abstract 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.

Non-small cell lung cancer (NSCLC) is characterized by a high mutational load. Accordingly, it is also among the tumor types responding to immune checkpoint blockade, likely through harnessing of the anti-tumor T cell response. However, the lung is continuously exposed to the outside environment, which may result in a continuous state of inflammation against outside pathogens unrelated to the tumor microenvironment. Therefore, further investigation into the T cell repertoire and T cell phenotypes across normal lung and tumor is warranted. We performed T cell receptor (TCR) sequencing on peripheral blood mononuclear cells (PBMC), normal lung, and tumor from 225 NSCLC patients, among which, 96 patients were also subjected to whole exome sequencing (WES) of PBMC, tumor and normal lung tissues. We further performed Cytometry by Time-of-Flight (CyTOF) on 10 NSCLC tumors and paired normal lung tissues to phenotype immune and T cell subsets. Comparison of the T cell repertoire showed 9% (from 4% to 15%) of T cell clones were shared between normal lung and paired tumor. Furthermore, among the top 100 clones identified in the tumor, on average 57 (from 0 to 95) were shared with paired normal lung tissue. Interestingly, T cell clonality was higher in the normal lung in 89% of patients suggesting potential differences in the immune response and immunogenicity. A substantial number of somatic mutations were also identified not only in NSCLC tumors (average 566; from 147 to 2819), but also in morphologically normal lung tissues (average 156; from 50 to 2481). CyTOF demonstrated striking differences in the immune infiltrate between normal lung and tumor, namely a lower frequency of PD-1+CD28+ T cells (both CD4+ and CD8+) in the normal lung (2.7% versus 3.0% in tumor). In addition, a unique GITR+ T cell subset (0.96%) was entirely restricted to the normal lung. Conversely, increases in regulatory T cell frequency (CD4+FoxP3+) were observed in the tumor (10.4% vs 1.7% in normal lung), further highlighting the differences in T cell phenotype and response across normal lung and tumor. These results suggest that a substantial proportion of infiltrating T cells in NSCLC tumors may be residential T cells associated with response to environmental factors. However, normal lung and NSCLC tumors carry T cells of distinct phenotypes including increases in immunosuppressive T cells within the tumor which may further highlight the differences in the anti-tumor immune response.

Abstract 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.

Abstract 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 (&gt;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&lt;0.0001) (Figure 1). 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.

&lt;p&gt;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.&lt;/p&gt;

&lt;p&gt;Table S1. Patient demographics and characteristics.&lt;/p&gt;

&lt;p&gt;Supplemental Figure Legends&lt;/p&gt;

&lt;p&gt;Table S1. Patient demographics and characteristics.&lt;/p&gt;

&lt;p&gt;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.&lt;/p&gt;

&lt;p&gt;Supplemental Figure Legends&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;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.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; 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. &lt;i&gt;Cancer Discov; 7(10); 1088–97. ©2017 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1047&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;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.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; 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. &lt;i&gt;Cancer Discov; 7(10); 1088–97. ©2017 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1047&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

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.

Our previous study has suggested that complex intratumor heterogeneity (ITH) was associated with increased risks of relapse of localized lung adenocarcinomas (LUAD). In this follow-up study, we performed multiregion whole exome sequencing on 30 Stage IA LUAD: 15 patients relapsed within 3 years post-surgery (cases) and 15 patients haven’t relapsed (≥ 5-year postsurgical follow up (controls). Cases and controls are 1:1 matched for tumor size, smoking, age at surgery, gender, ethnicity, etc. Shannon diversity index (SDI) was used to quantify ITH. 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 1.43 in cases vs 1.21 for controls, p = 0.03). Higher degree of genomic ITH was associated with shorter DFS (p=0.008) and shorter OS (p=0.01). 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). Majority of cancer gene mutations are clonal events during early carcinogenesis of LUAD. Complex genomic ITH 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.

The original version of this Article omitted the following from the Acknowledgements: Professor Stebbing sits on SABs for Celltrion, Singapore Biotech, Vor Biopharma, TLC Biopharmaceuticals and Benevolent AI, has consulted with Lansdowne partners, Vitruvian and Social Impact Capital and Chairs the Board of Directors for BB Biotech Healthcare Trust and Xerion Healthcare. This has now been corrected in both the PDF and HTML versions of the Article.

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.

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

Additional file 2.

Abstract 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 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 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. 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.

Abstract 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 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. Results We 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. 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.