ABSTRACT
Pancreatic ductal adenocarcinoma is a highly metastatic disease and macrophages support liver metastases. Efferocytosis, or engulfment of apoptotic cells by macrophages, is an essential process in tissue homeostasis and wound healing, but its role in metastasis is less well understood. Here, we found that the colonization of the hepatic metastatic site is accompanied by low-grade tissue injury and that efferocytosis-mediated clearance of parenchymal dead cells promotes macrophage reprogramming and liver metastasis. Mechanistically, progranulin expression in macrophages is necessary for efficient efferocytosis by controlling lysosomal acidification via cystic fibrosis transmembrane conductance regulator and the degradation of lysosomal cargo, resulting in LXRα/RXRα-mediated macrophage conversion and upregulation of arginase 1. Pharmacological blockade of efferocytosis or macrophage-specific genetic depletion of progranulin impairs macrophage conversion, improves CD8+ T cell functions, and reduces liver metastasis. Our findings reveal how hard-wired functions of macrophages in tissue repair contribute to liver metastasis and identify potential targets for prevention of pancreatic ductal adenocarcinoma liver metastasis.
Subject(s)
Carcinoma, Pancreatic Ductal , Liver Neoplasms , Macrophages , Pancreatic Neoplasms , Phagocytosis , Tumor Microenvironment , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Humans , Liver Neoplasms/secondary , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Animals , Mice , Macrophages/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Apoptosis , Lysosomes/metabolism , Arginase/metabolism , EfferocytosisABSTRACT
BACKGROUND: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumors at high risk of metastasis would have a significant impact on management. OBJECTIVE: To develop a robust and validated gene expression profile signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach. METHODS: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 nonmetastasizing and 86 metastasizing) were collected retrospectively from four centers. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets. RESULTS: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk. LIMITATIONS: This was a retrospective 4-center study and larger prospective multicenter studies are now required. CONCLUSION: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC.
Subject(s)
Carcinoma, Squamous Cell , Skin Neoplasms , Humans , Carcinoma, Squamous Cell/pathology , Prognosis , Retrospective Studies , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Transcriptome , Prospective Studies , Neoplasm StagingABSTRACT
The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.
Subject(s)
Colorectal Neoplasms , Animals , Humans , Mice , Adenosylhomocysteinase/genetics , Adenosylhomocysteinase/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Metabolomics , Mutation , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Specific cell types and, therefore, organs respond differently during aging. This is also true for the hematopoietic system, where it has been demonstrated that hematopoietic stem cells alter a variety of features, such as their metabolism, and accumulate DNA damage, which can lead to clonal outgrowth over time. In addition, profound changes in the bone marrow microenvironment upon aging lead to senescence in certain cell types such as mesenchymal stem cells and result in increased inflammation. This heterogeneity makes it difficult to pinpoint the molecular drivers of organismal aging gained from bulk approaches, such as RNA sequencing. A better understanding of the heterogeneity underlying the aging process in the hematopoietic compartment is, therefore, needed. With the advances of single-cell technologies in recent years, it is now possible to address fundamental questions of aging. In this review, we discuss how single-cell approaches can and indeed are already being used to understand changes observed during aging in the hematopoietic compartment. We will touch on established and novel methods for flow cytometric detection, single-cell culture approaches, and single-cell omics.
ABSTRACT
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.
Subject(s)
Adenocarcinoma/pathology , DNA Damage/genetics , DNA Repair/genetics , DNA Replication/genetics , Pancreatic Neoplasms/pathology , Adenocarcinoma/genetics , Adenocarcinoma/therapy , Biomarkers , Cell Culture Techniques , Cell Line, Tumor , Humans , Molecular Targeted Therapy , Organoids , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/therapy , Xenograft Model Antitumor AssaysABSTRACT
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 (GSK3ß) a key regulator of glycolysis. Pharmacological inhibition of GSK3ß 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 GSK3ß 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.
Subject(s)
Adenocarcinoma/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , GATA6 Transcription Factor/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Cell Line, Tumor , HumansABSTRACT
The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) ß-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.
Subject(s)
Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Receptor, Notch1/metabolism , Animals , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/mortality , Datasets as Topic , Disease Models, Animal , Gene Expression Profiling , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Male , Mice , Mutation , Neutrophil Activation/drug effects , Neutrophil Activation/genetics , Neutrophils/immunology , Prognosis , Proto-Oncogene Proteins p21(ras)/genetics , Receptor, Notch1/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Survival Analysis , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/metabolism , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
The genomic complexity of profound copy number aberrations has prevented effective molecular stratification of ovarian cancers. Here, to decode this complexity, we derived copy number signatures from shallow whole-genome sequencing of 117 high-grade serous ovarian cancer (HGSOC) cases, which were validated on 527 independent cases. We show that HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in known patterns of genomic aberration. Copy number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Measurement of signature exposures provides a rational framework to choose combination treatments that target multiple mutational processes.
Subject(s)
DNA Copy Number Variations , Mutation , Ovarian Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Female , Genomics/methods , Humans , Middle Aged , Neoplasm Recurrence, Local/genetics , Whole Genome Sequencing/methodsABSTRACT
This corrects the article DOI: 10.1038/nature21063.
ABSTRACT
The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
Subject(s)
Carcinoma, Neuroendocrine/genetics , Genome, Human/genetics , Genomics , Pancreatic Neoplasms/genetics , Base Sequence , Calmodulin-Binding Proteins/genetics , Chromatin Assembly and Disassembly/genetics , Chromosome Aberrations , DNA Copy Number Variations/genetics , DNA Glycosylases/genetics , DNA Mutational Analysis , DNA Repair/genetics , Female , Germ-Line Mutation/genetics , Humans , Male , RNA-Binding Protein EWS , RNA-Binding Proteins/genetics , TOR Serine-Threonine Kinases/metabolism , Telomere/genetics , Telomere/metabolismABSTRACT
Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , DNA Mismatch Repair/genetics , Mutation , Pancreatic Neoplasms/genetics , Transcriptome , Adult , Aged , Aged, 80 and over , DNA Mutational Analysis , Female , Genome , Humans , Male , Middle Aged , MutL Protein Homolog 1/genetics , MutS Homolog 2 Protein/genetics , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Acetamides/pharmacology , Acetamides/therapeutic use , Animals , Antigens, CD34/metabolism , Azepines/pharmacology , Azepines/therapeutic use , Cell Death/drug effects , Cell Differentiation/drug effects , DNA-Binding Proteins/metabolism , Female , Fusion Proteins, bcr-abl/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Imidazolines/pharmacology , Imidazolines/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Male , Mice , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/transplantation , Proteomics , Proto-Oncogene Proteins c-myc/deficiency , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Reproducibility of Results , Signal Transduction/drug effects , Transcriptome , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, 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.
Subject(s)
Genes, Neoplasm/genetics , Genome, Human/genetics , Genomics , Mutation/genetics , Pancreatic Neoplasms/classification , Pancreatic Neoplasms/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Pancreatic Ductal/classification , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Hepatocyte Nuclear Factor 3-beta/genetics , Hepatocyte Nuclear Factor 3-gamma/genetics , Histone Demethylases/genetics , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Humans , Mice , Nuclear Proteins/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Prognosis , Receptors, Cytoplasmic and Nuclear/genetics , Survival Analysis , Trans-Activators/genetics , Transcription Factors/genetics , Transcription, Genetic , Transcriptome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Zebrafish ProteinsABSTRACT
Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Brain Neoplasms/secondary , Gene Expression Profiling/methods , Genomics/methods , Biomarkers, Tumor/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/enzymology , DNA Mutational Analysis , Enzyme Activation , Gene Amplification , Gene Dosage , Gene Expression Regulation, Neoplastic , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Ligands , Molecular Targeted Therapy , Mutation , Phenotype , Phosphorylation , Precision Medicine , Predictive Value of Tests , Protein Kinase Inhibitors/therapeutic use , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Receptor, ErbB-4/genetics , Receptor, ErbB-4/metabolism , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Genome, Human/genetics , Ovarian Neoplasms/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cohort Studies , Cyclin E/genetics , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/genetics , DNA Methylation , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Female , Genes, BRCA1 , Genes, BRCA2 , Genes, Neurofibromatosis 1 , Germ-Line Mutation/genetics , Humans , Mutagenesis/genetics , Oncogene Proteins/genetics , Ovarian Neoplasms/drug therapy , PTEN Phosphohydrolase/genetics , Promoter Regions, Genetic/genetics , Retinoblastoma Protein/geneticsABSTRACT
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.
Subject(s)
DNA Mutational Analysis , Genome, Human/genetics , Genomics , Mutation/genetics , Pancreatic Neoplasms/genetics , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Animals , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , DNA Repair/genetics , Female , Genes, BRCA1 , Genes, BRCA2 , Genetic Markers/genetics , Genomic Instability/genetics , Genotype , Humans , Mice , Pancreatic Neoplasms/classification , Pancreatic Neoplasms/drug therapy , Platinum/pharmacology , Point Mutation/genetics , Poly(ADP-ribose) Polymerase Inhibitors , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/pathology , Carcinogenesis/genetics , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Gene Rearrangement/genetics , Genome, Human/genetics , Carcinogenesis/pathology , Chromosome Breakage , Chromosomes, Human/genetics , Humans , Mutation/geneticsABSTRACT
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.