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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. 1, 2, 3, 4, 1, 3, 5, 6, 7, 1, 1, 7, 3, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 2, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 3, 3, 3, 7, 3, 3, 3, 3, 3, 3, 3, 3, 3, 8, 3, 9, 3, 3, 3, 10, 11, 3, 11, 12, 3, 5, 13, 3, 14, 15, 16, 17, 18, 19, 7, 20, 21, 7, 22, 23, 24, 25, 25, 26, 27, 28, 29, 26, 30, 31, 30, 32, 32, 33, 31, 33, 34, 35 Australian Pancreatic Cancer Genome Initiative,., 3, 11, 26, 25, 30, 31, 7, 1, 2, 3, 5, 6, 7, § and 1, 7, §. Pancreatic cancer remains one of the most lethal of malignancies and a major health burden.
The Notebooks of Henry James by F. Matthiessen (Editor) in DJVU, FB3. PDF the-notebooks-henry-matthiessen-editor.pdf; DOC the-notebooks-henry-matthiessen. A summary and plot synopsis of the tale 'Brooksmith' by Henry James. Henry James Brooksmith. The following is.
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.
Pancreatic cancer (PC) has a median survival of 6 months and a 5-year survival that remains less than 5% despite 50 years of research and therapeutic development. It is the fourth commonest cause of cancer death in Western societies and is projected to be the second leading cause within a decade. As a consequence, there is an urgent need to better select patients for current therapies and develop novel therapeutic strategies.
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Recent exome and CNV analyses of pancreatic ductal adenocarcinoma have revealed a complex mutational landscape.Activating mutations of KRAS are near ubiquitous and inactivation of TP53, SMAD4 and CDKN2A occur at rates of 50%. The prevalence of recurrently mutated genes then drops to 10% for a handful of genes involved in chromatin modification, DNA damage repair and other mechanisms known to be important in carcinogenesis; however, a long tail of infrequently mutated genes dominates, resulting in significant intertumoural heterogeneity. Faced with this diversity, it is not surprising that therapeutic development using an unselected approach to patient recruitment for clinical trials has been challenging –. Somatic structural rearrangement of chromosomes represents a common class of mutation that is capable of causing gene disruption (such as deletion or rearrangement), gene activation (for example, copy number gain or amplification) and the formation of novel oncogenic gene products (gene fusions). Many of these events actively drive carcinogenesis, and in some instances present therapeutic targets.
Early karyotyping and more recent genomic sequencing of small numbers of primary tumours ( n = 3) and metastases ( n = 10) suggests that PDAC genomes contain widespread and complex patterns of chromosomal rearrangement. Here we performed deep whole-genome sequencing of 100 PDACs and show that structural variation (variation in chromosomal structure) is an important mechanism of DNA damage in pancreatic carcinogenesis. We classify PDAC into four subtypes based on structural variation profiles and implicate molecular mechanisms underlying some of these events. Finally, as proof of concept, we use a combination of structural variation, mutational signatures and gene mutations to define putative biomarkers of therapeutic responsiveness for platinum-based chemotherapy, which are current therapeutic options for PDAC –, and for therapeutics that target similar molecular mechanisms such as PARP inhibitors that are currently being tested in clinical trials.
Genomic landscape of pancreatic cancer Patients were recruited and consent obtained for genomic sequencing through participating institutions of the Australian Pancreatic Cancer Genome Initiative (APGI; ) as part of the International Cancer Genome Consortium (ICGC; ). Array-based CNV was analysed using GAP and tumour cellularity estimated with qPure. Whole-genome sequencing was performed on 100 primary PDACs with an epithelial cellularity of ≥ 40% ( n = 75), and complemented by cell lines derived from APGI participants ( n = 25) to an average depth of 65×, and compared to the germline (average depth 38×).
Mutations were detected using qSNP and GATK and indels called with Pindel and GATK. Point mutations and structural variation in PDAC A total of 857,971 somatic point mutations and small insertions and deletions were detected in the cohort: 7,888 were non-silent mutations in 5,424 genes ( and 4). Orthogonal validation of 3,000 exonic mutations estimated the accuracy of mutation calls at 95% (Methods). Consistent with previous estimates, the average mutational burden across the cohort was 2.64 per Mb (range 0.65–28.2 per Mb). Somatic structural variants were identified with the qSV package, which uses multiple lines of evidence to define events (discordant pairs, soft clipping and split reads). Events verified using an orthogonal sequencing method were also included (Methods and ). Where possible, these events were cross-referenced with CNV data (Methods).
In total, 11,868 somatic structural variants were detected at an average of 119 per individual (range 15–558) (Supplementary Table 5 and ). The majority of structural variants were intra-chromosomal (10,114) and were classified into 7 types: intra-chromosomal rearrangements (5,860), deletions (1,393), duplications (128), tandem duplications (179), inversions (1,629), fold-back inversions (579) and amplified inversions (346); inter-chromosomal translocations were less prevalent (1,754) (Supplementary Table 6). A total of 6,908 rearrangements directly disrupted gene sequences and 1,220 genes contained a breakpoint in 2 or more patients (Supplementary Table 7). Recurrent gene fusions were not detected: 1,236 structural variants led to the joining of two gene loci, however, only 183 of these events were fused in an orientation and frame that was capable of expressing a product, and none of these predicted fusion events occurred in more than one sample.
Genes affected by mutation and structural variation Commonly mutated genes that characterize PDAC ( KRAS, TP53, SMAD4 and CDKN2A), were reaffirmed as significant using MutSig analysis (Supplementary Table 8). Combining structural variation events with deleterious point mutations increased the prevalence of inactivation events for TP53 to 74% (3 structural variants and 71mutations), 31%for SMAD4 (9 structural variants and 22 mutations) and 35%for CDKN2A (11 structural variants and 24 mutations).
Two additional genes not previously described in human PDAC ( KDM6A and PREX2) had recurrent pathogenic mutations and structural variants at a rate of 10% or more. KDM6A is a SWI/SNF interacting partner that was identified in a pancreatic sleeping-beauty transposon mutagenesis screen, and is mutated in RCC and medulloblastoma. In our cohort, KDM6A was inactivated in 18% of patients, (4 frame shifts, 1 in-frame deletion and 2 missense mutations, 5 structural variants and 8 homozygous deletions).
In most cases ( n = 15), both alleles of KDM6A were affected. The RAC1 guanine nucleotide exchange factor PREX2, mutated in melanoma was inactivated in 10% of PDAC patients (1 frame shift, 1 splice site and 5 missense mutations, 2 structural variants and1 homozygous deletion). In addition, the tumour suppressor gene RNF43, originally identified in cystic tumours of the pancreas, was inactivated in 10% of PDAC patients (4 frameshift and 4 nonsense mutations, 2 structural variants). Two of these PDACs had an associated intraductal papillary mucinous neoplasm (IPMNs). Recent studies have suggested that loss of functional RNF43 may confer sensitivity to WNT inhibitors. Shows the prevalence of aberrations in key driver genes and pathways in PDAC; implicating structural variation as an important mutational mechanism in pancreatic carcinogenesis.
Locally rearranged subtype Subtype 2 was classified as ‘locally rearranged’ (30% of all samples). This subtype exhibited a significant focal event on one or two chromosomes. The group could be further divided into those with focal regions of gain/amplification and those that contained complex genomic rearrangements.
Approximately one-third of locally rearranged genomes contained regions of copy number gain that harboured known oncogenes (Supplementary Table 9). These included common focal amplifications in KRAS, SOX9 and GATA6 and often included therapeutic targets such as ERBB2, MET, CDK6, PIK3CA and PIK3R3, but at low individual prevalence (1–2% of patients) (Supplementary Table 9). The remaining local rearrangements involved complex genomic events such as breakage–fusion–bridge (BFB, n = 9) or chromothripsis, ( n = 15), which resulted in a ring chromosome in at least one case (ICGC0059) ( and ). Chromothripsis is linked to TP53 mutations in medullobastoma and acute myeloid leukaemia and here, 10/13 chromothriptic tumours had a TP53 mutation, 5 of which were bi-allelic. Five of these chromothriptic events occurred after chromosomal duplication suggesting that they are less likely to be driving carcinogenesis (Methods).
Genomic markers of defective DNA maintenance We mapped the relationship between the unstable subtype, mutations in BRCA pathway genes and a recently described mutational signature associated with deleterious mutations in BRCA1 or BRCA2 in breast, ovarian and pancreatic cancer. The majority of unstable tumours (10 of 14) fell within the top quintile of the BRCA signature when ranked by prevalence per Mb.
In addition, the top quintile of the BRCA signature was associated with deleterious mutations of BRCA1 ( n = 2), BRCA2 ( n = 7), and PALB2 ( n = 2) (Supplementary Table 10). Four of the BRCA2 mutations were germline in origin (3 frameshift and 1 nonsense), and in each case, the wild-type allele was inactivated in the tumour.
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A further 2 patients had somatic mutations in BRCA1 (both with splice site mutations), and another 3 had somatic BRCA2 mutations (1 indel and 2 splice site mutations). All deleterious BRCA1 and BRCA2 mutations had inactivation of the second allele. Three patients had pathogenic germline PALB2 mutations that were associated with the BRCA mutational signature.
One of these was a TGTT deletion, which is known to occur in pancreatic cancer (this tumour also had a somatic BRCA2 mutation), and the mutations of PALB2 in both the other 2 cases are associated with an inherited predisposition to breast cancer. Germline PALB2 mutation carriers did not have evidence of somatic loss of the second allele; however, heterozygous germline mutation of PALB2 appears sufficient to cause DNA replication and damage response defects. In contrast, tumours containing a somatic heterozygous silent mutation of BRCA2, a heterozygous intronic structural variation and 2 unclassified heterozygous missense mutations in BRCA1 (predicted to be benign or only possibly damaging by Polyphen2) were not associated with a high-ranking BRCA mutational signature (.
Defective DNA repair without BRCA pathway mutations Mutations in BRCA pathway genes accounted for approximately half of patients with a high BRCA mutational signature and/or an unstable genome. Hyper-methylation is known to play a role in silencing BRCA1, BRCA2 and PALB2 in some breast and ovarian cancers; however, high-density methylome array profiling of this cohort allowed us to exclude this as a contributing mechanism. Single instances of biallelic, inactivating, somatic mutation was observed for two genes known to induce genomic instability and chemosensitivity when inactivated: RPA1 ( ) (splice site and loss of heterozygosity (LOH)), and the DNA polymerase zeta catalytic unit/ REV3L (nonsense and LOH). We also detected mutations in other genes involved in DNA maintenance such as ATM, FANCM, XRCC4 and XRCC6 in tumours with an unstable genome or the BRCA mutational signature; however, they are yet to be causally linked to these genomic events or sensitivity to DNA-damaging agents. Putative genotypes of platinum responsiveness As the APGI was a prospective observational cohort study with extensive clinical follow-up, it was possible to track therapeutic responsiveness of participants that received chemotherapy when their disease recurred. At the time of analysis, 53 patients had documented recurrences and 25 received a variety of chemotherapeutic agents (Supplementary Table 11).
This analysis was complemented through therapeutic testing of patient-derived xenografts (PDXs) generated from APGI participants. Overall, 8 patients received a platinum-based therapy and 7 PDXs were treated with gemcitabine and cisplatin. Of 5 patients with unstable genomes and/or a high BRCA mutational signature burden (designated as ‘on-genotype’) 2 had exceptional responses (defined as complete radiological resolution of disease and normalization of CA19.9 levels ), and 2 had robust partial responses based on RECIST1.1 criteria , while 3 patients who did not have any of these characteristics (‘off-genotype’) did not respond.
These observations were supported by PDX studies where 2 of 3 on-genotype PDXs responded to cisplatin (one BRCA2 mutant responded and one carrying bi-allelic inactivation of RPA1, which notably retained RAD51 foci also responded. Another, with a mutational signature but not an unstable genome, and without a mutation in a BRCA pathway gene, did not respond.
This compares to no responses in the 4 PDXs in the off-genotype group ( and ). Combining patient and PDX response data, on-genotype tumours were associated with response to platinum-based therapy ( P = 0.0070, Fisher’s exact test, ) (Supplementary Table 11).