Craig Nourse

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.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

The analysis of T-cell antigens in long-term survivors of pancreatic ductal adenocarcinoma suggests that neoantigen immunogenicity and quality, not purely quantity, correlate with survival. A small percentage of patients with pancreatic cancer survive beyond five years, but the reason for their relative longevity remains uncertain. In this retrospective analysis, Vinod Balachandran et al. evaluate the immune mechanisms of long-term survival in human pancreatic cancer. The analysis shows that survival correlates with high mutation load in conjunction with increased infiltration of cytolytic T cells and polyclonal T-cell responses and that mutations at the tumour antigen MUC16 locus are enriched in long-term survivors. Additionally, patients with high predicted neoantigen–microbial cross-reactivity scores tended to live longest. The authors provide evidence that the quality rather than quantity of neoantigens determines survival. Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors1,2, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

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. The genomes of 102 primary pancreatic neuroendocrine tumours have been sequenced, revealing mutations in genes with functions such as chromatin remodelling, DNA damage repair, mTOR activation and telomere maintenance, and a greater-than-expected contribution from germ line mutations. Pancreatic neuroendocrine tumours (PanNETs) are the second most common epithelial neoplasm of the pancreas. Aldo Scarpa, Sean Grimmond and colleagues report whole-genome sequencing of 102 primary PanNETs and present analysis of their mutational signatures as part of the International Cancer Genome Consortium. They find frequent mutations in genes with functions that include chromatin remodelling, DNA damage repair, activation of mTOR signalling, and telomere maintenance. They also identify mutational signatures, including one resulting from inactivation of the DNA repair gene MUTYH, and report a larger than expected germline contribution to PanNET development.

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. The authors identify copy number signatures from shallow whole-genome sequencing of high-grade serous ovarian cancer (HGSOC) cases. HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in genomic aberration.

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) β-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.

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.

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. Leukaemic stem cells (LSCs) are responsible for BCR–ABL-driven chronic myeloid leukaemia relapse; here, p53 and MYC signalling networks are shown to regulate LSCs concurrently, and targeting both these pathways has a synergistic effect in managing the disease. Tyrosine kinase inhibitors are a first-line therapy in patients with chronic myeloid leukaemia (CML), where they target the oncogenic BCR-ABL fusion gene. However, relapse inevitably occurs, probably driven by a drug-resistant population of leukaemic stem cells (LSCs). This study uncovers the concurrent involvement of p53 and Myc signalling networks in regulating LSCs. The authors demonstrate that genetic and/or pharmacological targeting of both the p53 and c-Myc pathways achieves more effective disease neutralization in mouse and human cell models of CML.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

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. 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. Pancreatic ductal adenocarcinoma has a 5-year survival of <5%, with therapies offering only incremental benefit,1Vogelzang N.J. et al.J Clin Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar potentially due to the diversity of its genomic landscape.2Bailey P. et al.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1973) Google Scholar, 3Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1379) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar Recent reports link high mutation burden with response to immune checkpoint inhibitors in several cancer types.5Le D.T. et al.N Engl J Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6099) Google Scholar Defining tumors that are hypermutated with an increased mutation burden and understanding the underlying mechanisms in pancreatic cancer has the potential to advance therapeutic development, particularly for immunotherapeutic strategies. Whole genome sequencing (WGS, n = 180) and whole exome sequencing (n = 205) of 385 unselected predominantly sporadic pancreatic ductal adenocarcinoma (Supplementary Table 1) defined a mean mutation load of 1.8 and 1.1 mutation per megabase (Mb), respectively (Supplementary Table 2). Outlier analysis identified 20 tumors with the highest mutation burden (5.2%, 15 WGS and 5 exome) (Table 1 and Supplementary Figure 1A), 5 of which were considered extreme outliers and classified as hypermutated as they contained ≥12 somatic mutations/Mb, the defined threshold for hypermutation in colorectal cancer.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (5894) Google Scholar Immunohistochemistry for mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, and PMS2) identified 4 MMR-deficient tumors, all of which were hypermutated (n = 180, Figure 1).Table 1Clinical and Histologic Features and Proposed Etiology for Highly Mutated Pancreatic Ductal Adenocarcinoma Tumors (n = 20)Sample IDPersonal and family history of malignancyHistologyMutation load, mutations/MbIHC resultMSIsensor scoreKRAS mutationPredominant mutation signature (mutations/Mb)SV subtype (no. of events)Proposed etiologyHypermutation (extreme outliers) ICGC_0076aSample sequenced by WGS, other samples by exome sequencing.NoneMixed signet ring, mucinous and papillary adenocarcinoma38.55Absent MLH1 and PMS228.3p.G12VMMR (18.3)Scattered (131)MMR deficiency: >280 kb somatic homozygous deletion over MSH2. ICGC_0297aSample sequenced by WGS, other samples by exome sequencing.NoneUndifferentiated adenocarcinoma60.62Absent MSH2 and MSH627.33WTMMR (33.4)Scattered (75)MMR deficiency: Somatic MLH1 promoter hypermethylation. ICGC_0548aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, moderately differentiated30.13Absent MSH2 and MSH617.47WTMMR (16.6)Stable (49)MMR deficiency: >27 kb somatic inversion rearrangement disrupting MSH2. ICGC_0328aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma16.63Normal3.2p.G12DUnknown (11.9)Scattered (110)Cell line with signature: etiology unknown. ICGC_00901 FDR, father CRCDuctal adenocarcinoma, moderately differentiated12.9Absent MSH2 and MSH60.21p.G12CNANAMMR deficiency: somatic MSH2 splice site c.2006G>A.Highly mutated tumors ICGC_0054aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.52Normal0.01p.G12VHR deficiency (1.3)Unstable (310)HR deficiency: no germline or somatic cause found. ICGC_0290aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.54Not available0.07p.G12VHR deficiency (3.1)Unstable (558)HR deficiency: Germline BRCA2 mutation c.7180A>T, p.A2394*. Somatic CN-LOH. ICGC_0215aSample sequenced by WGS, other samples by exome sequencing.2 FDR lung cancer, 2 FDR prostate cancer. Previous CRC and melanomaDuctal adenocarcinoma, moderately differentiated6.27Normal0.01p.G12VHR deficiency (1.9)Scattered (111)HR deficiency: Germline ATM mutation c.7539_7540delAT, p.Y2514*. Somatic CN-LOH. ICGC_0324NoneDuctal adenocarcinoma, moderately differentiated6.24Normal0p.G12DNANAUndefined ICGC_0034aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.09Normal4.02p.G12DHR deficiency (3.4)Unstable (366)HR deficiency: Germline BRCA2 mutation c.5237_5238insT, p.N1747*. Somatic CN-LOH. ICGC_0131aSample sequenced by WGS, other samples by exome sequencing.Lung cancer after PCDuctal adenocarcinoma, moderately differentiated5.63Normal0p.G12DT>G at TT sites (3.0)Focal (147)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0006aSample sequenced by WGS, other samples by exome sequencing.1 FDR, father lung cancerAdenocarcinoma arising from IPMN, moderately differentiated5.29Normal0.01p.G12DHR deficiency (1.2)Unstable (211)HR deficiency: Somatic BRCA2 c.5351dupA, p.N1784KfsTer3. Somatic CN-LOH. ICGC_0321aSample sequenced by WGS, other samples by exome sequencing.2 FDR, mother and cousin breast cancerDuctal adenocarcinoma, poorly differentiated4.79Not available0p.G12DHR deficiency (2.1)Unstable (286)HR deficiency: Germline BRCA2 c.6699delT, p.F2234LfsTer7. Somatic CN loss- 1 copy. ICGC_0309aSample sequenced by WGS, other samples by exome sequencing.NoneAdenocarcinoma arising from IPMN, moderately differentiated4.74Normal0.03p.G12VT>G at TT sites (3.1)Unstable (232)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0005aSample sequenced by WGS, other samples by exome sequencing.1 FDR, mother CRCDuctal adenocarcinoma, poorly differentiated4.72Not available1p.G12VHR deficiency (1.1)Focal (95)HR deficiency: No germline or somatic cause found. ICGC_0016aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.61Normal3.03p.G12VHR deficiency (1.7)Unstable (447)HR deficiency: potentially linked to Somatic RPA1 c.273G>T, p.R91S ICGC_00461 FDR, brother PCDuctal adenocarcinoma, poorly differentiated4.3Normal0p.Q61HNANAUndefined GARV_0668aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.3Not available2.19p.G12VHR deficiency (1.6)Unstable (464)HR deficiency: Germline BRCA2 c.7068_7069delTC, p.L2357VfsTer2. Somatic CN loss - 1 copy. ICGC_0291NoneDuctal adenocarcinoma, well differentiated3.84Not available0.03p.G12RNANAHR deficiency: Somatic BRCA2 c.7283T>A, p.L2428*. ICGC_0256NoneDuctal adenocarcinoma, poorly differentiated3.72Not available0.06p.G12DNANAUndefinedCRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data.a Sample sequenced by WGS, other samples by exome sequencing. Open table in a new tab CRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data. KRAS mutation status and histopathologic characteristics have been associated with MMR-deficient pancreatic tumors.7Goggins M. et al.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Of the 4 MMR-deficient tumors in our cohort, 2 were KRAS wild-type; 3 had undifferentiated to moderately differentiated histology and one had a signet-ring component. These features were not predictive of MMR deficiency in our cohort, as 11 additional non−MMR-deficient tumors had a signet-ring cell component or colloid morphology, and 131 of 347 assessable tumors had poorly or undifferentiated histology. Mutational signature analysis can detect MMR deficiency indirectly based on the pattern of somatic mutations.8Alexandrov L.B. et al.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6213) Google Scholar An MMR-deficient signature dominated the MMR-deficient tumors (with WGS), and was minimal in MMR intact tumors (Supplementary Figure 1). In addition, microsatellite instability (MSI), a hallmark of MMR deficiency in colorectal cancer, was detected in all three MMR deficient tumors with WGS using MSIsensor9Niu B. Ye K. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (294) Google Scholar (Supplementary Table 2). MSI was not identified for the fourth MMR deficient sample potentially due to the reduced number of microsatellite loci in exome data. The underlying causes of MMR deficiency in the 4 cases were private somatic events. For 2 cases, MSH2 was disrupted by different structural rearrangements, 1 case contained a missense MSH2 mutation and the last, methylation of the MLH1 promoter (Figure 1). The missense mutation caused an MSH2 splice acceptor site mutation that alters the same nucleotide results in a pathogenic skipping of exon 13 in germline studies.10Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (346) Google Scholar Hypermethylation of the MLH1 promoter is the predominant mechanism of MSI in sporadic colon cancer.11Boland C.R. et al.Gastroenterology. 2010; 138: 2073-2087 e3Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar The remaining hypermutated tumor contained an intact MMR pathway, and was a cell line (ATCC, CRL-2551) with an unidentified mutational signature, therefore the high mutation burden in this sample may be the result of long-term cell culture. The 15 samples (11 WGS and 4 exome) identified in the outlier analysis with high mutation burden, but not hypermutated (∼4 to 12 mutations/Mb) contained no evidence of MMR deficiency. Mutational signature analysis of the WGS samples indicated homologous recombination (HR) repair deficiency as the most substantial (range, 1.0–3.4 mutations/Mb) contributor to the mutation burden for 8 WGS mutation load outlier tumors. In support of a HR defect4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar; 7 of these tumors contained high levels of genomic instability with >200 structural variants and mutations in genes involved in HR were present for 6 of 8 cases (Supplementary Table 2). In addition, 1 case that had undergone exome sequencing had a somatic BRCA2 nonsense mutation that likely contributed to HR deficiency in this case. A mutational signature associated with T>G mutations at TT sites previously described in other cancers, including esophageal cancer12Nones K. Waddell N. Wayte N. et al.Nat Commun. 2014; : 5Google Scholar was the major contributor (>3 mutations/Mb) in 2 samples. For these 2 and the remaining 4 cases, no potential causative event could be identified. Although germline defects in MMR genes are well reported in pancreatic cancer13Grant R.C. Selander I. et al.Gastroenterology. 2015; 148: 556-564Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar in our cohort, they did not contribute to MMR deficiency even in those with familial pancreatic cancer or a personal or family history of Lynch-related tumors. A germline truncating variant was detected in PMS2 in 1 case, but did not have loss of the second allele, had normal immunohistochemistry staining and did not display a MMR mutational signature (Supplementary Table 2). MMR deficiency is important in the evolution in a small, but meaningful proportion of pancreatic cancers with a prevalence of 1% (4 of 385) in our cohort. This is consistent with recent studies using the Bethesda polymerase chain reaction panel,14Laghi L. et al.PLoS One. 2012; 7: e46002Crossref PubMed Scopus (55) Google Scholar and with previous estimates of MSI prevalence of 2%−3%.15Nakata B. et al.Clin Cancer Res. 2002; 8: 2536-2540PubMed Google Scholar However, in tumors with low epithelial content that underwent exome sequencing, the sensitivity of somatic mutation detection is reduced, which will affect mutation burden and signature analysis. While cognizant of small numbers, immunohistochemistry was the most accurate in defining MMR due to multiple genomic mechanisms of MMR gene inactivation. Multiple methods to define MMR deficiency may be required for clinical trials that aim to recruit MMR-deficient participants to assess the potential efficacy of checkpoint inhibitors or other therapies in pancreatic cancer. Homologous recombination-deficient tumors, and those with a novel signature seen in esophageal cancer had an increased mutation burden, and need further evaluation as potential patient selection markers for clinical trials of checkpoint inhibitor and other therapies that target tumors with a high mutation burden. The authors would like to thank Cathy Axford, Deborah Gwynne, Mary-Anne Brancato, Clare Watson, Michelle Thomas, Gerard Hammond, and Doug Stetner for central coordination of the Australian Pancreatic Cancer Genome Initiative, data management, and quality control; Mona Martyn-Smith, Lisa Braatvedt, Henry Tang, Virginia Papangelis, and Maria Beilin for biospecimen acquisition; and Sonia Grimaldi and Giada Bonizzato of the ARC-Net Biobank for biospecimen acquisition. For a full list of contributors see Australian Pancreatic Cancer Genome Initiative: http://www.pancreaticcancer.net.au/apgi/collaborators. The cohort consisted of 385 patients with histologically verified pancreatic exocrine carcinoma, prospectively recruited between 2006 and 2013 through the Australian Pancreatic Cancer Genome Initiative (www.pancreaticcancer.net.au) as part of the International Cancer Genome Consortium.1Hudson T.J. et al.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1689) Google Scholar Ethical approval was granted at all treating institutions and individual patients provided informed consent upon entry to the study. The clinicopathologic information for the cohort is described in (Supplementary Table 1), and the global mutation profile has previously been reported for some of these tumors (Supplementary Table 2). Tumor and normal DNA were extracted after histologic review from fresh frozen tissue samples collected at the time of surgical resection or biopsy, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar Tumor cellularity was determined from single-nucleotide polymorphism array data using qpure.3Song S. et al.PLoS One. 2012; 7: e45835Crossref PubMed Scopus (85) Google Scholar Tumors with epithelial content ≥40% underwent WGS lower cellularity tumors underwent whole exome sequencing. DNA from patient-derived pancreas cell lines and matched normal was also extracted. Exome and WGS were performed using paired 100-bp reads on the Illumina HiSeq 2000, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar Regions of germline and somatic copy number change were detected using Illumina SNP BeadChips with GAP.5Popova T. et al.Genome Biol. 2009; 10 (R128−R128)Crossref PubMed Scopus (151) Google Scholar Somatic structural variants were identified from WGS reads using the qSV tool.4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar, 6Patch A.M. et al.Nature. 2015; 521: 489-494Crossref PubMed Scopus (930) Google Scholar Single nucleotide variants were called using 2 variant callers: qSNP7Kassahn K.S. et al.PLoS One. 2013; 8: e74380Crossref PubMed Scopus (52) Google Scholar and GATK.8McKenna A. et al.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14755) Google Scholar Mutations identified by both callers or, those that were unique to a caller but verified by an orthogonal sequencing approach, were considered high confidence and used in all subsequent analyses. Small indels (<200 bp) were identified using Pindel9Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1391) Google Scholar and each indel was visually inspected in the Integrative Genome Browser. The distribution of the total number of small somatic mutations (coding and noncoding single nucleotide and indel variants) identified per megabase for exome and WGS sequence data were analyzed separately. The group of samples with high mutation load, at the top of each distribution, were defined as the upper distribution outliers for mutations per megabase, that is, ≥75th centile + (1.5× interquartile range). The threshold for detecting outliers in the exome and WGS groups was 3.4 and 4.2 mutations/Mb, respectively. From within the highly mutated set of tumors, hypermutated samples were identified as those with a mutation rate exceeding the thresholds for extreme distribution outliers (≥75th centile + [5× interquartile range]) of 7.4 and 8.1 mutations/Mb for exome and WGS sequencing, respectively. MSIsensor was used to detect microsatellite instability by directly comparing microsatellite repeat lengths between paired normal and tumor sequencing data.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar A MSIsensor score of >3.5% of somatic microsatellites with repeat length shifts was the detection threshold used to indicate microsatellite instability as published for endometrial cancer.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar This correlated well with the 5 and 7 microsatellite panels recommended in the Bethesda guidelines.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar, 11Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Tissue microarrays were constructed using at least three 1-mm formalin-fixed, paraffin-embedded tumor cores. Immunohistochemistry for MSH6 and PMS2 proteins was performed on tissue microarray sections as a screen for MMR deficiency due to MMR proteins forming heterodimers with concordant mismatch repair loss (ie, loss of MLH1 and PMS2 or loss of MSH2 and MSH6).12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar Immunohistochemistry on full tumor sections for MSH2, MLH1, MSH6, and PMS2 was performed in those with abnormal staining in core sections. The immunohistochemistry was performed as described previously12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar and scored by a senior pathologist. Somatic mutational signatures were extracted from the whole genome sequenced samples using the framework described previously.13Alexandrov L.B. et al.Cell Rep. 2013; 3: 246-259Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar High confidence somatic substitutions were classified by the substitution change and sequence context, that is, the type of immediately neighboring bases to the variant. The framework processes the counts of somatic mutations at each context within each sample using non-negative factorization to produce the different signature profiles that are present in the data. The profiles identified were matched against reported signatures from the Cancer of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic/signatures). The major contributory signatures, defined as the mutational signature with the highest number of contributing somatic substitution variants, is reported for highly mutated whole genome samples. Bisulfite-converted whole-genome amplified DNA was hybridized to Infinium Human Methylation 450K Beadchips according to the manufacturers protocol (Illumina). Methylation arrays were performed on DNA from 174 pancreatic ductal adenocarcinoma samples, which were compared to DNA from 29 adjacent nonmalignant pancreata. A subset of the methylation data has been published previously.14Nones K. et al.Int J Cancer. 2014; 135: 1110-1118Crossref PubMed Scopus (156) Google Scholar We examined the data for evidence of tumor-specific hypermethylation of the promoter region of MLH1 and MSH2 genes. The methylation array data have been deposited into the International Cancer Genome Consortium data portal (dcc.icgc.org, project PACA-AU). Download .xlsx (.08 MB) Help with xlsx files Supplementary Tables 1 and 2

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.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.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.Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Abstract 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 &lt; 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. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

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

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

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.

The TPD52 (tumor protein D52)-like proteins are small coiled-coil motif-bearing proteins which were first identified though their expression in human breast carcinoma. TPD52-like proteins are known to interact in hetero-and homomeric fashions, but there are no known heterologous binding partners for these proteins. We now report the cloning of a novel member of the MAL proteolipid family, named MAL2, though its interaction with a TPD52L2 bait in a yeast two-hybrid screen. MAL2 is predicted to be 176 residues (19 kDa) with four transmembrane domains and is 35.8% identical to MAL, a proteolipid required in apical vesicle transport. The MAL2 prey bound all TPD52-like baits tested in the yeast two-hybrid system and in vitro translation of MAL2 produced a single 19-kDa 35S-labeled protein which specifically bound full-length GST–Tpd52 in GST pull-down assays. The gene MAL2, which was localized to human chromosomal band 8q23 and shown to consist of four exons, is predominantly expressed in human kidney, lung, and liver. Our study has therefore identified a novel member of the MAL proteolipid family and potentially implicates TPD52-like proteins in vesicle transport.

Abstract Background Intraductal Papillary Mucinous Neoplasms (IPMN) remain the largest subtype of cystic pancreatic cancer precursors. Cancer risk has been observed to vary between histological grades and histological subtypes. Spatial characterisation has revealed variation in immune cell distribution disease subtypes. Methods A cohort of 14 surgically resected IPMN tumors across a range of histological grades (Low-grade LG, high-grade HG and invasive IPMN cancers IPMN-PDAC) and subtypes (gastric, intestinal, pancreaticobiliary) underwent multimodal spatial interrogation. After expert histopathological annotation; tissue sections underwent regional whole transcriptome analysis with TempO-seq and 10x Visium. Extensive region analysis with NanoString GeoMx employing segmentation using staining for epithelium (PanCK) fibroblast stroma (aSMA) was performed. A tissue microarray was then constructed, and representative cores underwent single-cell and subcellular spatial transcriptomic analysis with NanoString CosMx. Raw count data and digital images were exported and analysed using Seurat, Giotto, SPATA2 and custom R pipelines. Region deconvolution, Harmony integration, Leiden Clustering, Gene Set Enrichment Analysis, Trajectory Mapping, and Moran’s I Analysis were performed. Results TempO-seq revealed up-regulation of canonical oncogene expression in all IPMN-PDAC cases as compared to LG and HG IPMN. There were no statistically significant differences in expression comparing LG and HG IPMN lesions. Visium differential gene expression and clustering models identified cancer epithelium, stroma and lymphatic components that correlated with histopathological annotations. Immune cell rich spots were identified via gene ontology of top 50 marker genes. The spatial trajectory of B-cell expression signatures in IPMN-PDAC showed greater concentration and less variability than HG tumors, Moran’s I -0.34 vs 0.12 (P = 0.021). B-cells in HG IPMN were showed greater concentration and less variability than LG IPMN, Moran’s I -0.15 vs 0.02 (P=0.05). Much of this concentration was in identified lymphoid aggregates or tertiary lymphoid structures. The spatial trajectory of T-cell expression. The spatial trajectory of macrophage expression showed greater concentration and less variability than in LG and HG tumors. GeoMx analysis of stromal regions identified up regulation of B cell signatures in IPMN PDAC and HG IPMN when compared to LG IPMN. Markers associated with TLS formation were found to be significantly upregulated including CXCL13, CXCR5 and LAMP3. Pancreaticobiliary subtypes were found to have the least concentrated B-cell distribution across subtypes. Conclusions Immune cell composition was found to vary across histological grades and subtypes of IPMN. A paradoxical relationship was observed between T-cell infiltrates and B-cell and macrophage populations. The greater proportion of tertiary lymphoid structures, and high levels of TLS specific gene expression, may indicate a propensity for b-cell activation in IPMN tumors which progress to malignancy. Citation Format: Andrew J. Cameron, Assya Legrini, Colin S. Wood, Craig Nourse, Yoana Doncheva, Claire Kennedy Dietrich, Colin Nixon, Jennifer Hay, Fraser Duthie, Pawel Herzyk, Jennifer Morton, Nigel B. Jamieson. Multiomic modelling of pancreatic IPMN stroma reveals distinct tertiary lymphoid structure distribution: Mapping the transcriptomic landscape via regional bulk, single-cell and subcellular approaches [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A028.

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.

D52 proteins are emerging as signalling molecules which may be regulators of cell proliferation. Having previously reported the existence of the human D52 gene family, comprising the hD52 and hD53 genes expressed in human breast carcinoma, we report the identification of a novel human gene hD54 (TPD52L2), which represents a third D52 gene family member. In situ mapping placed the hD54 gene on human chromosome 20q13.2–q13.3, a localization distinct from those of both hD52 and hD53 genes. The identified hD54 cDNAs predicted three hD54 isoforms, suggesting that alternatively-spliced transcripts may be produced from D52-like genes. This was confirmed by directly sequencing reverse transcriptase–polymerase chain reaction (RT–PCR) products amplified from D52-like gene transcripts expressed in developing and adult rat tissues, and by performing sequence analyses of the expressed sequence tag divisions of nucleotide databases. Alternative splicing of sequences encoding two regions, termed ins2 and ins3, was identified in one or more D52-like genes, with these alternative splicing events being differentially regulated. The functional consequences of alternative splicing were examined by characterizing the protein–protein interactions mediated by a truncated hD53 isoform within the yeast two-hybrid system. This hD53 isoform displayed altered interaction capabilities with respect to those of full-length hD53, suggesting that alternative splicing within the D52 gene family functions in part to alter the protein–protein interaction capabilities of encoded isoforms.

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.

BackgroundMetastasis 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.ObjectiveTo develop a robust and validated gene expression profile signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach.MethodsArchival 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.ResultsA 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.LimitationsThis was a retrospective 4-center study and larger prospective multicenter studies are now required.ConclusionThe 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC. 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. To develop a robust and validated gene expression profile signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach. 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. 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. This was a retrospective 4-center study and larger prospective multicenter studies are now required. The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC.

The resurgence of tuberculosis worldwide has closely mirrored the HIV pandemic. In regions like sub-Saharan Africa, a large proportion of individuals are co-infected with Mycobacterium tuberculosis and HIV. Macrophages are the reservoir host cells for both pathogens, however the interactions between both pathogens in co-infected cells remain poorly understood. Thus, the global gene responses of primary human macrophages following productive co-infection with highly purified HIV and M. tuberculosis were analyzed using cDNA microarrays. A broad range of genes was up-regulated in response to co-infection or M. tuberculosis infection of primary macrophages, including those encoding pro-inflammatory chemokines and cytokines, their receptors, signalling associated genes, type I IFN signalling genes and genes of the tryptophan degradation pathway. Real-time RT-PCR analysis confirmed up-regulation of a wide variety of genes including indoleamine 2,3 dioxygenase and Sp110 in M. tuberculosis and co-infected samples. Downstream analysis confirmed significant elevation of the chemokines CCL3, CCL4 and CCL8 in M. tuberculosis and co-infected culture supernatants. In contrast, the changes seen in gene expression following HIV infection alone were fewer in number and significantly less in magnitude. Thus, the effects of M. tuberculosis infection on global gene expression dominated the effects of HIV-1 in co-infected primary human macrophages.

This corrects the article DOI: 10.1038/nature21063.

NHS genetics centres in Scotland sought to investigate the Genomics England 100,000 Genomes Project diagnostic utility to evaluate genome sequencing for in rare, inherited conditions. Four regional services recruited 999 individuals from 394 families in 200 rare phenotype categories, with negative historic genetic testing. Genome sequencing was performed at Edinburgh Genomics, and phenotype and sequence data were transferred to Genomics England for variant calling, gene-based filtering and variant prioritisation. NHS Scotland genetics laboratories performed interpretation, validation and reporting. New diagnoses were made in 23% cases - 19% in genes implicated in disease at the time of variant prioritisation, and 4% from later review of additional genes. Diagnostic yield varied considerably between phenotype categories and was minimal in cases with prior exome testing. Genome sequencing with gene panel filtering and reporting achieved improved diagnostic yield over previous historic testing but not over now routine trio-exome sequence tests. Re-interpretation of genomic data with updated gene panels modestly improved diagnostic yield at minimal cost. However, to justify the additional costs of genome vs exome sequencing, efficient methods for analysis of structural variation will be required and / or cost of genome analysis and storage will need to decrease.

Abstract Genomic complexity from profound copy-number aberration has prevented effective molecular stratification of ovarian and other cancers. Here we present a method for copy-number signature identification that decodes this complexity. We derived eight signatures using 117 shallow whole-genome sequenced high-grade serous ovarian cancer cases, which were validated on a further 497 cases. Mutational processes underlying the copy-number signatures were identified, including breakage-fusion-bridge cycles, homologous recombination deficiency and whole-genome duplication. We show that most tumours are heterogeneous and harbour multiple signature exposures. We also demonstrate that copy number signatures predict overall survival and changes in signature exposure observed in response to chemotherapy suggest potential treatment strategies.

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 Intratumor heterogeneity and phenotypic plasticity drive tumour progression and therapy resistance. Oncogene dosage variation contributes to cell state transitions and phenotypic heterogeneity, thereby providing a substrate for somatic evolution. Nonetheless, the genetic mechanisms underlying phenotypic heterogeneity are still poorly understood. Here, we show that extrachromosomal DNA (ecDNA) is a major source of high-level focal amplification in key oncogenes and a major contributor of MYC heterogeneity in pancreatic ductal adenocarcinoma (PDAC). We demonstrate that ecDNA can drive exceptionally high dosage of MYC and afford cancer cells rapid adaptation to microenvironmental changes. The continued maintenance of extrachromosomal MYC is uniquely ensured by the presence of the selective pressure. We also show that MYC dosage affects cell morphology and dependence of cancer cells on stromal niche factors, with the highest MYC levels correlating with squamous-like phenotypes. Our work provides the first detailed analysis of ecDNAs in PDAC and describes a new genetic mechanism driving MYC heterogeneity in PDAC.

The identification of molecularly defined subgroups of Pancreatic ductal Adenocarcinoma (PDAC) has the potential to transform clinical practice. There is now a growing consensus that PDAC can be divided into transcriptomic subtypes with 2 broad linages 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 linage specification, HNF4A and GATA6, switch metabolic profiles from Classical (Pancreatic) to predominantly Squamous, with GSK3B a key regulator of glycolysis. Pharmacological inhibition of GSK3B results in selective sensitivity in the Squamous subtype, however a subset of these Squamous PDCLs acquired rapid drug tolerance. Using chromatin accessibility maps, we identify subtype specific chromatin landscapes and unique promoter usage between subpopulations of Squamous PDCLs can affect drug tolerance. Our findings demonstrate that a chromatin-based framework can be used to identify subtypes of PDAC that are indistinguishable by gene expression profiles which could refine patient selection for precision medicine.

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.

ABSTRACT 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. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that 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, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo . We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy. Abstract Figure STATEMENT OF SIGNIFICANCE We define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

Abstract BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate &amp;lt;0.05. RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.