Search

SV loads in non-BRCA1/2 HR genes per sample by SV type

Diagnostic performance of urine biomarkers in discriminating pancreatic adenocarcinoma all stages (A-C) and stage I-II (D-F) from chronic pancreatitis patients. A, ROC curves of PDAC (n=143) versus CP (n=62) patients for individual urine biomarkers in the training set (70% of the data). B, ROC curves of PDAC versus CP patients for the panel in the training set and in the independent validation set (30% of the data, PDAC n=49, CP n=30). C, Summary table. D, ROC curves of individual urine biomarkers in training dataset (70%, PDAC stage I-II n= 56, CP=66). E, ROC curves of the panel in training and validation (PDAC stage I-II n=15, CP n=26) dataset. F, Summary table. Cnorm, creatinine-normalised, creat, creatinine, AUC: area under the curve SN: sensitivity, SP: specificity with 95% Confidence Interval (CI). SN and SP in the validation set were derived for optimal cutpoint determined in the training dataset.

A full list of mutations identified in SCC and MCT samples

Kaplan Meier overall survival curves by PIK3CA, CDKN2A and TP53 allelic mutation state

Figures S12-S14: Flow cytometry - Ly6C:Ly6G; Flow cytometry - F4:80;iNOS;CD206; Flow cytometry - gating strategy

Figures S5-S9: Survival of mice bearing Trp53-/- and Trp53-/-:Brca2-/- tumors; Disease distribution and ascites production in mice bearing Trp53-/-:Brca2-/- tumors; CD3 staining in Trp53-/-:Brca2-/- tumors; Quantification of lymphoid aggregates by absolute area and percentage tumor area; CD8 staining in Trp53-/-:Brca2-/- tumors

Expression of the protein biomarkers in pancreatic cancer tissues. A, Immunohistochemical analysis of REG1A: i) REG1A in poorly differentiated PDAC, ii) luminal REG1A in malignant glands. B, TFF1: i) heterogenous expression in cancer,, ii) luminal TFF1 in malignant gland. C, LYVE1 expression in the scattered lymphatic vessels i) in the muscle layer and ii) in the stroma surrounding malignant gland. D, The biomarker levels during monitoring of pancreatic adenocarcinoma patients: LYVE1, REG1A and TFF1 were measured using ELISA in urine samples collected before surgery and during the patient's follow up. Each point represents log-transformed ELISA values at a particular time point (x-axis).

Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynecological malignancy of poor prognosis.Experimental design: We performed copy number, mutational state, and zygosity analysis of 151 genes in SCC arising in MCT (n = 25) using next-generation sequencing. The presence of high-/intermediate-risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome.Results: MCT had a low mutation burden with a mean of only one mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%), and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8 of 20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic.Conclusions: Ovarian SCC arising in MCT has a high mutational burden, with TP53 mutation the most common abnormality. The presence of TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs. Clin Cancer Res; 23(24); 7633–40. ©2017 AACR.

Figures S2-S4: Transcription of Cdkn1a in CRISPR control clones; Sanger sequencing of Brca2 exon 3; RAD51 focus formation following irradiation

Exploratory comparison of plasma CA19.9 and the urine biomarker panel in discriminating early pancreatic adenocarcinoma from chronic pancreatitis patients. A, ROC curves of the biomarker panel with corresponding plasma CA19.9 alone and in combination comparing CP urine (n=50), and urines from PDAC stages I-II (n=71) and, I-IIA (n=16) (B). C, Summary table. AUC: area under the curve, SN: sensitivity, SP: specificity with 95% Confidence Interval (CI). SN and SP in the validation set were derived for optimal cutpoint determined in the training dataset.

Supplementary Methods and legends for Supplementary tables and figures

Figure S1: Sanger sequencing data for Trp53 exon 5 in the 4 Trp53-/- ID8 clones

Full description of next generation sequencing, quantitative IHC and flow cytometry

Supplementary Figure Legends from Identification of a Three-Biomarker Panel in Urine for Early Detection of Pancreatic Adenocarcinoma

Cohort per experiment

Urine biomarker concentrations in different tumours. Scatter dot plots of urine LYVE1, REG1A and plasma CA19.9 in different hepatobiliary pathologies and early stages of pancreatic adenocarcinoma (I-IIA, n=16) and I-II (n=71). The level of TFF1 protein was not measured in these samples due to substantial modifications made to the original ELISA assay by the source company at the moment of this analysis. IPMN (n=33): intraductal papillary mucinous neoplasm, AMP (n=26): ampullary cancer, NET (n=18): neuroendocrine tumour, CHL (n=24): cholangiocarcinoma, DuCA (n=16): duodenal cancer. Bars indicate median and IQR values. Upper bars: Kruskal-Wallis/Dunn's post test, *: p0.05 (no adjustment for multiplicity was made).

Purpose: The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSC), and its relationship to treatment response.Experimental Design: We obtained pre- and posttreatment omental biopsies and blood samples from a total of 54 patients undergoing platinum-based NACT and 6 patients undergoing primary debulking surgery. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC, flow cytometry, electrochemiluminescence assays, and RNA sequencing and related our findings to the histopathologic treatment response.Results: There was evidence of T-cell activation in omental biopsies after NACT: CD4+ T cells showed enhanced IFNγ production and antitumor Th1 gene signatures were increased. T-cell activation was more pronounced with good response to NACT. The CD8+ T-cell and CD45RO+ memory cell density in the tumor microenvironment was unchanged after NACT but biopsies showing a good therapeutic response had significantly fewer FoxP3+ T regulatory (Treg) cells. This finding was supported by a reduction in a Treg cell gene signature in post- versus pre-NACT samples that was more pronounced in good responders. Plasma levels of proinflammatory cytokines decreased in all patients after NACT. However, a high proportion of T cells in biopsies expressed immune checkpoint molecules PD-1 and CTLA4, and PD-L1 levels were significantly increased after NACT.Conclusions: NACT may enhance host immune response but this effect is tempered by high/increased levels of PD-1, CTLA4, and PD-L1. Sequential chemoimmunotherapy may improve disease control in advanced HGSC. Clin Cancer Res; 22(12); 3025–36. ©2016 AACR.

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53–/–) or double (Trp53–/–;Brca2–/–) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53–/–;Brca2–/– mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53–/– cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118–29. ©2016 AACR.

Total patient cohort

Correlation of the three urinary biomarkers and plasma CA19.9 (CA19.9p). A, Correlation plots (Navy blue: Healthy; Turquoise: chronic pancreatitis (CP); Purple: pancreatic adenocarcinoma (PDAC). B, Pearson correlation coefficients and corresponding significance (NS: non-significant, *: p

PDS cohort

List of all putative functional variants in ID8

Purpose:The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated.Experimental Design:Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma (N = 205) together with matched RNA sequencing for the majority of tumors (N = 150), we have comprehensively characterized mutation and expression at BRCA1/2.Results:In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types.Conclusions:These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

Supplementary methods and supplementary figures

Expression of non-redundant proteins identified by GeLC-MS/MS from males and females in healthy controls, chronic pancreatitis and pancreatic adenocarcinoma patients.

Legends for supplementary figures

Urine proteome analysis. A, schematic outline of the study. B, classification of total identified proteins according to sub-cellular localisation and C, functional activity determined by Ingenuity Pathway Analysis. H: healthy, CP: chronic pancreatitis, PDAC: pancreatic ductal adenocarcinoma, GeLC/MS/MS: SDS-PAGE-Liquid Chromatography-Tandem Mass Spectrometry.

The drivers of recurrence and resistance in ovarian high grade serous carcinoma (HGSC) remain unclear. We established BriTROC-1 to investigate the acquisition of resistance by collecting tumour biopsies from women with recurrent ovarian HGSC that had relapsed following at least one line of platinum-based chemotherapy. Patients underwent biopsy or secondary debulking surgery, with tumour samples fixed in methanol-based fixative. Normal and tumour DNA samples underwent tagged-amplicon panel sequencing. Tumour DNA underwent shallow whole genome sequencing (sWGS). Tissue microarrays (TMA), created from diagnosis samples, were stained for CD3, CD8, CD20 and FoxP3. 276 patients were recruited (209 platinum-sensitive, 67 platinum-resistant). Panel sequencing showed close concordance between diagnosis and relapse, with only 4 discordant cases, and no revertant mutations inBRCA1orBRCA2were identified in relapse samples. CN signatures were strongly correlated with immune cell infiltration. There was very strong concordance in copy number (CN) between diagnosis and relapse, with no significant difference in purity, ploidy or focal somatic CN alterations, even when stratified by platinum sensitivity or prior chemotherapy lines. Small increases in CN signature 3 and 7 exposure were observed between diagnosis and relapse across the whole cohort but were not present in matched sample pairs. Diagnosis samples from patients with primary platinum resistance had increased rates ofCCNE1andKRASamplification and CN signature 6 exposure. The HGSC genome is remarkably stable between diagnosis and relapse and acquired chemotherapy resistance does not select for common copy number drivers. We have identified new genomic events at diagnosis, includingKRASamplification and CN signature 6 exposure, that are associated with primary platinum resistance.

High grade serous ovarian carcinoma (HGSOC) is characterised by poor outcome and extreme chromosome instability (CIN). Therapies targeting centrosome amplification (CA), a key mediator of chromosome missegregation and CIN, may have significant clinical utility in HGSOC. However, the prevalence of CA in HGSOC, its relationship to genomic biomarkers of CIN and its potential impact on therapeutic response have not been defined. Using high-throughput multi-regional microscopy on 287 clinical HGSOC tumour tissues and 73 ovarian cancer cell lines, we show that CA through centriole overduplication is a highly recurrent and heterogeneous feature of HGSOC and is strongly associated with CIN and genome subclonality. Cell-based studies showed that high prevalence CA is phenocopied in ovarian cancer cell lines, and that high CA is associated with increased multi-treatment resistance; most notably to paclitaxel which is the most common treatment used in HGSOC. CA in HGSOC may therefore present a potential driver of tumour evolution and a powerful biomarker for response to standard-of-care treatment.

Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumour immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells whilst suppressing tumour-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/-null ID8 ovarian tumours and resulted in tumour burden reduction.These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumour growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.Abstract Figure

PurposeHigh grade serous carcinoma (HGSC) is the commonest type of ovarian cancer. Nearly all HGSC cases are diagnosed at late stage and it is not clear whether early stage HGSC has unique characteristics compared to late stage tumours.Experimental DesignWe analysed samples from 45 patients with FIGO stage I - IIA HGSC - 40 from the pathology archives of three large UK cancer centres and 5 from the BriTROC-1 study. We performed shallow whole genome sequencing (sWGS) and targeted next generation sequencing to investigate somatic mutations and copy number alterations. We compared results to 51 stage IIIC/IV HGSC patients from the BriTROC-1 study.ResultsThere was no difference in median age between the early stage (median 61.3 years, range 40-84) and late stage (median 62.3 years, range 34-76) patients at diagnosis. TP53 mutations were near-universal (92% early stage, 100% late stage samples) and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2, or focal copy number alterations between early- and late-stage cohorts. There were also no unique amplifications or deletions in either cohort. However, median ploidy was greater in late stage (median 3.1) than early stage (median 2.0) samples. In addition, there were higher numbers of breakpoints per 10MB and per chromosome arm and higher absolute copy number in late stage than early stage cohorts; early stage samples had longer segment length. Overall copy number signature exposures were significantly different between early and late stage samples with greater signature 3 exposure in early stage and greater signature 4 in late stage. Both simplex plot and unsupervised hierarchical clustering suggested that a subset of late stage samples retain early stage appearances with high signature 3 and co-clustering with the early stage samplesConclusionsThese data suggest that there are no unique mutations or focal copy number alterations in early stage HGSC. However, whole genome duplication is significantly more common in late-stage disease, suggesting evolution during disease progression. However, a subset of late stage HGSC retains early-stage features, which are associated with improved overall survival.

PURPOSE Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. The gold standard for detecting copy number changes in tumor cells is fluorescence in situ hybridization (FISH) using locus-specific probes that are imaged as fluorescent spots. However, spot counting often does not perform well on solid tumor tissue sections due to partially represented or overlapping nuclei. MATERIALS AND METHODS To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes or a homogeneous Poisson point process model for automated spot counting. RESULTS We benchmarked the performance of FrenchFISH against previous approaches using a controlled simulation scenario and tested it experimentally in 12 ovarian carcinoma FFPE-tissue sections for copy number alterations at three loci (c-Myc, hTERC, and SE7). FrenchFISH outperformed standard spot counting with 74% of the automated counts having < 1 copy number difference from the manual counts and 17% having < 2 copy number differences, while taking less than one third of the time of manual counting. CONCLUSION FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue by both speeding up and improving the accuracy of spot count estimates.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumour type with limited treatment options. OCS is hypothesised to develop via the combination theory from a single progenitor, resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). We show OCS from 18 women to be monoclonal through analysis of DNA variants from isolated carcinoma and sarcoma components. RNA sequencing indicated the carcinoma components were more mesenchymal when compared with pure ovarian carcinomas, supporting the conversion theory. We used pre-clinical OCS models to test the efficacy of microtubule-targeting drugs, including eribulin, which has been shown to reverse EMT characteristics. We demonstrated that microtubule inhibitors, vinorelbine and eribulin, were more effective than standard-of-care platinum-based chemotherapy. Eribulin reduced mesenchymal characteristics, N-MYC expression and cholesterol biosynthesis. Finally, eribulin induced a strong immune response, supporting immunotherapy combinations in the clinic.

PURPOSE: The abundance and effects of structural variation at BRCA1/2 in tumours are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated. EXPERIMENTAL DESIGN: Exploiting a large collection of whole genome sequencing data from high grade serous ovarian carcinoma (N=205) together with matched RNA-seq for the majority of tumours (N=150), we have comprehensively characterised mutation and expression at BRCA1/2. RESULTS: In addition to the known spectrum of short somatic mutations (SSMs), we discover that multi-megabase structural variants (SVs) are a frequent, unappreciated source of BRCA1/2 disruption in these tumours, and we find a genome wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affect a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring homologous recombination repair deficiency (HRD) and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Further, these SVs are abundant and disruptive in other cancer types. CONCLUSIONS: These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification.

Oncolytic viruses (OVs) can trigger profound innate and adaptive immune responses, which have the potential both to potentiate and reduce the activity of OVs. Natural killer (NK) cells can mediate potent anti-viral and anti-tumoral responses, but there are no data on the role of NK cells in oncolytic adenovirus activity. Here, we have used two different oncolytic adenoviruses—the Ad5 E1A CR2-deletion mutant dl922-947 (group C) and the chimeric Ad3/Ad11p mutant enadenotucirev (group B)—to investigate the effect of NK cells on overall anti-cancer efficacy in ovarian cancer. Because human adenoviruses do not replicate in murine cells, we utilized primary human NK cells from peripheral blood and ovarian cancer ascites. Our results show that dl922-947 and enadenotucirev do not infect NK cells, but induce contact-dependent activation and anti-cancer cytotoxicity against adenovirus-infected ovarian cancer cells. Moreover, manipulation of NK receptors DNAM-1 (DNAX accessory molecule-1) and TIGIT (T cell immunoreceptor with Ig and ITIM domains) significantly influences NK cytotoxicity against adenovirus-infected cells. Together, these results indicate that NK cells act to increase the activity of oncolytic adenovirus in ovarian cancer and suggest that strategies to augment NK activity further via the blockade of inhibitory NK receptor TIGIT could enhance therapeutic potential of OVs. Keywords: Ovarian cancer, Oncolytic virus, Adenovirus, NK cell, DNAM-1, TIGIT

BACKGROUND: Investigating tumour evolution and acquired chemotherapy resistance requires analysis of sequential tumour material. We describe the feasibility of obtaining research biopsies in women with relapsed ovarian high-grade serous carcinoma (HGSC).METHODS: Women with relapsed ovarian HGSC underwent either image-guided biopsy or intra-operative biopsy during secondary debulking, and samples were fixed in methanol-based fixative. Tagged-amplicon sequencing was performed on biopsy DNA.RESULTS: We screened 519 patients in order to enrol 220. Two hundred and two patients underwent successful biopsy, 118 of which were image-guided. There were 22 study-related adverse events (AE) in the image-guided biopsies, all grades 1 and 2; pain was the commonest AE. There were pre-specified significant AE in 3/118 biopsies (2.5%). 87% biopsies were fit-for-purpose for genomic analyses. Median DNA yield was 2.87 μg, and was higher in biopsies utilising 14 G or 16 G needles compared to 18 G. TP53 mutations were identified in 94.4% patients.CONCLUSIONS: Obtaining tumour biopsies for research in relapsed HGSC is safe and feasible. Adverse events are rare. The large majority of biopsies yield sufficient DNA for genomic analyses-we recommend use of larger gauge needles and methanol fixation for such biopsies, as DNA yields are higher but with no increase in AEs.British Journal of Cancer advance online publication, 30 March 2017; doi:10.1038/bjc.2017.86 www.bjcancer.com.

There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53–/–) or double (Trp53–/–;Brca2–/–) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53–/–;Brca2–/– mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53–/– cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118–29. ©2016 AACR.

Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. Imaging of spots generated using fluorescence in situ hybridisation (FISH) of locus specific probes is routinely used to detect copy number changes in tumour nuclei. However, it often does not perform well on solid tumour tissue sections, where partially represented or overlapping nuclei are common. To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes; or a homogenous Poisson Point Process model for automated spot counting. We benchmarked the performance of FrenchFISH against previous approaches in a controlled simulation scenario and exemplify its use in 12 ovarian cancer FFPE-tissue sections, for which we assess copy number alterations in three loci (c-Myc, hTERC and SE7). We show that FrenchFISH outperforms standard spot counting approaches and that the automated spot counting is significantly faster than manual without loss of performance. FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue.

Purpose: The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSC), and its relationship to treatment response. Experimental Design: We obtained pre- and posttreatment omental biopsies and blood samples from a total of 54 patients undergoing platinum-based NACT and 6 patients undergoing primary debulking surgery. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC, flow cytometry, electrochemiluminescence assays, and RNA sequencing and related our findings to the histopathologic treatment response. Results: There was evidence of T-cell activation in omental biopsies after NACT: CD4+ T cells showed enhanced IFNγ production and antitumor Th1 gene signatures were increased. T-cell activation was more pronounced with good response to NACT. The CD8+ T-cell and CD45RO+ memory cell density in the tumor microenvironment was unchanged after NACT but biopsies showing a good therapeutic response had significantly fewer FoxP3+ T regulatory (Treg) cells. This finding was supported by a reduction in a Treg cell gene signature in post- versus pre-NACT samples that was more pronounced in good responders. Plasma levels of proinflammatory cytokines decreased in all patients after NACT. However, a high proportion of T cells in biopsies expressed immune checkpoint molecules PD-1 and CTLA4, and PD-L1 levels were significantly increased after NACT. Conclusions: NACT may enhance host immune response but this effect is tempered by high/increased levels of PD-1, CTLA4, and PD-L1. Sequential chemoimmunotherapy may improve disease control in advanced HGSC. Clin Cancer Res; 22(12); 3025–36. ©2016 AACR.

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis., Highlights • A mechanistic link between the tRNA repertoire and cancer progression • Selective control of the secretome by levels of the initiator methionine tRNA • A link between the tRNAome and ECM secretion from stromal fibroblasts • A new transgenic model recapitulating a key aspect of the cancer tRNAome, Clarke et al. show that levels of the initiator tRNAiMet are increased in carcinoma-associated fibroblasts. Using a transgenic approach to recapitulate this in vivo and quantitative mass spectrometry to characterize the stromal secretome, they show that elevated tRNAiMet drives production of a type II collagen-rich ECM to drive tumor progression.

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

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 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.

Tumours with profound copy-number aberration elude molecular stratification due to their genomic complexity. By representing this complexity as a mixture of copy-number signatures, we provide molecular explanations for differing clinical outcomes. Here we present a method for copy-number signature identification, deriving eight signatures in 117 shallow whole-genome sequenced high-grade serous ovarian cancers (HGSOC), which validated on independent cohorts of 95 deep whole-genome sequenced, and 402 SNP array-profiled cases. Three copy-number signatures predicted longer overall survival, while the others predicted poorer outcome. We found evidence for the mutational processes giving rise to copy-number change for six of the eight signatures via correlations with other genomic features. Our results provide insights into the pathogenesis of HGSOC by uncovering multiple mutational processes that shape genomes following TP53 mutation. Importantly, our work shows that most HGSOC have a mixture of mutational processes suggesting that targeting a single mutator phenotype may be therapeutically suboptimal.

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922‐947 (E1A CR2‐deleted), dl1520 (E1B‐55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel‐resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non‐CAR receptors to infect cells, are also significantly more effective in paclitaxel‐resistant ovarian cell models. Inhibition of CDK4/6 using PD‐0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.