Generation of an Inhibitory NK Cell Subset by TGF-ß1/IL-15 Polarization.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined in vitro conditions that could polarize human NK cells into an inhibitory subset. TGF-ß1 has been shown to induce regulatory T cells in vitro and in vivo; we therefore investigated if TGF-ß1 could also induce immunosuppressive NK-like cells. First, we found that TGF-ß1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells in vitro in a TGF-ß-dependent manner. Interestingly, TGF-ß1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells in vitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-ß1-rich environments.

Identification of antigenic epitopes recognized by tumor infiltrating lymphocytes in high grade serous ovarian cancer by multi-omics profiling of the auto-antigen repertoire.

Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reducing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched auto-antibody repertoires. Focusing on MHC class I epitopes recognized by CD8+ T cells, HLA-binding epitopes were identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides (MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) population expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly found in the auto-antibody and MAP repertoires and CD8+ TILs recognizing epitopes from these antigens were detected, although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multiple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a range of self-antigens targeted by HGSC TILs.

The Prevent Ovarian Cancer Program (POCP): Identification of women at risk for ovarian cancer using complementary recruitment approaches.

BACKGROUND: Up to 20% of high-grade serous ovarian carcinomas (HGSOC) are hereditary; however, historical uptake of genetic testing is low. We used a unique combination of approaches to identify women in Ontario, Canada, with a first-degree relative (FDR) who died from HGSOC without prior genetic testing, and offer them multi-gene panel testing. METHODS: From May 2015-Sept 2019, genetic counseling and testing was provided to eligible participants. Two recruitment strategies were employed, including self-identification in response to an outreach campaign and direct targeting of FDRs of deceased HGSOC patients treated at our institution. The rate of pathogenic variants (PV) in established/potential ovarian cancer risk genes and the benefits/challenges of each approach were assessed. RESULTS: A total of 564 women enrolled in response to our outreach campaign (n = 473) or direct recruitment (n = 91). Mean age at consent was 52 years and 96% did not meet provincial testing criteria. Genetic results were provided to 528 individuals from 458 families. The rate of PVs in ovarian cancer risk genes was highest when FDRs were diagnosed with HGSOC <60 years (9.4% vs. 3.9% ≥ 60y, p = 0.0160). Participants in the outreach vs. direct recruitment cohort had a similar rate of PVs; however, uptake of genetic testing (97% vs. 89%; p = 0.0036) and study completion (95% vs. 87%; p = 0.0062) rates were higher in the former. Eleven participants with pathogenic variants have completed risk-reducing gynecologic surgery, with one stage I HGSOC and two breast cancers identified. CONCLUSION: Overall PV rates in this large cohort were lower than expected; however, we provide evidence that genetic testing criteria in Ontario should include individuals with a deceased FDR diagnosed with HGSOC <60 years of age.

High-resolution epigenomic liquid biopsy for noninvasive phenotyping in pregnancy.

OBJECTIVE: We explored the potential of genome-wide epigenomic liquid biopsy for the comprehensive analysis of cell-free DNA (cfDNA) methylation signatures in maternal plasma in early gestation. METHOD: We used solution phase hybridization for targeted region capture of bisulfite-converted DNA obtained from plasma of pregnant women in early gestation and nonpregnant female controls. RESULTS: Targeted sequencing of ~80.5 Mb of the plasma methylome generated an average read depth across all 17 plasma samples of ~42x. We used these data to explore the pregnancy-specific characteristics of cfDNA methylation in plasma and found that pregnancy resulted in clearly detectable global alterations in DNA methylation patterns that were influenced by genomic location. We analyzed similar, previously published, data from first-trimester maternal leukocyte populations and gestational age-matched chorionic villus (CV) and confirmed that tissue-specific DNA methylation signatures in these samples had a significant influence on global and gene-specific methylation in the plasma of pregnant women. CONCLUSION: We describe an approach for targeted epigenomic liquid biopsy in pregnancy and discuss our findings in the context of noninvasive prenatal testing with respect to phenotypic pregnancy monitoring and the early detection of complex gestational phenotypes such as preeclampsia and preterm birth.

Towards an Equitable Digital Society: Artificial Intelligence (AI) and Corporate Digital Responsibility (CDR).

In the digital era, we witness the increasing use of artificial intelligence (AI) to solve problems, while improving productivity and efficiency. Yet, inevitably costs are involved with delegating power to algorithmically based systems, some of whose workings are opaque and unobservable and thus termed the "black box". Central to understanding the "black box" is to acknowledge that the algorithm is not mendaciously undertaking this action; it is simply using the recombination afforded to scaled computable machine learning algorithms. But an algorithm with arbitrary precision can easily reconstruct those characteristics and make life-changing decisions, particularly in financial services (credit scoring, risk assessment, etc.), and it could be difficult to reconstruct, if this was done in a fair manner reflecting the values of society. If we permit AI to make life-changing decisions, what are the opportunity costs, data trade-offs, and implications for social, economic, technical, legal, and environmental systems? We find that over 160 ethical AI principles exist, advocating organisations to act responsibly to avoid causing digital societal harms. This maelstrom of guidance, none of which is compulsory, serves to confuse, as opposed to guide. We need to think carefully about how we implement these algorithms, the delegation of decisions and data usage, in the absence of human oversight and AI governance. The paper seeks to harmonise and align approaches, illustrating the opportunities and threats of AI, while raising awareness of Corporate Digital Responsibility (CDR) as a potential collaborative mechanism to demystify governance complexity and to establish an equitable digital society.

Non-invasive epigenomic molecular phenotyping of the human brain via liquid biopsy of cerebrospinal fluid and next generation sequencing.

Our goal was to undertake a genome-wide epigenomic liquid biopsy of cerebrospinal fluid (CSF) for the comprehensive analysis of cell-free DNA (cfDNA) methylation signatures in the human central nervous system (CNS). Solution-phase hybridization and massively parallel sequencing of bisulfite converted human DNA was employed to compare methylation signatures of cfDNA obtained from CSF with plasma. Recovery of cfDNA from CSF was relatively low (68-840 pg/mL) compared to plasma (2720-8390 pg/mL) and cfDNA fragments from CSF were approximately 20 bp shorter than their plasma-derived counterparts. Distributions of CpG methylation signatures were significantly altered between CSF and plasma, both globally and at the level of functional elements including exons, introns, CpG islands, and shores. Sliding window analysis was used to identify differentially methylated regions. We found numerous gene/locus-specific differences in CpG methylation between cfDNA from CSF and plasma. These loci were more frequently hypomethylated in CSF compared to plasma. Differentially methylated CpGs in CSF were identified in genes related to branching of neurites and neuronal development. Using the GTEx RNA expression database, we found clear association between tissue-specific gene expression in the CNS and cfDNA methylation patterns in CSF. Ingenuity pathway analysis of differentially methylated regions identified an enrichment of functional pathways related to neurobiology. In conclusion, we present a genome-wide analysis of DNA methylation in human CSF. Our methods and the resulting data demonstrate the potential of epigenomic liquid biopsy of the human CNS for molecular phenotyping of brain-derived DNA methylation signatures.

Rare variants and biological pathways identified in treatment-refractory depression.

Individuals diagnosed with major depressive disorder not responding to at least two adequate treatments are defined as treatment-refractory major depressive disorder (TR-MDD). Some TR-MDD patients have altered metabolic phenotypes that may be pharmacologically reversed. The characterization of these phenotypes and their underlying etiologies is paramount, particularly their genetic components. In this study, TR-MDD patients (n = 124) were recruited and metabolites were quantified in their cerebrospinal fluid (CSF) and peripheral blood. Three sub-categories of deficiencies were examined, namely 5-methyltetrahydrofolte (in CSF; n = 13), tetrahydrobiopterin (in CSF; n = 11), and abnormal acylcarnitine profiles (in peripheral blood; n = 8). Whole exome sequencing was performed on genomic DNA from the entire TR-MDD cohort and exonic variant allele frequencies for cases were compared to a control cohort (1:5 matching on ancestry). Low frequency, damaging alleles were identified and used for in silico pathway analyses. Three association signals for TR-MDD approached genome-wide significance on chromosomes 22, 7, and 3. Three risk-associated variants from a prior depression study were replicated. Relevant biological pathways were identified that contained an enrichment of rare, damaging variants in central nervous system (CNS)-specific pathways, including neurotransmitter receptors, potassium channels, and synapse transmission. Some TR-MDD patients had rare variants in genes that were previously associated with other psychiatric disorders, psychiatric endophenotypes, CNS structural defects, and CNS-related cellular and molecular functions. Exome analysis of metabolically phenotyped TR-MDD patients has identified potentially functional gene pathways and low frequency, deleterious gene variants for further investigation. Further studies in larger cohorts of biochemically phenotyped TR-MDD patients are desirable to extend and confirm these findings.

High-resolution analysis of the human placental DNA methylome in early gestation.

BACKGROUND/OBJECTIVE: We communicate high-read-depth bisulfite sequencing analysis of the chorionic villus (CV) DNA methylome from samples obtained between 11 and 13 weeks gestation and samples of gestationally age-matched maternal blood cells (MBC). METHODS: This was achieved through solution-phase targeted region capture (84 Mb) of bisulfite converted human DNA. RESULTS: We identified biphasic distribution of methylation in CV and MBC genomes. We found greater numbers of intermediate methylated sites (20%-80% methylated) in CV and greater number of high methylation sites in MBC and investigated distributions of these in promoters, introns, exons, CpG islands, CpG islands shores, and enhancers. We identified differentially methylated sites distinguishing CV and MBC. These are less likely to occur in CpG islands (CGIs), particularly those that exist outside promoters, exons, and introns. We found that gene promoter and gene body methylation patterns are associated with mRNA transcriptional profiles in CV. Despite the relative hypomethylation of CV genomes, we found that these contain DM regions that are more likely to be hypermethylated in CV relative to MBC. CONCLUSIONS: Our data provide novel insight into the structure and organization of the CV epigenome, which may inform future studies of placental biology and noninvasive prenatal phenotyping.

A Genomically Characterized Collection of High-Grade Serous Ovarian Cancer Xenografts for Preclinical Testing.

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture-based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient-derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patient's tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.

International Society of Gynecological Pathologists (ISGyP) Endometrial Cancer Project: Guidelines From the Special Techniques and Ancillary Studies Group.

The aim of this article is to propose guidelines and recommendations in problematic areas in pathologic reporting of endometrial carcinoma (EC) regarding special techniques and ancillary studies. An organizing committee designed a comprehensive survey with different questions related to pathologic features, diagnosis, and prognosis of EC that was sent to all members of the International Society of Gynecological Pathologists. The special techniques/ancillary studies group received 4 different questions to be addressed. Five members of the group reviewed the literature and came up with recommendations and an accompanying text which were discussed and agreed upon by all members of the group. Twelve different recommendations are made. They address the value of immunohistochemistry, ploidy, and molecular analysis for assessing prognosis in EC, the value of steroid hormone receptor analysis to predict response to hormone therapy, and parameters regarding applying immunohistochemistry and molecular tests for assessing mismatch deficiency in EC.

TP53 mutations in high grade serous ovarian cancer and impact on clinical outcomes: a comparison of next generation sequencing and bioinformatics analyses.

OBJECTIVE: Mutations in TP53 are found in the majority of high grade serous ovarian cancers, leading to gain of function or loss of function of its protein product, p53, involved in oncogenesis. There have been conflicting reports as to the impact of the type of these on prognosis. We aim to further elucidate this relationship in our cohort of patients. METHODS: 229 patients with high grade serous ovarian cancer underwent tumor profiling through an institutional molecular screening program with targeted next generation sequencing. TP53 mutations were classified using methods previously described in the literature. Immunohistochemistry on formalin-fixed paraffin embedded tissue was used to assess for TP53 mutation. Using divisive hierarchal clustering, we generated patient clusters with similar clinicopathologic characteristics to investigate differences in outcomes. RESULTS: Six different classification schemes of TP53 mutations were studied. These did not show an association with first platinum-free interval or overall survival. Next generation sequencing reliably predicted mutation in 80% of cases, similar to the proportion detected by immunohistochemistry. Divisive hierarchical clustering generated four main clusters, with cluster 3 having a significantly worse prognosis (p<0.0001; log-rank test). This cluster had a higher concentration of gain of function mutations and these patients were less likely to have undergone optimal debulking surgery. CONCLUSIONS: Different classifications of TP53 mutations did not show an impact on outcomes in this study. Immunohistochemistry was a good predictor for TP53 mutation. Cluster analysis showed that a subgroup of patients with gain of function mutations (cluster 3) had a worse prognosis.

Very-low-dose twice-daily aspirin maintains platelet inhibition and improves haemostasis during dual-antiplatelet therapy for acute coronary syndrome.

Higher aspirin doses may be inferior in ticagrelor-treated acute coronary syndrome (ACS) patients and reducing bleeding risk whilst maintaining antithrombotic benefits could improve outcomes. We characterized the pharmacodynamics of a novel dual-antiplatelet-therapy regimen consisting of very-low-dose twice-daily (BD) aspirin with standard-dose ticagrelor. A total of 20 ticagrelor-treated ACS patients entered a randomized crossover to take aspirin 20 mg BD (12-hourly) during one 14-day period and 75 mg once-daily (OD) in the other. After 14 days of treatment, serum thromboxane (TX)B2 and light-transmittance aggregometry were assessed pre- and 2 h post-morning-dose, bleeding time was measured post-dose, and TXA2 and prostacyclin stable metabolites were measured in urine collected 2 h post-morning-dose. Data are expressed as mean ± SD. After 14 days treatment, serum TXB2 levels were significantly greater 2 h post-dosing with aspirin 20 mg BD vs. 75 mg OD (3.0 ± 3.6 ng/mL vs. 0.8 ± 1.9 ng/mL; p = 0.018) whereas pre-dosing levels were not significantly different (3.5 ± 4.1 ng/mL vs. 2.5 ± 3.1 ng/mL, p = 0.23). 1-mmol/L arachidonic acid-induced platelet aggregation was similarly inhibited by both regimens pre-dose (8.5 ± 14.3% vs. 5.1 ± 3.6%, p = 0.24) and post-dose (8.7 ± 14.2% vs. 6.6 ± 5.3%; p = 0.41). Post-dose bleeding time was shorter with 20 mg BD (680 ± 306 s vs. 834 ± 386 s, p = 0.02). Urinary prostacyclin and TX metabolite excretion were not significantly different. In conclusion, compared to aspirin 75 mg OD, aspirin 20 mg BD provided consistent inhibition of platelet TXA2 release and aggregation, and improved post-dose hemostasis, in ticagrelor-treated ACS patients. Further studies are warranted to assess whether this regimen improves the balance of clinical efficacy and safety.

Evolution of genetic assessment for BRCA-associated gynaecologic malignancies: a Canadian multisociety roadmap.

The landscape of genetic testing in ovarian cancer patients has changed dramatically in recent years. The therapeutic benefits of poly ADP-ribose polymerase (PARP) inhibitors in treatment of BRCA1/2-related ovarian cancers has resulted in an increased demand and urgency for genetic testing results, while technological developments have led to widespread use of multi-gene cancer panels and development of tumour testing protocols. Traditional genetic counselling models are no longer sustainable and must evolve to match the rapid evolution of genetic testing technologies and developments in personalized medicine. Recently, representatives from oncology, clinical genetics, molecular genetics, pathology, and patient advocacy came together to create a national multi-disciplinary Canadian consortium. By aligning stakeholder interests, the BRCA Testing to Treatment (BRCA TtoT) Community of Practice aims to develop a national strategy for tumour and germline BRCA1/2 testing and genetic counselling in women with ovarian cancer. This article serves to provide an overview of the recent evolution of genetic assessment for BRCA1/2-associated gynecologic malignancies and outline a Canadian roadmap to facilitate change, improve genetic testing rates, and ultimately improve outcomes for hereditary ovarian cancer patients and their families.

Glycaemic index, glycaemic load and dietary fibre characteristics of two commercially available fruit smoothies.

In light of the updated Eatwell Guide and the corresponding change in the consumption of fruit smoothies, the aim of this study was to measure the glycaemic index and load of two commercial fruit smoothies and to investigate the retention of dietary fibre following production. In vitro analysis was performed to identify fibre material (cellulose and pectins) using calcofluor staining and immunocytochemical labelling. A repeated measures cross-over study was conducted (n 10) to determine the glycaemic index (GI) and glycaemic load (GL) of the smoothies. Results showed that dietary fibre was still present in the smoothies after processing (16.9-17.5% cellular material by dry weight). The GI was low for both smoothies (39 and 36), whereas the GL was medium and borderline-low, respectively (11.4 and 9.7). The retention of fibre in these smoothies may have a potential positive effect on glycaemic response and may contribute to daily fibre requirements.

Novel EPC1 gene fusions in endometrial stromal sarcoma.

Endometrial stromal sarcoma encompasses a heterogeneous group of uterine mesenchymal neoplasms, which are currently divided into low-grade and high-grade subtypes. Low-grade endometrial stromal sarcoma is morphologically bland; molecularly, these tumors frequently contain JAZF1-SUZ12, JAZF1-PHF1, and EPC1-PHF1 fusions. In contrast, high-grade endometrial stromal sarcoma is characterized by morphologically undifferentiated neoplasms with high-grade nuclear features; these tumors likewise appear to be genetically diverse with YWHAE-NUTM2 and ZC3H7B-BCOR representing the most frequent gene fusions. Herein, we describe two novel EPC1 fusion genes in endometrial stromal sarcoma: EPC1-SUZ12 and EPC1-BCOR. Both tumors were characterized be an aggressive clinical course.

Implementing a Cervical Sentinel Lymph Node Biopsy Program: Quality Improvement in Gynaecologic Oncology.

OBJECTIVE: Sentinel lymph node (SLN) biopsy is becoming a reasonable alternative to pelvic lymphadenectomy in early-stage cervical cancer. It is therefore imperative that centres without prior experience are able to successfully implement the procedure. The objectives of the current study were to (1) describe the process of implementing an SLN biopsy program with a novel peer mentorship component and (2) assess post-program quality improvement metrics, including SLN detection rate (DR) and diagnostic parameters. METHODS: An institutional SLN biopsy protocol was developed collaboratively by gynaecologic oncology, nuclear medicine, and pathology departments at University Health Network, Toronto, Ontario. All decisions were based on the best evidence available. Newly diagnosed, early-stage cervical cancer patients undergoing primary surgery were then recruited prospectively for SLN biopsy with combined technique, followed by pelvic lymphadenectomy to evaluate key quality indicators, including SLN DR, sensitivity, and negative predictive value. Surgeons with previous SLN biopsy experience mentored surgeons unfamiliar with the technique. Interim analyses and multidisciplinary rounds were regularly carried out to identify failures of technique or protocol. RESULTS: Thirty-nine patients (median age 42) were enrolled in the study between August 2010 and February 2014. The median number of SLNs and total pelvic lymph nodes removed per patient were 3 and 19, respectively. SLN DRs were 92% per patient (36/39), 88.5% per hemipelvis (69/78), and 85% bilaterally (33/39). SLN biopsy correctly identified seven of eight hemipelvises with nodal metastases, yielding a sensitivity of 88% (95% CI 0.47 to 1.00) and a false negative rate of 12% (95% CI 0 to 0.53). Surgeons undergoing peer mentorship (n = 3) performed as effectively (DR 100%) as surgeons (n = 2) with prior experience (DR 85%). CONCLUSIONS: This study provides a model upon which other centres can adopt and validate cervical SLN biopsy. High SLN DRs and accurate identification of lymph node metastases can be achieved by focusing on multidisciplinary collaboration, knowledge translation with creation of evidence-based protocols, peer mentorship, and ongoing quality control.

Loss of SMARCA4 (BRG1) protein expression as determined by immunohistochemistry in small-cell carcinoma of the ovary, hypercalcaemic type distinguishes these tumours from their mimics.

AIMS: Molecular investigation of small-cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) has revealed that it is a monogenetic tumour characterized by alteration of SMARCA4 (BRG1), encoding a member of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex. A large majority of cases show loss of expression of the corresponding SMARCA4/BRG1 protein. Furthermore, three cases of SCCOHT with retained SMARCA4 protein expression showed loss of SMARCB1/INI1 expression. The aim of this study was to assess the sensitivity and specificity of loss of SMARCA4 expression as a diagnostic test for SCCOHT. METHODS AND RESULTS: We performed SMARCA4 and SMARCB1 staining in 245 tumours, many of which were potentially in the differential diagnosis of SCCOHT. We also stained 56 cases of SCCOHT for SMARCA4 and 37 of these for SMARCB1. Fifty-four of the SCCOHT cases showed complete absence of SMARCA4 expression. The two cases with retained expression showed molecular alteration of SMARCA4. Of the 217 other neoplasms with interpretable staining, all retained SMARCA4 expression. Although the majority showed diffuse, strong nuclear expression, a heterogeneous, typically weak staining pattern was present in 13% of cases. All 37 cases of SCCOHT tested and all other neoplasms, apart from three malignant rhabdoid tumours, showed retained nuclear SMARCB1 expression. Loss of SMARCA4 expression had a sensitivity of 96.55% and specificity of 100%. CONCLUSIONS: Loss of SMARCA4 expression is sensitive and specific for SCCOHT. Although some mimics show heterogeneous expression, there is retention of nuclear staining in at least a part of the tumour; therefore, only complete loss of staining should be regarded as being supportive of SCCOHT.

Frequent somatic mutations of the telomerase reverse transcriptase promoter in ovarian clear cell carcinoma but not in other major types of gynaecological malignancy.

Up-regulated expression of telomerase reverse transcriptase (TERT) and subsequent maintenance of telomere length are essential in tumour development. Recent studies have implicated somatic gain-of-function mutations at the TERT promoter as one of the mechanisms that promote transcriptional activation of TERT; however, it remains unclear whether this genetic abnormality is prevalent in gynaecological neoplasms. We performed mutational analysis in a total of 525 gynaecological cancers, and correlated TERT promoter mutations with clinicopathological features. With the exception of ovarian clear cell carcinomas, in which mutations were found in 37 (15.9%) of 233 cases, the majority of gynaecological malignancies were wild-type. TERT promoter mutation does not appear to be an early event during oncogenesis, as it was not detected in the contiguous endometriosis associated with ovarian clear cell carcinoma. Ovarian clear cell carcinoma cell lines with TERT promoter mutations exhibited higher TERT mRNA expression than those with wild-type sequences (p = 0.0238). TERT promoter mutation tended to be mutually exclusive with loss of ARID1A protein expression (p = 4.4 × 10(-9) ) and PIK3CA mutation (p = 0.0019) in ovarian clear cell carcinomas. No associations with disease-specific survival were observed for ovarian clear cell carcinoma. The above results, in conjunction with our previous report showing longer telomeres in ovarian clear cell carcinomas relative to other types of ovarian cancer, suggests that aberrations in telomere biology may play an important role in the pathogenesis of ovarian clear cell carcinoma.

Retinoblastoma pathway deregulatory mechanisms determine clinical outcome in high-grade serous ovarian carcinoma.

Alterations in the retinoblastoma pathway are frequent in ovarian/tubal high-grade serous cancers, but the mechanism of deregulation and the impact on patient outcome are poorly understood. A cohort of 334 high-grade serous carcinomas was studied by immunohistochemical analysis of RB1, p16, cyclin D1, cyclin E1, and Ki67. Additional detailed analyses including RB1 allelic deletion (n=42), mutation (n=75), methylation (n=31), and SNP array analyses (n=75) were performed on cases with clinical parameters, including age, debulking status, treatment, and clinical outcome. p16/RB1 expression results yielded three distinct clinically relevant subgroups upon multivariable analysis controlling for stage, debulking status, and treatment types: p16 homogeneous/RB1+ with the shortest progression-free survival (median 15 months (95% CI: 13-18); P=0.016) compared with the p16 heterogeneous/RB1+ subgroup (median 22 months (95% CI: 16-32)) and the p16 homogeneous/RB1- subgroup (median 20 months (95% CI: 15-24)). Patients in the p16 homo/RB1- subgroup showed a significant increase in overall survival (>60 months; P=0.013), which suggests an increase in sensitivity to cytotoxic agents. Analyses of Rb pathway mechanistic differences among these groups revealed frequent RB1 genomic alterations such as RB1 allelic loss and/or large spanning deletions (83%) in the p16 homo/RB1- subgroups, also indicating that RB1 deletions are frequent in high-grade serous carcinoma. CCNE1 gene gains/amplifications were frequent in the p16 homogeneous/RB1+ subgroup (68%) and cyclin D1 protein overexpression was predominantly characteristic of the p16 heterogeneous/RB1+ subgroup. These subcategories occur early in tumor progression and are seen with similar frequency in the cancer precursor lesion, serous tubal intra-epithelial carcinoma. Overall, this study uniquely identifies multiple non-synonymous mechanisms of retinoblastoma pathway deregulation that correlate with significantly different clinical outcomes. Furthermore, deregulations identified in precursor lesions suggest a key role of this pathway in serous tumor development. Recognition of these categories may identify patients with increased sensitivity to chemotherapy and new opportunities for novel therapeutics.

Molecular characterization of mucinous ovarian tumours supports a stratified treatment approach with HER2 targeting in 19% of carcinomas.

Mucinous ovarian carcinomas (MCs) typically do not respond to current conventional therapy. We have previously demonstrated amplification of HER2 in 6 of 33 (18.2%) mucinous ovarian carcinomas (MCs) and presented anecdotal evidence of response with HER2-targeted treatment in a small series of women with recurrent HER2-amplified (HER2+) MC. Here, we explore HER2 amplification and KRAS mutation status in an independent cohort of 189 MCs and 199 mucinous borderline ovarian tumours (MBOTs) and their association to clinicopathological features. HER2 status was assessed by immunohistochemistry (IHC), FISH, and CISH, and interpreted per ASCO/CAP guidelines, with intratumoural heterogeneity assessment on full sections, where available. KRAS mutation testing was performed with Sanger sequencing. Stage and grade were associated with recurrence on both univariate and multivariate analysis (p < 0.001). Assessment of HER2 status revealed overexpression/amplification of HER2 in 29/154 (18.8%) MCs and 11/176 (6.2%) MBOTs. There was excellent agreement between IHC, FISH, and CISH assessment of HER2 status (perfect concordance of HER2 0 or 1+ IHC with non-amplified status, and 3+ IHC with amplified status). KRAS mutations were seen in 31/71 (43.6%) MCs and 26/33 (78.8%) MBOTs, and were near mutually exclusive of HER2 amplification. In the 189 MC cases, a total of 54 recurrences and 59 deaths (53 of progressive disease) were observed. Within MCs, either HER2 amplification/overexpression or KRAS mutation was associated with decreased likelihood of disease recurrence (p = 0.019) or death (p = 0.0041) when compared to cases with neither feature. Intratumoural heterogeneity was noted in 26% of HER2-overexpressing cases. These data support the stratification of MCs for the testing of new treatments, with HER2-targeted therapy as a viable option for HER2+ advanced or recurrent disease. Further research is required to delineate the molecular and clinical features of the ∼34% of MC cases with neither HER2 amplification nor KRAS mutations.