Genome-wide association analyses of ovarian cancer patients undergoing primary debulking surgery identify candidate genes for residual disease.

Survival from ovarian cancer depends on the resection status after primary surgery. We performed genome-wide association analyses for resection status of 7705 ovarian cancer patients, including 4954 with high-grade serous carcinoma (HGSOC), to identify variants associated with residual disease. The most significant association with resection status was observed for rs72845444, upstream of MGMT, in HGSOC (p = 3.9 × 10-8). In gene-based analyses, PPP2R5C was the most strongly associated gene in HGSOC after stage adjustment. In an independent set of 378 ovarian tumours from the AGO-OVAR 11 study, variants near MGMT and PPP2R5C correlated with methylation and transcript levels, and PPP2R5C mRNA levels predicted progression-free survival in patients with residual disease. MGMT encodes a DNA repair enzyme, and PPP2R5C encodes the B56γ subunit of the PP2A tumour suppressor. Our results link heritable variation at these two loci with resection status in HGSOC.

Proteogenomic analysis of enriched HGSOC tumor epithelium identifies prognostic signatures and therapeutic vulnerabilities.

We performed a deep proteogenomic analysis of bulk tumor and laser microdissection enriched tumor cell populations from high-grade serous ovarian cancer (HGSOC) tissue specimens spanning a broad spectrum of purity. We identified patients with longer progression-free survival had increased immune-related signatures and validated proteins correlating with tumor-infiltrating lymphocytes in 65 tumors from an independent cohort of HGSOC patients, as well as with overall survival in an additional 126 HGSOC patient cohort. We identified that homologous recombination deficient (HRD) tumors are enriched in pathways associated with metabolism and oxidative phosphorylation that we validated in independent patient cohorts. We further identified that polycomb complex protein BMI-1 is elevated in HR proficient (HRP) tumors, that elevated BMI-1 correlates with poor overall survival in HRP but not HRD HGSOC patients, and that HRP HGSOC cells are uniquely sensitive to BMI-1 inhibition.

Targeted and shallow whole genome sequencing identifies therapeutic opportunities in p53abn endometrial cancers.

PURPOSE: Shallow whole genome sequencing (sWGS) can detect copy number (CN) aberrations. In high-grade serous ovarian (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers (ECs) to identify additional prognostic stratification and therapeutic opportunities. EXPERIMENTAL DESIGN: sWGS and targeted panel sequencing was performed on formalin-fixed paraffin-embedded p53abn ECs. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. RESULTS: In 187 p53abn ECs, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. 22% potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with a high ploidy state, and CCNE1, ERBB2 and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. 28% of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. 34% of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on immunohistochemistry, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations. CONCLUSIONS: sWGS and targeted sequencing identified therapeutic opportunities in 75% of p53abn EC patients. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn ECs.

Cell State of Origin Impacts Development of Distinct Endometriosis-Related Ovarian Carcinoma Histotypes.

Clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are ovarian carcinoma histotypes, which are both thought to arise from ectopic endometrial (or endometrial-like) cells through an endometriosis intermediate. How the same cell type of origin gives rise to two morphologically and biologically different histotypes has been perplexing, particularly given that recurrent genetic mutations are common to both and present in nonmalignant precursors. We used RNA transcription analysis to show that the expression profiles of CCOC and ENOC resemble those of normal endometrium at secretory and proliferative phases of the menstrual cycle, respectively. DNA methylation at the promoter of the estrogen receptor (ER) gene (ESR1) was enriched in CCOC, which could potentially lock the cells in the secretory state. Compared with normal secretory-type endometrium, CCOC was further defined by increased expression of cysteine and glutathione synthesis pathway genes and downregulation of the iron antiporter, suggesting iron addiction and highlighting ferroptosis as a potential therapeutic target. Overall, these findings suggest that while CCOC and ENOC arise from the same cell type, these histotypes likely originate from different cell states. This "cell state of origin" model may help to explain the presence of histologic and molecular cancer subtypes arising in other organs. SIGNIFICANCE: Two cancer histotypes diverge from a common cell of origin epigenetically locked in different cell states, highlighting the importance of considering cell state to better understand the cell of origin of cancer.

Opportunistic Salpingectomy Between 2017 and 2020: A Descriptive Analysis.

OBJECTIVES: Opportunistic salpingectomy (OS) is the removal of fallopian tubes during another pelvic surgery for the purpose of ovarian cancer prevention. Herein, we describe the rates of OS at the time of hysterectomy and tubal sterilization between 2017 and 2020. METHODS: This study uses the Canadian Institute of Health Information's Discharge Abstract Database and National Ambulatory Care Reporting System for all Canadian provinces and territories except for Quebec between the fiscal years 2017 and 2020. A descriptive analysis on all people aged 15 years and older who had hysterectomies or tubal sterilizations was conducted to determine the proportion of hysterectomies that included bilateral salpingectomy (OS) and the proportion of tubal sterilizations that were OS compared to tubal ligation. RESULTS: There were 174 006 people included in the study. The proportion of hysterectomies that included OS increased from 31.7% in 2017 to 39.9% by 2020. With respect to tubal sterilizations, rates of OS increased from 26.3% of all tubal sterilizations in 2017 to 42.5% in 2020. British Columbia remained the jurisdiction with the highest rates of OS, but rates increased significantly in many jurisdictions, particularly at the time of tubal sterilization. CONCLUSION: The rates of OS have continued to increase in all Canadian jurisdictions following the official Society of Obstetricians and Gynaecologists of Canada recommendation to consider OS in 2015. Assuming that all tubal ligations could have been OS and 75% of hysterectomies with ovarian conservation could have included OS, our data indicate 76 932 missed opportunities for ovarian cancer prevention.

Biallelic Dicer1 Mutations in the Gynecologic Tract of Mice Drive Lineage-Specific Development of DICER1 Syndrome-Associated Cancer.

DICER1 is an RNase III enzyme essential for miRNA biogenesis through cleaving precursor-miRNA hairpins. Germline loss-of-function DICER1 mutations underline the development of DICER1 syndrome, a rare genetic disorder that predisposes children to cancer development in organs such as lung, gynecologic tract, kidney, and brain. Unlike classical tumor suppressors, the somatic "second hit" in DICER1 syndrome-associated cancers does not fully inactivate DICER1 but impairs its RNase IIIb activity only, suggesting a noncanonical two-hit hypothesis. Here, we developed a genetically engineered conditional compound heterozygous Dicer1 mutant mouse strain that fully recapitulates the biallelic DICER1 mutations in DICER1 syndrome-associated human cancers. Crossing this tool strain with tissue-specific Cre strains that activate Dicer1 mutations in gynecologic tract cells at two distinct developmental stages revealed that embryonic biallelic Dicer1 mutations caused infertility in females by disrupting oviduct and endometrium development and ultimately drove cancer development. These multicystic tubal and intrauterine tumors histologically resembled a subset of DICER1 syndrome-associated human cancers. Molecular analysis uncovered accumulation of additional oncogenic events (e.g., aberrant p53 expression, Kras mutation, and Myc activation) in murine Dicer1 mutant tumors and validated miRNA biogenesis defects in 5P miRNA strand production, of which, loss of let-7 family miRNAs was identified as a putative key player in transcriptomic rewiring and tumor development. Thus, this DICER1 syndrome-associated cancer model recapitulates the biology of human cancer and provides a unique tool for future investigation and therapeutic development. SIGNIFICANCE: Generation of a Dicer1 mutant mouse model establishes the oncogenicity of missense mutations in the DICER1 RNase IIIb domain and provides a faithful model of DICER1 syndrome-associated cancer for further investigation.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool.

OBJECTIVES: Despite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. METHODS: DNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. RESULTS: The new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. CONCLUSION: ProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Alanine supplementation exploits glutamine dependency induced by SMARCA4/2-loss.

SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.

Genomic characterization of DICER1-associated neoplasms uncovers molecular classes.

DICER1 syndrome is a tumor predisposition syndrome that is associated with up to 30 different neoplastic lesions, usually affecting children and adolescents. Here we identify a group of mesenchymal tumors which is highly associated with DICER1 syndrome, and molecularly distinct from other DICER1-associated tumors. This group of DICER1-associated mesenchymal tumors encompasses multiple well-established clinicopathological tumor entities and can be further divided into three clinically meaningful classes designated "low-grade mesenchymal tumor with DICER1 alteration" (LGMT DICER1), "sarcoma with DICER1 alteration" (SARC DICER1), and primary intracranial sarcoma with DICER1 alteration (PIS DICER1). Our study not only provides a combined approach to classify DICER1-associated neoplasms for improved clinical management but also suggests a role for global hypomethylation and other recurrent molecular events in sarcomatous differentiation in mesenchymal tumors with DICER1 alteration. Our results will facilitate future investigations into prognostication and therapeutic approaches for affected patients.

Molecular subtype stratified outcomes according to adjuvant therapy in endometrial cancer.

OBJECTIVES: Recent data support the predictive implications of molecular subtype assignment in endometrial cancer (EC). Our objective was to retrospectively assess clinical outcomes according to adjuvant treatment received within EC molecular subtypes. METHODS: Clinical outcomes (disease-specific and progression-free survival DSS/PFS) of EC patients from a single institution and population-based cohorts that had undergone molecular classification were assessed with respect to adjuvant therapy received and 2016 ESMO risk group. RESULTS: 2472 ECs were assessed; 184 (7.4%) POLEmut, 638 (25.8%) MMRd, 1223 (49.5%) NSMP and 427 (17.3%) p53abn. N = 774 (34.6%) of the cohort were ESMO 2016 high risk and 109 (4.8%) were advanced or metastatic. In patients with MMRd EC, assessed across and within stage, there was no observed benefit in DSS or PFS with the addition of chemotherapy +/- radiation compared to radiation alone in ESMO high risk (p = 0.694) or ESMO high, advanced, metastatic risk groups combined (p = 0.852). In patients with p53abn EC, adjuvant chemotherapy given with radiation was associated with significantly longer DSS compared to radiation alone in ESMO high risk (p = 0.007) and ESMO high, advanced and metastatic risk groups combined (p = 0.015), even when restricted to stage I disease (p < 0.001) and when compared in serous vs. non-serous histotypes (p = 0.009). CONCLUSIONS: Adjuvant chemotherapy is associated with more favorable outcomes for patients with p53abn EC, including stage I disease and non-serous histotypes, but does not appear to add benefit within MMRd ECs for any stage of disease, consistent with PORTEC-3 molecular subanalysis. Prospective trials, assessing treatment efficacy within molecular subtype are needed, however these 'real-world' data should be considered when discussing adjuvant treatment with patients.

p53-Abnormal "Fields of Dysplasia" in Human Papillomavirus-Independent Vulvar Squamous Cell Carcinoma Impacts Margins and Recurrence Risk.

Abnormal p53 (p53abn) immunohistochemical (IHC) staining patterns can be found in vulvar squamous cell carcinoma (VSCC) and differentiated vulvar intraepithelial neoplasia (dVIN). They can also be found in the adjacent skin that shows morphology that falls short of the traditional diagnostic threshold for dVIN. Vulvectomy specimens containing human papillomavirus-independent p53abn VSCC with margins originally reported as negative for invasive and in situ disease were identified. Sections showing the closest approach by invasive or in situ neoplasia to margins were stained with p53 IHC stains. We evaluated the following: (1) detection of morphologically occult p53abn in situ neoplasia, (2) rates of margin status change after p53 IHC staining, and (3) effect of p53abn IHC staining at margins on the 2-year local recurrence rates. Seventy-three human papillomavirus-independent p53abn VSCCs were included. Half (35/73, 48%) had documented an in situ lesion in the original report. The use of p53 IHC staining identified 21 additional cases (29%) with the p53abn in situ lesions that were originally unrecognized. The histology of in situ lesions in the p53abn "field" varied and became more subtle (morphologically occult) farther away from the VSCC. Fifteen (21%) cases had a morphologically occult and previously unrecognized p53abn in situ lesion present at a resection margin, which conferred an increased risk of local recurrence (5/7 [71.4%] vs 6/22 [27.3%], P = .036). The p53abn in situ lesions at a margin were confirmed to have TP53 mutations by sequencing. p53 IHC staining identified morphologically occult p53abn in situ lesions surrounding human papillomavirus-independent VSCC. p53abn IHC staining at a margin was associated with a 3-fold increased risk of local recurrence.

Grade and Estrogen Receptor Expression Identify a Subset of No Specific Molecular Profile Endometrial Carcinomas at a Very Low Risk of Disease-Specific Death.

Endometrial carcinoma (EC) can be divided into 4 prognostic molecular subtypes, and no specific molecular profile (NSMP) type is the most commonly occurring type (∼50%). Although described as having an intermediate to favorable prognosis, this subtype encompasses pathologically and molecularly diverse tumors. We aimed to identify factors associated with outcomes within the NSMP ECs that might be used to stratify prognosis and direct treatment. Clinicopathologic, immunohistochemical, and genetic features of a large series of NSMP EC were used to identify parameters that could identify the subset associated with a very favorable outcome (disease-specific death rate <5% at 5 years, termed low-risk NSMP). A total of 1110 NSMP ECs were profiled. In a univariate analysis, stage, grade, lymphovascular invasion, estrogen receptor (ER) and progesterone receptor (PR) expression, L1CAM overexpression, and mutations in PIK3CA were associated with disease-specific survival. Two critical features, grade and ER expression, identified a low-risk NSMP subset (grade 1-2, ER-positive [>1%], 84% of cases), which showed a 5-year disease-specific death rate of 1.6% across all stages and 1.4% within stage I. The remaining cases (high-risk NSMPs, grade 3, and/or ER-negative status) were responsible for most of the disease-specific deaths (disease-specific death rate at 5 years, 22.9%; hazard ratio compared with that of low-risk NSMPs: 16.3; 95% CI, 8.4-31.7). Within NSMP EC, the low-risk and high-risk categories were of prognostic significance independent of the stage on a multivariate analysis. Low-grade and ER-positive NSMP ECs are a homogeneous low-risk group associated with an exceptionally favorable prognosis in which de-escalation and/or endocrine therapy strategies can be applied. Grade 3 and/or ER-negative status identifies a high-risk NSMP subset, including rare high-grade histotypes (eg, clear cell, dedifferentiated, and mesonephric-like), responsible for most NSMP-related deaths. Subclassification of NSMPs allows for the category of low-risk EC molecular subtypes to be dramatically expanded because it now includes both POLEmut and the much more common low-risk NSMP EC.

Single-cell transcriptomic analysis of endometriosis.

Endometriosis is a common condition in women that causes chronic pain and infertility and is associated with an elevated risk of ovarian cancer. We profiled transcriptomes of >370,000 individual cells from endometriomas (n = 8), endometriosis (n = 28), eutopic endometrium (n = 10), unaffected ovary (n = 4) and endometriosis-free peritoneum (n = 4), generating a cellular atlas of endometrial-type epithelial cells, stromal cells and microenvironmental cell populations across tissue sites. Cellular and molecular signatures of endometrial-type epithelium and stroma differed across tissue types, suggesting a role for cellular restructuring and transcriptional reprogramming in the disease. Epithelium, stroma and proximal mesothelial cells of endometriomas showed dysregulation of pro-inflammatory pathways and upregulation of complement proteins. Somatic ARID1A mutation in epithelial cells was associated with upregulation of pro-angiogenic and pro-lymphangiogenic factors and remodeling of the endothelial cell compartment, with enrichment of lymphatic endothelial cells. Finally, signatures of ciliated epithelial cells were enriched in ovarian cancers, reinforcing epidemiologic associations between these two diseases.

Profiling the immune landscape in mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients. METHODS: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration. RESULTS: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates. CONCLUSION: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies.

A tumor-restricted glycoform of podocalyxin is a highly selective marker of immunologically cold high-grade serous ovarian carcinoma.

Introduction: Targeted-immunotherapies such as antibody-drug conjugates (ADC), chimeric antigen receptor (CAR) T cells or bispecific T-cell engagers (eg, BiTE®) all aim to improve cancer treatment by directly targeting cancer cells while sparing healthy tissues. Success of these therapies requires tumor antigens that are abundantly expressed and, ideally, tumor specific. The CD34-related stem cell sialomucin, podocalyxin (PODXL), is a promising target as it is overexpressed on a variety of tumor types and its expression is consistently linked to poor prognosis. However, PODXL is also expressed in healthy tissues including kidney podocytes and endothelia. To circumvent this potential pitfall, we developed an antibody, named PODO447, that selectively targets a tumor-associated glycoform of PODXL. This tumor glycoepitope is expressed by 65% of high-grade serous ovarian carcinoma (HGSOC) tumors. Methods: In this study we characterize these PODO447-expressing tumors as a distinct subset of HGSOC using four different patient cohorts that include pre-chemotherapy, post-neoadjuvant chemotherapy (NACT) and relapsing tumors as well as tumors from various peritoneal locations. Results: We find that the PODO447 epitope expression is similar across tumor locations and negligibly impacted by chemotherapy. Invariably, tumors with high levels of the PODO447 epitope lack infiltrating CD8+ T cells and CD20+ B cells/plasma cells, an immune phenotype consistently associated with poor outcome. Discussion: We conclude that the PODO447 glycoepitope is an excellent biomarker of immune "cold" tumors and a candidate for the development of targeted-therapies for these hard-to-treat cancers.

Copy Number Variants Are Ovarian Cancer Risk Alleles at Known and Novel Risk Loci.

BACKGROUND: Known risk alleles for epithelial ovarian cancer (EOC) account for approximately 40% of the heritability for EOC. Copy number variants (CNVs) have not been investigated as EOC risk alleles in a large population cohort. METHODS: Single nucleotide polymorphism array data from 13 071 EOC cases and 17 306 controls of White European ancestry were used to identify CNVs associated with EOC risk using a rare admixture maximum likelihood test for gene burden and a by-probe ratio test. We performed enrichment analysis of CNVs at known EOC risk loci and functional biofeatures in ovarian cancer-related cell types. RESULTS: We identified statistically significant risk associations with CNVs at known EOC risk genes; BRCA1 (PEOC = 1.60E-21; OREOC = 8.24), RAD51C (Phigh-grade serous ovarian cancer [HGSOC] = 5.5E-4; odds ratio [OR]HGSOC = 5.74 del), and BRCA2 (PHGSOC = 7.0E-4; ORHGSOC = 3.31 deletion). Four suggestive associations (P < .001) were identified for rare CNVs. Risk-associated CNVs were enriched (P < .05) at known EOC risk loci identified by genome-wide association study. Noncoding CNVs were enriched in active promoters and insulators in EOC-related cell types. CONCLUSIONS: CNVs in BRCA1 have been previously reported in smaller studies, but their observed frequency in this large population-based cohort, along with the CNVs observed at BRCA2 and RAD51C gene loci in EOC cases, suggests that these CNVs are potentially pathogenic and may contribute to the spectrum of disease-causing mutations in these genes. CNVs are likely to occur in a wider set of susceptibility regions, with potential implications for clinical genetic testing and disease prevention.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Significance of p53 and presence of differentiated vulvar intra-epithelial neoplasia (dVIN) at resection margin in early stage human papillomavirus-independent vulvar squamous cell carcinoma.

OBJECTIVE: Vulvar squamous cell carcinoma and in situ lesions can be stratified by human papillomavirus (HPV) and TP53 status into prognostic risk groups using p16 and p53 immunohistochemistry. We assessed the significance of vulvar squamous cell carcinoma resection margin positivity for either differentiated vulvar intra-epithelial neoplasia (dVIN) or abnormal p53 immunohistochemistry, and other pathologic variables, in a cohort of patients with HPV-independent (HPV-I) p53 abnormal (p53abn) vulvar squamous cell carcinomas. METHODS: Patients with stage I-II HPV-I p53abn vulvar squamous cell carcinoma with negative invasive margins who did not receive adjuvant radiation from a single institution were included. Tumors underwent margin reassessment using p53 immunohistochemistry. Cases were segregated into (1) morphologic dVIN at margin; or (2) abnormal p53 immunohistochemistry staining at margin without morphologic dVIN (p53abn immunohistochemistry); or (3) margins negative by morphology and p53 immunohistochemistry. Clinicopathologic/outcome data were collected. RESULTS: A total of 51 patients were evaluated: (1) 12 with dVIN on margin; (2) 12 with p53abn immunohistochemistry on margin without morphologic dVIN; and (3) 27 with margins negative for morphologic dVIN and p53abn immunohistochemistry. The recurrence rate for patients with dVIN or p53abn immunohistochemistry on the margin was equally high at 75% each, compared with 33% with margins negative for morphologic dVIN and p53abn immunohistochemistry (p=0.009). On multivariate analysis, positive in situ margins maintained an association with disease recurrence (p=0.03) whereas invasive margin distance (radial and deep), lymphovascular invasion, and tumor size did not. CONCLUSIONS: Patients with stage I-II HPV-I vulvar squamous cell carcinoma with margins positive for either dVIN or p53abn immunohistochemistry without morphologic dVIN showed increased disease recurrence, regardless of invasive margin distance. These findings show that p53 immunohistochemistry is a useful adjunct for evaluating margin status in HPV-I vulvar squamous cell carcinoma and may support repeat excision for positive in situ margins (dVIN or p53abn immunohistochemistry).