A population-based analysis of germline BRCA1 and BRCA2 testing among ovarian cancer patients in an era of histotype-specific approaches to ovarian cancer prevention.

BACKGROUND: Identifying female carriers of BRCA1 and BRCA2 mutations is imperative for prevention of ovarian cancer and breast cancer. There are five major histologic subtypes of ovarian cancer and high grade serous cancer (the most common) is reported in 75-100% of BRCA1 and BRCA2 mutation carriers. We examined histology-based referral to the Hereditary Cancer Program following an educational prevention campaign recommending BRCA1 and BRCA2 mutation screening for all high-grade serous cancer patients. METHODS: We conducted a population-based retrospective study in the province of British Columbia, Canada that included all patients visiting the Hereditary Cancer Program for genetic counselling for BRCA1 and BRCA2 mutation between 2001 and 2014. We examined the difference in rates of BRCA1 and BRCA2 testing between serous cancer patients and endometrioid and clear cell cancer patients using a differences in differences analysis. We also calculated the mean number of family members tested for every BRCA1 and BRCA2 identified ovarian cancer patient before and after the educational campaign. RESULTS: There were 5712 women tested for a BRCA1 and BRCA2 mutation at the HCP between 2001 and 2014, 887 of which had previously received a diagnosis of ovarian cancer. By 2013, 43% of serous cancer patients were being tested for BRCA1 and BRCA2 mutations compared with 20% of endometrioid and clear cell patients (p < 0.001). The mean number of family members tested for each BRCA1 and BRCA2 positive ovarian cancer patient increased after the educational campaign from 2.54 to 3.27 (p = 0.071), and the number of family members identified as BRCA positive also increased significantly. CONCLUSIONS: Recommendations for histology-based referral significantly increased the likelihood of serous cancer patients being tested for BRCA mutations. There was also an increase in the number of carrier tests performed for each BRCA1 and BRCA2 index ovarian cancer patient.

Does MMR status in endometrial cancer influence response to adjuvant therapy?

OBJECTIVE: Mismatch repair (MMR) deficiency occurs in 20-40% of endometrial cancers but its therapeutic implication remains uncertain. Our objective was to compare clinical outcomes after adjuvant therapy between MMR deficient and proficient endometrial cancers from a population-based study. METHODS: This was a retrospective population-based cohort study of all endometrial cancers from the Vancouver Coastal Health authority region from 2011 to 2016, for which adjuvant therapy (radiotherapy and/or chemotherapy) was administered. Primary outcome measure was recurrence rates, expressed per 100 person-years (p100 py). Progression free survival (PFS) and overall survival (OS) rates were compared using Kaplan-Meier method and log-rank tests, and covariates were evaluated using Cox proportional hazards regression. RESULTS: There were 535 patients who received adjuvant therapy (radiotherapy and/or chemotherapy), including 162 (30.3%) and 373 (69.7%) with MMR-deficient and proficient tumors, respectively. Demographic variables were similar except MMR-deficient patients were younger (62.0 vs. 64.8, p = 0.01). Patients with MMR-deficient tumors were more likely to have endometrioid histotype (85.8% vs. 61.4%), more likely to have Stage I disease (62.3% vs 54.7%), and LVSI (65.4% vs. 53.4%) compared to those with MMR-proficient tumors. There was a trend for MMR-proficient group to have higher recurrence rates (10.7 p100 py vs 5.9 p100 py) and MMR deficiency was associated with better OS and PFS, but on multivariable analysis, MMR status was no longer significant. CONCLUSION: Women with MMR-deficient endometrial cancers who receive adjuvant therapy have a lower rate of recurrence compared to those with MMR-proficient cancers. However, on multivariable analysis, MMR status does not remain associated with differences in PFS or OS.

Concurrent ARID1A and ARID1B inactivation in endometrial and ovarian dedifferentiated carcinomas.

Dedifferentiated carcinoma of the endometrium or the ovary is an aggressive epithelial malignancy that comprises an endometrioid carcinoma together with an undifferentiated carcinoma. We recently reported that inactivation of BRG1 or INI1, core subunits of the switch/sucrose non-fermenting (SWI/SNF) complex, was the likely molecular event underlying dedifferentiation in about half of dedifferentiated carcinomas. In this study, we performed a genomic screen that included other members of the SWI/SNF complex to better delineate the molecular basis in the remainder of these tumours. We identified concurrent inactivating mutations involving ARID1A and ARID1B in 12 of 24 BRG1/INI1-intact, 0 of 3 INI1-deficient and 0 of 16 BRG1-deficient dedifferentiated carcinomas. All ARID1A and ARID1B co-mutated tumours displayed loss of ARID1A expression in the undifferentiated component with 11 of 12 tumours also displaying absent staining in the endometrioid component. ARID1B expression was absent in the undifferentiated component in all 12 tumours, whereas the corresponding endometrioid component showed intact expression. Clinically, ARID1A/ARID1B co-inactivated tumours showed similar aggressive behaviour to BRG1 or INI1-inactivated tumours. Given that ARID1A and ARID1B are the only known DNA-binding subunits of the SWI/SNF-A complex, additional inactivation of ARID1B in an ARID1A-deficient background appears to represent an alternative mechanism of disruption of SWI/SNF-mediated transcriptional regulation, resulting in arrested cellular differentiation in endometrial and ovarian endometrioid cancer.

Loss of switch/sucrose non-fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas.

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low-grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of eight dedifferentiated carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core ATPase of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of four tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these four SMARCA4-mutated cases, whereas the corresponding low-grade endometrioid component showed retained SMARCA4 expression. An expanded survey of other members of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of two SMARCA4-intact tumors, and all SMARCA4- or SMARCB1-deficient tumors showed concomitant loss of expression of SMARCA2. We subsequently examined the expression of SMARCA2, SMARCA4, and SMARCB1 in an additional set of 22 centrally reviewed dedifferentiated carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated carcinomas examined showed either concurrent SMARCA4 and SMARCA2 loss (37%) or concurrent SMARCB1 and SMARCA2 loss (13%) in the undifferentiated component. The loss of SMARCA4 or SMARCB1 was mutually exclusive. All 31 grade 3 endometrioid carcinomas showed intact expression of these core SWI/SNF proteins. The majority (73%) of the SMARCA4/SMARCA2-deficient and half of SMARCB1/SMARCA2-deficient undifferentiated component developed in a mismatch repair-deficient molecular context. The observed spatial association between SWI/SNF protein loss and histologic dedifferentiation suggests that inactivation of these core SWI/SNF proteins may contribute to the development of dedifferentiated endometrial carcinoma.

Vulvar Acanthosis With Altered Differentiation (VAAD): Report of a Case With Progression to Poorly Differentiated Carcinoma Over a 5-Yr Period.

Vulvar acanthosis with altered differentiation is an uncommon proliferation of the vulvar squamous epithelium that is typically seen in association with verrucous carcinoma, and may represent an early phase of non-HPV-related squamous neoplastic transformation. We report a case of vulvar acanthosis with altered differentiation that, over a 5-yr period, progressed first to verrucous carcinoma in association with well-differentiated invasive squamous cell carcinoma and then, after treatment with radiotherapy, to poorly differentiated carcinoma with a component of anaplastic carcinoma. This case supports the concept of vulvar acanthosis with altered differentiation as a premalignant lesion, with potential to progress to invasive carcinoma.

FOXL2 molecular testing in ovarian neoplasms: diagnostic approach and procedural guidelines.

A single, recurrent somatic point mutation (402C→G) in FOXL2 has been described in almost all adult-type granulosa cell tumors but not other ovarian neoplasms. Histopathological features of adult-type granulosa cell tumors can be mimicked by a variety of other tumors, making diagnosis of adult-type granulosa cell tumor challenging. It has been suggested that molecular testing for FOXL2 mutation might be a useful tool in the diagnosis of adult-type granulosa cell tumors. The aim of this study was to demonstrate how testing for the FOXL2 mutation can be used in a gynecological pathology consultation service and to establish clear procedural guidelines for FOXL2 testing. Immunohistochemistry for FOXL2 was done using an anti-FOXL2 polyclonal antiserum. If immunohistochemistry was positive, FOXL2 mutation status was subsequently analyzed using a TaqMan assay. A dilution experiment was done to assess the sensitivity and minimum tumor cellularity requirements for our TaqMan assay. Twenty problematic cases were assessed, where the differential diagnosis after the initial investigations included adult-type granulosa cell tumors. Differential diagnoses included: thecoma, Sertoli-Leydig cell tumor, juvenile granulosa cell tumor, endometrial stromal sarcoma and others. In all cases, FOXL2 immunohistochemistry was positive and in six samples the FOXL2 mutation was detected, thus confirming a diagnosis of adult-type granulosa cell tumor. The TaqMan assay was able to reliably detect the FOXL2 mutation with input DNA in the range of 2.5-20 ng, and with a minimum of 25% tumor cell nuclei. The analysis of the FOXL2 mutational status in clinical samples is a useful diagnostic tool in situations where the differential diagnosis is between adult-type granulosa cell tumor and other ovarian tumors. The TaqMan assay requires a minimum of 2.5 ng DNA, with optimal assay performance for 5 to 10 ng DNA input. Laser capture or needle-macrodissection should be undertaken to enrich samples with tumor cell content below 25%.

Concurrent RB1 loss and BRCA-deficiency predicts enhanced immunological response and long-term survival in tubo-ovarian high-grade serous carcinoma.

Background: Somatic loss of the tumour suppressor RB1 is a common event in tubo-ovarian high-grade serous carcinoma (HGSC), which frequently co-occurs with alterations in homologous recombination DNA repair genes including BRCA1 and BRCA2 (BRCA). We examined whether tumour expression of RB1 was associated with survival across ovarian cancer histotypes (HGSC, endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous carcinoma (LGSC)), and how co-occurrence of germline BRCA pathogenic variants and RB1 loss influences long-term survival in a large series of HGSC. Patients and methods: RB1 protein expression patterns were classified by immunohistochemistry in epithelial ovarian carcinomas of 7436 patients from 20 studies participating in the Ovarian Tumor Tissue Analysis consortium and assessed for associations with overall survival (OS), accounting for patient age at diagnosis and FIGO stage. We examined RB1 expression and germline BRCA status in a subset of 1134 HGSC, and related genotype to survival, tumour infiltrating CD8+ lymphocyte counts and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cell lines with and without BRCA1 mutations to model co-loss with treatment response. We also performed genomic analyses on 126 primary HGSC to explore the molecular characteristics of concurrent homologous recombination deficiency and RB1 loss. Results: RB1 protein loss was most frequent in HGSC (16.4%) and was highly correlated with RB1 mRNA expression. RB1 loss was associated with longer OS in HGSC (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.66-0.83, P = 6.8 ×10-7), but with poorer prognosis in ENOC (HR 2.17, 95% CI 1.17-4.03, P = 0.0140). Germline BRCA mutations and RB1 loss co-occurred in HGSC (P < 0.0001). Patients with both RB1 loss and germline BRCA mutations had a superior OS (HR 0.38, 95% CI 0.25-0.58, P = 5.2 ×10-6) compared to patients with either alteration alone, and their median OS was three times longer than non-carriers whose tumours retained RB1 expression (9.3 years vs. 3.1 years). Enhanced sensitivity to cisplatin (P < 0.01) and paclitaxel (P < 0.05) was seen in BRCA1 mutated cell lines with RB1 knockout. Among 126 patients with whole-genome and transcriptome sequence data, combined RB1 loss and genomic evidence of homologous recombination deficiency was correlated with transcriptional markers of enhanced interferon response, cell cycle deregulation, and reduced epithelial-mesenchymal transition in primary HGSC. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA mutation was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

The evolution of endometrial carcinoma classification through application of immunohistochemistry and molecular diagnostics: past, present and future.

Uterine cancer was first subclassified based on anatomic site, separating those tumours arising from the endometrium from cervical cancers. There was then further subclassification of endometrial cancers based on cell type, and this correlated with the Type I and Type II categories identified through the epidemiological studies of Bokhman, with endometrioid carcinoma corresponding (approximately) to Type I and serous carcinoma to Type II. These histotypes are not clearly separable in practice, however, with considerable interobserver variability in histotype diagnosis, especially for high-grade tumours. There followed studies of immunomarkers and then mutational studies of single genes, in attempts to improve subclassification. While these have revealed significant differences in protein expression and mutation profiles between endometrioid and serous carcinomas, there is also considerable overlap, so that there remain challenges in subclassification of endometrial carcinoma. Gene panel testing, using next-generation sequencing, was applied to endometrial cancers and highlighted that there are tumours that show genetic alterations intermediate between classic Type I/endometrioid and Type II/serous carcinomas. The Cancer Genome Atlas studies of endometrioid and serous carcinoma offered revolutionary insight into the subclassification of endometrial carcinoma, i.e. that there are four distinct categories of endometrial carcinoma, rather than two, based on genomic architecture. In this review, we provide an overview of immunohistochemical and molecular markers in endometrial carcinoma and comment on the important future directions in endometrial carcinoma subclassification arising from The Cancer Genome Atlas results.

Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells.

Akt/PKB is a serine/threonine kinase that promotes tumor cell growth by phosphorylating transcription factors and cell cycle proteins. There is particular interest in finding tumor-specific substrates for Akt to understand how this protein functions in cancer and to provide new avenues for therapeutic targeting. Our laboratory sought to identify novel Akt substrates that are expressed in breast cancer. In this study, we determined that activated Akt is positively correlated with the protein expression of the transcription/translation factor Y-box binding protein-1 (YB-1) in primary breast cancer by screening tumor tissue microarrays. We therefore questioned whether Akt and YB-1 might be functionally linked. Herein, we illustrate that activated Akt binds to and phosphorylates the YB-1 cold shock domain at Ser102. We then addressed the functional significance of disrupting Ser102 by mutating it to Ala102. Following the stable expression of Flag:YB-1 and Flag:YB-1 (Ala102) in MCF-7 cells, we observed that disruption of the Akt phosphorylation site on YB-1 suppressed tumor cell growth in soft agar and in monolayer. This correlated with an inhibition of nuclear translocation by the YB-1(Ala102) mutant. In conclusion, YB-1 is a new Akt substrate and disruption of this specific site inhibits tumor cell growth.

Molecularly Defined Adult Granulosa Cell Tumor of the Ovary: The Clinical Phenotype.

The histopathologic features of adult granulosa cell tumors (AGCTs) are relatively nonspecific, resulting in misdiagnosis of other cancers as AGCT, a problem that has not been well characterized. FOXL2 mutation testing was used to stratify 336 AGCTs from three European centers into three categories: 1) FOXL2 mutant molecularly defined AGCT (MD-AGCT) (n = 256 of 336), 2) FOXL2 wild-type AGCT (n = 17 of 336), 3) misdiagnosed other tumor types (n = 63 of 336). All statistical tests were two-sided. The overall and disease-specific survival of the misdiagnosed cases was lower than in the MD-AGCTs (P < .001). The misdiagnosed cases accounted for 71.9% of disease-specific deaths within five years. In the population-based cohort, overall survival of MD-AGCT patients was not different from age-matched, population-based controls. Even though 35.2% of all the MD-AGCT patients in our study experienced a relapse, AGCT is usually an indolent disease. The historical, premolecular data underpinning our clinical understanding of AGCT was likely skewed by inclusion of misdiagnosed cases, and future management strategies should reflect the potential for surgical cure and long survival even after relapse.

Cyclin D1 as a diagnostic immunomarker for endometrial stromal sarcoma with YWHAE-FAM22 rearrangement.

Endometrial stromal sarcoma (ESS) characterized by YWHAE-FAM22 genetic fusion is histologically higher grade and clinically more aggressive than ESS with JAZF1-SUZ12 or equivalent genetic rearrangements, hence it is clinically important to recognize this subset of ESS. To identify diagnostic immunomarkers for this biologically defined ESS subset, we compared gene expression profiles between YWHAE-FAM22 ESS and JAZF1-rearranged ESS. These studies showed consistent upregulation of cyclin D1 in YWHAE-FAM22 ESS compared with JAZF1-SUZ12 ESS. Immunohistochemically, the high-grade round cell component of all 12 YWHAE-FAM22 ESS demonstrated diffuse (≥70%) moderate to strong nuclear cyclin D1 staining, and this diffuse positivity was not seen in 34 ESSs with JAZF1 and equivalent genetic rearrangements or in 21 low-grade ESS with no demonstrable genetic rearrangements. In a series of 243 non-ESS pure uterine mesenchymal and mixed epithelial-mesenchymal tumors, only 2 of 8 undifferentiated endometrial sarcomas with nuclear uniformity and 1 of 80 uterine leiomyosarcomas demonstrate diffuse cyclin D1 immunoreactivity. Both cyclin D1-positive undifferentiated endometrial sarcomas showed diffuse strong CD10 staining, which is consistently absent in the high-grade round cell component of YWHAE-FAM22 ESS. The low-grade spindle cell component of YWHAE-FAM22 ESS showed a spatially heterogenous cyclin D1 staining pattern that was weaker and less diffuse overall. Our findings indicate that cyclin D1 is a sensitive and specific diagnostic immunomarker for YWHAE-FAM22 ESS. When evaluating high-grade uterine sarcomas, cyclin D1 can be included in the immunohistochemical panel as an indicator of YWHAE-FAM22 ESS.

High-Grade Serous Carcinoma Involving Fallopian Tube, Ovary and Peritoneum.

The focus of this review is high-grade serous carcinoma (HGSC); for the purposes of this review, the term "pelvic SC" is used for HGSC that could be considered, based on historical definitions, to have arisen from ovary, fallopian tube, or peritoneum. These assignments of primary site are arbitrary and there is evidence that the distal fallopian tube is the site of origin of many pelvic HGSCs. The diagnosis of HGSC can be made readily based on routine histomorphologic examination in most cases; however, a variety of neoplasms can resemble HGSC. Thus, we review the key features of pelvic SC, current concepts of its pathogenesis, histopathological diagnostic criteria, discuss differential diagnosis, and review diagnostic ancillary studies that can be used in practice.