Jessica N. McAlpine

Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling

High‐grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment‐resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2–91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole‐genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug‐resistance mechanisms.

Use of mutation profiles to refine the classification of endometrial carcinomas.

The classification of endometrial carcinomas is based on pathological assessment of tumour cell type; the different cell types (endometrioid, serous, carcinosarcoma, mixed, undifferentiated, and clear cell) are associated with distinct molecular alterations. This current classification system for high‐grade subtypes, in particular the distinction between high‐grade endometrioid (EEC‐3) and serous carcinomas (ESC), is limited in its reproducibility and prognostic abilities. Therefore, a search for specific molecular classifiers to improve endometrial carcinoma subclassification is warranted. We performed target enrichment sequencing on 393 endometrial carcinomas from two large cohorts, sequencing exons from the following nine genes: ARID1A, PPP2R1A, PTEN, PIK3CA, KRAS, CTNNB1, TP53, BRAF, and PPP2R5C. Based on this gene panel, each endometrial carcinoma subtype shows a distinct mutation profile. EEC‐3s have significantly different frequencies of PTEN and TP53 mutations when compared to low‐grade endometrioid carcinomas. ESCs and EEC‐3s are distinct subtypes with significantly different frequencies of mutations in PTEN, ARID1A, PPP2R1A, TP53, and CTNNB1. From the mutation profiles, we were able to identify subtype outliers, ie cases diagnosed morphologically as one subtype but with a mutation profile suggestive of a different subtype. Careful review of these diagnostically challenging cases suggested that the original morphological classification was incorrect in most instances. The molecular profile of carcinosarcomas suggests two distinct mutation profiles for these tumours: endometrioid‐type (PTEN, PIK3CA, ARID1A, KRAS mutations) and serous‐type (TP53 and PPP2R1A mutations). While this nine‐gene panel does not allow for a purely molecularly based classification of endometrial carcinoma, it may prove useful as an adjunct to morphological classification and serve as an aid in the classification of problematic cases. If used in practice, it may lead to improved diagnostic reproducibility and may also serve to stratify patients for targeted therapeutics. Copyright

14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma

14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t(10;17) genomic rearrangement, leading to fusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE–FAM22 fusion oncoproteins was demonstrated by immunoblot in t(10;17)-bearing frozen tumor and cell line samples. YWHAE–FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE–FAM22 maintains a structurally and functionally intact 14-3-3ε (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE–FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE–FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions.

A brief mindfulness-based cognitive behavioral intervention improves sexual functioning versus wait-list control in women treated for gynecologic cancer

GOAL The goal of this study was to evaluate a mindfulness-based cognitive behavioral intervention for sexual dysfunction in gynecologic cancer survivors compared to a wait-list control group. METHODS Thirty-one survivors of endometrial or cervical cancer (mean age 54.0, range 31-64) who self-reported significant and distressing sexual desire and/or sexual arousal concerns were assigned either to three, 90-minute mindfulness-based cognitive behavior therapy sessions or two months of wait-list control prior to entering the treatment arm. Validated measures of sexual response, sexual distress, and mood, as well as laboratory-evoked physiological and subjective sexual arousal were assessed at pre-, one month post-, and 6-months following treatment. RESULTS There were no significant effects of the wait-list condition on any measure. Treatment led to significant improvements in all domains of sexual response, and a trend towards significance for reducing sexual distress. Perception of genital arousal during an erotic film was also significantly increased following the intervention despite no change in physiologically-measured sexual arousal. CONCLUSIONS A brief mindfulness-based intervention was effective for improving sexual functioning. Geographic restrictions permitted only a select sample of survivors to participate, thus, the generalizability of the findings is limited. Future studies should aim to develop online modalities for treatment administration to overcome this limitation.

HER2 overexpression and amplification is present in a subset of ovarian mucinous carcinomas and can be targeted with trastuzumab therapy

BackgroundThe response rate of ovarian mucinous carcinomas to paclitaxel/carboplatin is low, prompting interest in targeted molecular therapies. We investigated HER2 expression and amplification, and the potential for trastuzumab therapy in this histologic subtype of ovarian cancer.MethodsHER2 status was tested in 33 mucinous carcinomas and 16 mucinous borderline ovarian tumors (BOT)). Five cases with documented recurrence and with tissue from the recurrence available for testing were analyzed to determine whether HER2 amplification status changed over time. Three prospectively identified recurrent mucinous ovarian carcinomas were assessed for HER2 amplification and patients received trastuzumab therapy with conventional chemotherapy.ResultsAmplification of HER2 was observed in 6/33 (18.2%) mucinous carcinomas and 3/16 (18.8%) BOT. HER2 amplification in primary mucinous carcinomas was not associated with an increased likelihood of recurrence. The prospectively identified recurrent mucinous carcinomas showed overexpression and amplification of HER2; one patients tumor responded dramatically to trastuzumab in combination with conventional chemotherapy, while another patient experienced an isolated central nervous system recurrence after trastuzumab therapy.ConclusionHER2 amplification is relatively common in ovarian mucinous carcinomas (6/33, 18.2%), although not of prognostic significance. Trastuzumab therapy is a treatment option for patients with mucinous carcinoma when the tumor has HER2 amplification and overexpression.

Opportunistic salpingectomy: uptake, risks, and complications of a regional initiative for ovarian cancer prevention

OBJECTIVE The purpose of this study was to assess the uptake and perioperative safety of bilateral salpingectomy (BS) as an ovarian cancer risk-reduction strategy in low-risk women after a regional initiative that was aimed at general gynecologists in the province of British Columbia, Canada. STUDY DESIGN This population-based retrospective cohort study evaluated 43,931 women in British Columbia from 2008-2011 who underwent hysterectomy that was performed with and without BS or bilateral salpingo-oophorectomy or who underwent surgical sterilization by means of BS or tubal ligation. Parameters that were examined include patient age, operating time, surgical approach, indication, length of hospital stay, and perioperative complications. RESULTS There was an increase in the uptake of hysterectomy with BS (5-35%; P < .001) and BS for sterilization (0.5-33%; P < .001) over the study period, particularly in women <50 years old. Minimal additional surgical time is required for hysterectomy with BS (16 minutes; P < .001) and BS for sterilization (10 minutes; P < .001) compared with hysterectomy alone or tubal ligation, respectively. No significant differences were observed in the risks of hospital readmission or blood transfusions in women who underwent hysterectomy with BS (adjusted odds ratio [aOR], 0.91; 95% confidence interval [CI], 0.75-1.10; and aOR, 0.86; 95% CI, 0.67-1.10, respectively) or BS for sterilization (aOR, 0.8; 95% CI, 0.56-1.21; and aOR, 0.75; 95% CI, 0.32-1.73, respectively). From 2008-2011 the proportion of hysterectomies with BS performed by open laparotomy decreased from 77-44% with uptake in laparoscopic, vaginal, and combined procedures (P < .001). CONCLUSION After our 2010 educational initiative, there has been a shift in surgical paradigm in our province. This cancer prevention approach does not increase the risk of operative/perioperative complications and appears both feasible and safe.

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.

TITAN: inference of copy number architectures in clonal cell populations from tumor whole-genome sequence data

The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational landscapes. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting evolutionary dynamics of copy number alterations (CNA) and loss of heterozygosity (LOH) in whole-genome sequencing data remain underdeveloped. We present a novel probabilistic model, TITAN, to infer CNA and LOH events while accounting for mixtures of cell populations, thereby estimating the proportion of cells harboring each event. We evaluate TITAN on idealized mixtures, simulating clonal populations from whole-genome sequences taken from genomically heterogeneous ovarian tumor sites collected from the same patient. In addition, we show in 23 whole genomes of breast tumors that the inference of CNA and LOH using TITAN critically informs population structure and the nature of the evolving cancer genome. Finally, we experimentally validated subclonal predictions using fluorescence in situ hybridization (FISH) and single-cell sequencing from an ovarian cancer patient sample, thereby recapitulating the key modeling assumptions of TITAN.

Prophylactic salpingectomy and delayed oophorectomy as an alternative for BRCA mutation carriers.

OBJECTIVE: Prophylactic bilateral salpingo-oophorectomy is advised for women with BRCA mutations, but there are adverse consequences of premature menopause. The majority of BRCA-associated ovarian cancers appear to arise in the fallopian tube; therefore, salpingectomy may be an alternative to bilateral salpingo-oophorectomy. We compared the costs and benefits of salpingectomy with bilateral salpingo-oophorectomy among BRCA mutation carriers. METHODS: We developed a Markov Monte Carlo simulation model to compare three strategies for risk reduction in women with BRCA mutations: 1) bilateral salpingo-oophorectomy; 2) bilateral salpingectomy; and 3) bilateral salpingectomy with delayed oophorectomy. Net health benefits were measured in years-of-life expectancy and quality-adjusted life-year expectancy, and the primary outcome was the incremental cost-effectiveness ratio. The model estimated the number of future breast and ovarian cancers and cardiovascular deaths attributed to premature menopause with each strategy. RESULTS: Bilateral salpingo-oophorectomy was associated with the lowest cost and highest life expectancy compared with the other two strategies. When quality-of-life measures were included, salpingectomy followed by delayed oophorectomy yielded the highest quality-adjusted life expectancy with incremental cost-effectiveness ratios of

A clinically applicable molecular-based classification for endometrial cancers.

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