Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression.

The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p53(25,26), is severely compromised for transactivation of most p53 target genes, and, moreover, p53(25,26) cannot induce G(1)-arrest or apoptosis in response to acute DNA damage. Surprisingly, p53(25,26) retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p53(25,26,53,54) mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression-a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression.

Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. METHODS: We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complex class I [MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). RESULTS: Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. CONCLUSIONS: Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

TP53 as a Diagnostic Aid in the Distinction of Ovarian Mucinous Borderline Tumors From Mucinous Carcinoma.

Ovarian mucinous borderline tumors (MBTs) are clinically managed as benign neoplasms while the management of ovarian mucinous carcinomas (MC) is dependent on tumor stage. Despite the standardization of sampling of ovarian mucinous neoplasms, limited interobserver reproducibility between MBT and MC persists. Based on our recent finding that abnormal TP53 expression is associated with unfavorable outcome in MBT, we hypothesized that TP53 status might improve the reproducible distinction of MBT from MC. A virtual slide set of 85 consecutive ovarian mucinous neoplasms received at a single institution, with each case represented by 3 full sections, were reviewed by 3 pathologists in 2 iterations. The initial assessment was based solely on morphologic review, while the second iteration was performed with knowledge of TP53 status. The reproducibility of a trinary categorization (MBT, MBT with intraepithelial carcinoma [IEC], MC) significantly improved from a κ of 0.60 based on the initial morphologic assessment to a κ of 0.76 (t-test, P =0.0042) after consideration of TP53 immunohistochemistry (IHC) results. Six out of 85 patients died of disease, and in 2 of them, at least 1 pathologist assessed MBT with IEC and not MC even after integration of TP53 IHC. With the integration of TP53 IHC, substantial interobserver agreement for MBT and MC can be reached, particularly in cases with an uncertain degree of confluent growth. TP53 IHC can also be used to highlight and support the presence of IEC in MBT, however, discordances remained in 2 cases with adverse outcome.

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.

EWSR1-WT1 gene fusions in neoplasms other than desmoplastic small round cell tumor: a report of three unusual tumors involving the female genital tract and review of the literature.

Desmoplastic small round cell tumor (DSRCT) is a high-grade round cell sarcoma that typically arises in the abdominopelvic cavity of young males, co-expresses keratins and desmin, and carries a pathognomonic EWSR1-WT1 gene fusion. The EWSR1-WT1 gene fusion is generally considered specific for DSRCT, although there are two reports of this fusion in tumors otherwise lacking features of DSRCT. We report three female genital tract tumors with EWSR1-WT1 fusions but showing morphologic and immunohistochemical features incompatible with DSRCT. The tumors occurred in the uterine cervix, uterine corpus/ovaries, and vagina, respectively, of 46, 30, and 20-year-old women. Two tumors consisted of a sheet-like to fascicular proliferation of relatively uniform spindled to occasionally more epithelioid cells arrayed about thick-walled, hyalinized, and capillary-sized vessels, with distinctive areas of pseudovascular change, and absence of desmoplastic stroma. The third tumor resembled a monomorphic spindle cell sarcoma with necrosis. All had diffuse desmin and variable but more limited keratin expression, two of three expressed smooth muscle actin, and all were negative for h-caldesmon, CD10, estrogen receptor, myogenin, N-terminus WT-1, and S100 protein. One patient received neoadjuvant chemotherapy and radiation therapy followed by resection and is disease-free 42 months after diagnosis. Another patient was managed by resection only and is disease-free 9 months after initial diagnosis. The remaining patient recently underwent resection of multifocal pelvic disease. Comprehensive differential gene expression analysis on two tumors compared to two classic DSRCTs with known EWSR1-WT1 fusions resulted in 1726 genes that were differentially expressed (log2 fold change >2 or < -2) and statistically significant (FDR < 5%). In combination with previous reports, our findings suggest pleiotropy of the EWSR1-WT1 fusion is possible and not limited to DSRCT. Subsets of non-DSRCT EWSR1-WT1 positive tumors may represent discrete entities, but further study is necessary.

Re-assigning the histologic identities of COV434 and TOV-112D ovarian cancer cell lines.

OBJECTIVE: The development of effective cancer treatments depends on the availability of cell lines that faithfully recapitulate the cancer in question. This study definitively re-assigns the histologic identities of two ovarian cancer cell lines, COV434 (originally described as a granulosa cell tumour) and TOV-112D (originally described as grade 3 endometrioid carcinoma), both of which were recently suggested to represent small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), based on their shared gene expression profiles and sensitivity to EZH2 inhibitors. METHODS: For COV434 and TOV-112D, we re-reviewed the original pathology slides and obtained clinical follow-up on the patients, when available, and performed immunohistochemistry for SMARCA4, SMARCA2 and additional diagnostic markers on the original formalin-fixed, paraffin-embedded (FFPE) clinical material, when available. For COV434, we further performed whole exome sequencing and validated SMARCA4 mutations by Sanger sequencing. We studied the growth of the cell lines at baseline and upon re-expression of SMARCA4 in vitro for both cell lines and evaluated the serum calcium levels in vivo upon injection into immunodeficient mice for COV434 cells. RESULTS: The available morphological, immunohistochemical, genetic, and clinical features indicate COV434 is derived from SCCOHT, and TOV-112D is a dedifferentiated carcinoma. Transplantation of COV434 into mice leads to increased serum calcium level. Re-expression of SMARCA4 in either COV434 and TOV-112D cells suppressed their growth dramatically. CONCLUSIONS: COV434 represents a bona fide SCCOHT cell line. TOV-112D is a dedifferentiated ovarian carcinoma cell line.

Comprehensive genomic profiles of small cell lung cancer.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice.

The principal reason for failure of targeted cancer therapies is the emergence of resistant clones that regenerate the tumor. Therapeutic efficacy therefore depends on not only how effectively a drug inhibits its target, but also the innate or adaptive functional redundancy of that target and its attendant pathway. In this regard, the Myc transcription factors are intriguing therapeutic targets because they serve the unique and irreplaceable role of coordinating expression of the many diverse genes that, together, are required for somatic cell proliferation. Furthermore, Myc expression is deregulated in most-perhaps all-cancers, underscoring its irreplaceable role in proliferation. We previously showed in a preclinical mouse model of non-small-cell lung cancer that systemic Myc inhibition using the dominant-negative Myc mutant Omomyc exerts a dramatic therapeutic impact, triggering rapid regression of tumors with only mild and fully reversible side effects. Using protracted episodic expression of Omomyc, we now demonstrate that metronomic Myc inhibition not only contains Ras-driven lung tumors indefinitely, but also leads to their progressive eradication. Hence, Myc does indeed serve a unique and nondegenerate role in lung tumor maintenance that cannot be complemented by any adaptive mechanism, even in the most aggressive p53-deficient tumors. These data endorse Myc as a compelling cancer drug target.

Clinical and pathological associations of PTEN expression in ovarian cancer: a multicentre study from the Ovarian Tumour Tissue Analysis Consortium.

BACKGROUND: PTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study. METHODS: Tumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran-Mantel-Haenszel tests. RESULTS: Downregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65-0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression (p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC (p value < 0.0001). Heterogeneous expression of PTEN was more prevalent in advanced HGSOC (p value = 0.019) and associated with higher CD8 counts (p value = 0.0016). CONCLUSIONS: PTEN loss is a frequent driver in ovarian carcinoma associating distinctly with expression of hormonal receptors and CD8+ TIL counts in HGSOC and CCOC histotypes.

Practical roles for molecular diagnostic testing in ovarian adult granulosa cell tumour, Sertoli-Leydig cell tumour, microcystic stromal tumour and their mimics.

Within the last decade, molecular advances have provided insights into the genetics of several ovarian sex cord-stromal tumours that have otherwise been enigmatic. Chief among these advances are the identification of FOXL2, DICER1 and CTNNB1 mutations in adult granulosa cell tumours, Sertoli-Leydig cell tumours (SLCTs), and microcystic stromal tumours (MCSTs), respectively. As access to molecular diagnostic laboratories continues to become more widely available, the potential roles for tumour mutation testing in the pathological diagnosis of these tumours merit discussion. Furthermore, links to inherited cancer susceptibility syndromes may exist for some women with SLCT (DICER1 syndrome) and MCST [familial adenomatous polyposis (FAP)]. This review will address practical issues in deciding when and how to apply mutation testing in the diagnosis of these three sex cord-stromal tumours. The pathologist's role in recommending referral for formal risk assessment for DICER1 syndrome and FAP will also be discussed.

Short-term organoid culture for drug sensitivity testing of high-grade serous carcinoma.

OBJECTIVE: Cancer patient-derived organoids (PDOs) grow as three dimensional (3D) structures in the presence of extracellular matrix and have been found to represent the original tumor's genetic complexity. In addition, PDOs can be grown and subjected to drug sensitivity testing in a shorter time course and with lesser expense than patient-derived xenograft models. Many patients with recurrent ovarian cancer develop malignant effusions that become refractory to chemotherapy. Since these same patients often present for palliative aspiration of ascites or pleural effusions, there is a potential opportunity to obtain tumor specimens in the form of multicellular spheroids (MCS) present in malignant effusion fluids. Our objective was to develop a short duration culture of MCS from ovarian cancer malignant effusions in conditions selected to support organoid growth and use them as a platform for empirical drug sensitivity testing. METHODS: In this study, malignant effusion specimens were collected from patients with high-grade serous ovarian carcinoma (HGSOC). MCS were recovered and subjected to culture conditions designed to support organoid growth. In a subset of specimens, RNA-sequencing was performed at two time points during the short-term culture to determine changes in transcriptome in response to culture conditions. Organoid induction was also characterized in these specimens using Ki67 staining and histologic analysis. Drug sensitivity testing was performed on all specimens. RESULTS: Our model describes organoids formed within days of primary culture, which can recapitulate the histological features of malignant ascites fluid and can be expanded for at least 6 days. RNA-seq analysis of four patient specimens showed that within 6 days of culture, there was significant up-regulation of genes related to cellular proliferation, epithelial-mesenchymal transition, and KRAS signaling pathways. Drug sensitivity testing identified several agents with therapeutic potential. CONCLUSIONS: Short duration organoid culture of MCS from HGSOC malignant effusions can be used as a platform for empiric drug sensitivity testing. These ex vivo models may be helpful in screening new or existing therapeutic agents prior to individualized treatment options.

The P72R Polymorphism in R248Q/W p53 Mutants Modifies the Mutant Effect on Epithelial to Mesenchymal Transition Phenotype and Cell Invasion via CXCL1 Expression.

High-grade serous carcinoma (HGSC), the most lethal subtype of epithelial ovarian cancer (EOC), is characterized by widespread TP53 mutations (>90%), most of which are missense mutations (>70%). The objective of this study was to investigate differential transcriptional targets affected by a common germline P72R SNP (rs1042522) in two p53 hotspot mutants, R248Q and R248W, and identify the mechanism through which the P72R SNP affects the neomorphic properties of these mutants. Using isogenic cell line models, transcriptomic analysis, xenografts, and patient data, we found that the P72R SNP modifies the effect of p53 hotspot mutants on cellular morphology and invasion properties. Most importantly, RNA sequencing studies identified CXCL1 a critical factor that is differentially affected by P72R SNP in R248Q and R248W mutants and is responsible for differences in cellular morphology and functional properties observed in these p53 mutants. We show that the mutants with the P72 SNP promote a reversion of the EMT phenotype to epithelial characteristics, whereas its R72 counterpart promotes a mesenchymal transition via the chemokine CXCL1. These studies reveal a new role of the P72R SNP in modulating the neomorphic properties of p53 mutants via CXCL1, which has significant implications for tumor invasion and metastasis.

High Frequency of Ovarian Cyst Development in Vhl2B/+;Snf5+/- Mice.

The new paradigm of mutations in chromatin-modifying genes as driver events in the development of cancers has proved challenging to resolve the complex influences over disease phenotypes. In particular, impaired activities of members of the SWI/SNF chromatin remodeling complex have appeared in an increasing variety of tumors. Mutations in SNF5, a member of this ubiquitously expressed complex, arise in almost all cases of malignant rhabdoid tumor in the absence of additional genetic alterations. Therefore, we studied how activation of additional oncogenic pathways might shift the phenotype of disease driven by SNF5 loss. With the use of a genetically engineered mouse model, we examined the effects of a hypomorphic Vhl2B allele on disease phenotype, with a modest up-regulation of the hypoxia response pathway. Snf5+/-;Vhl2B/+ mice did not demonstrate a substantial difference in overall survival or a change in malignant rhabdoid tumor development. However, a high percentage of female mice showed complex hemorrhagic ovarian cysts, a phenotype rarely found in either parental mouse strain. These lesions also showed mosaic expression of SNF5 by immunohistochemistry. Therefore, our studies implicate that modest changes in angiogenic regulation interact with perturbations of SWI/SNF complex activity to modulate disease phenotypes.

Bartholin Gland Carcinoma: Clinicopathologic Features, Including p16 Expression and Clinical Outcome.

Bartholin gland carcinomas are rare forms of vulvar malignancy and it is unclear what proportion is associated with high-risk human papilloma virus (HPV) infection. Our hospital archives were searched for all cases of Bartholin gland carcinoma from 1984 to 2017 (n=16). We excluded 3 adenoid cystic carcinomas, which were the subject of a previous study, leaving 13 cases. We reviewed all slides and performed immunostains for p16 as a surrogate biomarker for high-risk HPV. There were 12 squamous cell carcinomas (SCCs), including 1 SCC with transitional-like morphology and 1 papillary SCC, and 1 adenocarcinoma. All SCCs showed diffuse and intense p16 expression consistent with the presence of HPV. The single case of poorly differentiated adenocarcinoma showed patchy staining. Patient age ranged from 38 to 72 yr (mean, 58.3 yr). Most tumors were low stage. All patients were treated with radical vulvectomy and inguinofemoral lymphadenectomy. Mean clinical follow-up was 53.7 mo (range, 3-181 mo), 9 patients were free of disease (75%), recurrence occurred in 3 cases, with death due to disease in 2 of the patients with recurrence, including the single patient with adenocarcinoma. All SCC of Bartholin gland expressed p16 diffusely and intensely regardless of histologic features and grade. Our results support the etiologic role of HPV in the pathogenesis of SCC of Bartholin gland. In this small study we observed SCC as the predominant histotype, and most tumors presented at early stage and were associated with relatively favorable outcomes.

Tubo-Ovarian Transitional Cell Carcinoma and High-grade Serous Carcinoma Show Subtly Different Immunohistochemistry Profiles.

Tubo-ovarian transitional cell carcinoma (TCC) is grouped with high-grade serous carcinoma (HGSC) in the current World Health Organization classification. TCC is associated with BRCA mutations and a better prognosis compared with HGSC. Previous papers examining the immunohistochemical features of TCC have studied limited numbers of samples. No marker reflecting the biological difference between TCC and HGSC is known. We collected a large cohort of TCC to determine whether TCC and HGSC could be distinguished by immunohistochemistry. A tissue microarray was built from 89 TCC and a control cohort of 232 conventional HGSC. Immunohistochemistry was performed, scored, and statistically analyzed for routine markers of HGSC and urothelial tumors: PAX8, WT1, p53, p16, ER, p63, and GATA3. Using scoring cutoffs commonly employed in clinical practice, the immunohistochemical profile of TCC was indistinguishable from HGSC for all markers. However, more detailed scoring criteria revealed statistically significant differences between the 2 groups of tumors with respect to ER, PAX8, and WT1. HGSC showed more diffuse and intense staining for PAX8 (P=0.004 and 0.001, respectively) and WT1 (P=0.002 and 0.002, respectively); conversely, TCC showed more intense staining for ER (P=0.007). TCC and HGSC therefore show subtle differences in their immunohistochemical profiles which might reflect underlying (epi)genetic differences. Further studies using proteomic analysis will focus on the identification of differentially expressed proteins that might serve as markers of TCC-like differentiation, which could help explain biologic differences between TCC and HGSC and might identify other cases of HGSC with a better prognosis.

Endogenous Myc maintains the tumor microenvironment.

The ubiquitous deregulation of Myc in human cancers makes it an intriguing therapeutic target, a notion supported by recent studies in Ras-driven lung tumors showing that inhibiting endogenous Myc triggers ubiquitous tumor regression. However, neither the therapeutic mechanism nor the applicability of Myc inhibition to other tumor types driven by other oncogenic mechanisms is established. Here, we show that inhibition of endogenous Myc also triggers ubiquitous regression of tumors in a simian virus 40 (SV40)-driven pancreatic islet tumor model. Such regression is presaged by collapse of the tumor microenvironment and involution of tumor vasculature. Hence, in addition to its diverse intracellular roles, endogenous Myc serves an essential and nonredundant role in coupling diverse intracellular oncogenic pathways to the tumor microenvironment, further bolstering its credentials as a pharmacological target.

L1CAM further stratifies endometrial carcinoma patients with no specific molecular risk profile.

BACKGROUND: The newly developed Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) has consistently been shown to be prognostically significant in endometrial carcinomas (EC). Recently, we and others have demonstrated L1 cell-adhesion molecule (L1CAM) to be a significant indicator of high-risk disease in EC. In the current study, it was our aim to determine the prognostic significance of aberrant L1CAM expression in ProMisE subgroups in a large, single centre, population-based EC cohort. METHODS: ProMisE (POLE; MMR-D; p53 wt/NSMP; p53 abn) classification results from a cohort of 452 EC were available for analysis. L1CAM expression was studied by immunohistochemistry on whole slides. Correlations between clinicopathological data and survival were calculated. RESULTS: Expression of L1CAM was most frequent in p53 abnormal tumours (80%). L1CAM status was predictive of worse outcome among tumours with no specific molecular profile (p53 wt/NSMP) (p < 0.0001). Among p53 wt/NSMP EC, L1CAM remained a significant prognosticator for disease-specific survival after multivariate analysis (p = 0.035). CONCLUSION: L1CAM status was able to significantly stratify risk among tumours of the large p53 wt/NSMP ProMisE subgroup of EC. Furthermore, our study confirms a highly significant correlation between mutation-type p53 immunostaining and abnormal L1CAM expression in EC.

DICER1 hot-spot mutations in ovarian gynandroblastoma.

AIMS: Gynandroblastoma is a rare ovarian sex cord-stromal tumour characterised by the presence of both male (Sertoli and/or Leydig cells) and female (granulosa cells) components. We investigated the mutational status of DICER1, FOXL2 and AKT1 genes at hot-spot regions that are known to be the key driving events in the development of Sertoli-Leydig cell tumour (SLCT), adult granulosa cell tumour (aGCT) and juvenile granulosa cell tumour (jGCT), respectively, to gain insights into the molecular pathogenesis of gynandroblastoma. METHODS AND RESULTS: Sixteen cases of gynandroblastoma were studied. All contained SLCT or Sertoli cell tumour components. aGCT and jGCT components were identified in seven and 10 cases, respectively, with one presenting both components. Heterozygous hot-spot mutations in the RNase IIIb domain of DICER1 were discovered in three cases, including one case with heterologous mucinous elements, all of which were composed of moderately or poorly differentiated SLCT and jGCT components, and harboured the mutations in both histological components. None of the 16 cases displayed mutations at the p.C134W (c.402C→G) of FOXL2 or within the pleckstrin-homology domain of AKT1. All cases showed FOXL2 immunostaining in both male and female components. CONCLUSION: DICER1 hot-spot mutation is the key-driving event in a subset of gynandroblastomas containing components of SLCT and jGCT. Gynandroblastomas composed of SLCT and jGCT may represent morphological variants of SLCT. The molecular basis of gynandroblastoma containing a component of aGCT is different from pure aGCT.

The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcaemic type.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.