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.

Primary clear cell carcinoma of the vulva: A case report.

Clear cell carcinoma (CCC) of the vulva is extremely rare. We report a case of a 54-year-old woman who presented with a 5 cm mass of the mons pubis. She underwent needle biopsy demonstrating CCC. She then underwent radical vulvectomy with bilateral inguinofemoral lymph node dissection. Surgical pathology revealed CCC of the vulva with lymphovascular space invasion (LVSI) and metastatic carcinoma in 1/7 inguinal lymph nodes. The patient has a history of endometriosis, raising suspicion that her CCC could have arisen from endometriosis in the mons. She completed adjuvant treatment with cisplatin and concurrent external beam radiation therapy with radiographic evidence of complete response. However, short-interval imaging demonstrated multi-focal recurrence, which was confirmed with supraclavicular lymph node biopsy. She then completed 8 cycles carboplatin, paclitaxel, and biosimilar bevacizumab-bvzr with favorable response on imaging. She was continued on bevacizumab maintenance. She was later started on pembroluzimab for disease progression based on new mediastinal adenopathy and worsening retroperitoneal lymphadenopathy. She received eight cycles of pembrolizumab with ongoing disease progression before enrolling in hospice and discontinuing cancer-directed treatment. As described in the related literature which we summarize here, the majority of reported cases of vulvar CCC arise from endometriosis implants at the site of prior episiotomy or from the Bartholin's gland. This patient had clinical history of endometriosis; prior tissue sampling was not performed to support the diagnosis. Given the absence of data regarding this rare type of primary vulvar cancer, treatment of this patient's disease was based on existing data specific to squamous cell carcinoma of the vulva and extrapolated from treatment guidelines for CCC of the ovary and endometrium. Continued research is needed on this rare form of vulvar carcinoma to determine the risk factors, prognostic factors, and treatment recommendations specific to this disease.

The Prognostic Effect of Immune Cell Infiltration Depends on Molecular Subtype in Endometrioid Ovarian Carcinomas.

PURPOSE: Endometrioid ovarian carcinoma (ENOC) is the second most-common type of ovarian carcinoma, comprising 10%-20% of cases. Recently, the study of ENOC has benefitted from comparisons to endometrial carcinomas including defining ENOC with four prognostic molecular subtypes. Each subtype suggests differential mechanisms of progression, although tumor-initiating events remain elusive. There is evidence that the ovarian microenvironment may be critical to early lesion establishment and progression. However, while immune infiltrates have been well studied in high-grade serous ovarian carcinoma, studies in ENOC are limited. EXPERIMENTAL DESIGN: We report on 210 ENOC, with clinical follow-up and molecular subtype annotation. Using multiplex IHC and immunofluorescence, we examine the prevalence of T-cell lineage, B-cell lineage, macrophages, and populations with programmed cell death protein 1 or programmed death-ligand 1 across subtypes of ENOC. RESULTS: Immune cell infiltrates in tumor epithelium and stroma showed higher densities in ENOC subtypes with known high mutation burden (POLEmut and MMRd). While molecular subtypes were prognostically significant, immune infiltrates were not (overall survival P > 0.2). Analysis by molecular subtype revealed that immune cell density was prognostically significant in only the no specific molecular profile (NSMP) subtype, where immune infiltrates lacking B cells (TILB minus) had inferior outcome (disease-specific survival: HR, 4.0; 95% confidence interval, 1.1-14.7; P < 0.05). Similar to endometrial carcinomas, molecular subtype stratification was generally superior to immune response in predicting outcomes. CONCLUSIONS: Subtype stratification is critical for better understanding of ENOC, in particular the distribution and prognostic significance of immune cell infiltrates. The role of B cells in the immune response within NSMP tumors warrants further study.

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.

The impact of whole genome and transcriptome analysis (WGTA) on predictive biomarker discovery and diagnostic accuracy of advanced malignancies.

In this study, we evaluate the impact of whole genome and transcriptome analysis (WGTA) on predictive molecular profiling and histologic diagnosis in a cohort of advanced malignancies. WGTA was used to generate reports including molecular alterations and site/tissue of origin prediction. Two reviewers analyzed genomic reports, clinical history, and tumor pathology. We used National Comprehensive Cancer Network (NCCN) consensus guidelines, Food and Drug Administration (FDA) approvals, and provincially reimbursed treatments to define genomic biomarkers associated with approved targeted therapeutic options (TTOs). Tumor tissue/site of origin was reassessed for most cases using genomic analysis, including a machine learning algorithm (Supervised Cancer Origin Prediction Using Expression [SCOPE]) trained on The Cancer Genome Atlas data. WGTA was performed on 652 cases, including a range of primary tumor types/tumor sites and 15 malignant tumors of uncertain histogenesis (MTUH). At the time WGTA was performed, alterations associated with an approved TTO were identified in 39 (6%) cases; 3 of these were not identified through routine pathology workup. In seven (1%) cases, the pathology workup either failed, was not performed, or gave a different result from the WGTA. Approved TTOs identified by WGTA increased to 103 (16%) when applying 2021 guidelines. The histopathologic diagnosis was reviewed in 389 cases and agreed with the diagnostic consensus after WGTA in 94% of non-MTUH cases (n = 374). The remainder included situations where the morphologic diagnosis was changed based on WGTA and clinical data (0.5%), or where the WGTA was non-contributory (5%). The 15 MTUH were all diagnosed as specific tumor types by WGTA. Tumor board reviews including WGTA agreed with almost all initial predictive molecular profile and histopathologic diagnoses. WGTA was a powerful tool to assign site/tissue of origin in MTUH. Current efforts focus on improving therapeutic predictive power and decreasing cost to enhance use of WGTA data as a routine clinical test.

SMARCA4 biology in alveolar rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and phenocopies a muscle precursor that fails to undergo terminal differentiation. The alveolar subtype (ARMS) has the poorest prognosis and represents the greatest unmet medical need for RMS. Emerging evidence supports the role of epigenetic dysregulation in RMS. Here we show that SMARCA4/BRG1, an ATP-dependent chromatin remodeling enzyme of the SWI/SNF complex, is prominently expressed in primary tumors from ARMS patients and cell cultures. Our validation studies for a CRISPR screen of 400 epigenetic targets identified SMARCA4 as a unique factor for long-term (but not short-term) tumor cell survival in ARMS. A SMARCA4/SMARCA2 protein degrader (ACBI-1) demonstrated similar long-term tumor cell dependence in vitro and in vivo. These results credential SMARCA4 as a tumor cell dependency factor and a therapeutic target in ARMS.

Accurate Distinction of Ovarian Clear Cell From Endometrioid Carcinoma Requires Integration of Phenotype, Immunohistochemical Predictions, and Genotype: Implications for Lynch Syndrome Screening.

Ovarian clear cell carcinoma (OCCC) and ovarian endometrioid carcinoma (OEC) are both associated with endometriosis but differ in histologic phenotype, biomarker profile, and survival. Our objectives were to refine immunohistochemical (IHC) panels that help distinguish the histotypes and reassess the prevalence of mismatch repair deficiency (MMRd) in immunohistochemically confirmed OCCC. We selected 8 candidate IHC markers to develop first-line and second-line panels in a training set of 344 OCCC/OEC cases. Interobserver reproducibility of histotype diagnosis was assessed in an independent testing cohort of 100 OCC/OEC initially without and subsequently with IHC. The prevalence of MMRd was evaluated using the testing cohort and an expansion set of 844 ovarian carcinomas. The 2 prototypical combinations (OCCC: Napsin A+/HNF1B diffusely+/PR-; OEC: Napsin A-/HNF1B nondiffuse/PR+) occurred in 75% of cases and were 100% specific. A second-line panel (ELAPOR1, AMACR, CDX2) predicted the remaining cases with 83% accuracy. Integration of IHC improved interobserver reproducibility (κ=0.778 vs. 0.882, P<0.0001). The prevalence of MMRd was highest in OEC (11.5%, 44/383), lower in OCCC (1.7%, 5/297), and high-grade serous carcinomas (0.7%, 5/699), and absent in mucinous (0/126) and low-grade serous carcinomas (0/50). All 5 MMRd OCCC were probable Lynch syndrome cases with prototypical IHC profile but ambiguous morphologic features: 3/5 with microcystic architecture and 2/5 with intratumoral stromal inflammation. Integration of first-line and second-line IHC panels increases diagnostic precision and enhances prognostication and triaging for predisposing/predictive molecular biomarker testing. Our data support universal Lynch syndrome screening in all patients with OEC when the diagnosis of other histotypes has been vigorously excluded.

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.

STING pathway expression in low-grade serous carcinoma of the ovary: an unexpected therapeutic opportunity?

Ovarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype-specific treatment modalities, especially for women with widespread and chemo-resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS-STING pathway that mediates innate immune defence against infectious DNA-containing pathogens and also detects tumour-derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low-grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high-grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low-grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.

Effect of the p53 P72R Polymorphism on Mutant TP53 Allele Selection in Human Cancer.

BACKGROUND: TP53 mutations occur in more than 50% of cancers. We sought to determine the effect of the intragenic P72R single nucleotide polymorphism (SNP; rs1042522) on the oncogenic properties of mutant p53. METHODS: P72R allelic selection in tumors was determined from genotype calls and a Gaussian distributed mixture model. The SNP effect on mutant p53 was determined in p53-negative cancer cell lines. RNA-sequencing, chromatin immunoprecipitation, and survival analysis were performed to describe the SNP effect. All statistical tests were 2-sided. RESULTS: Among 409 patients with germline heterozygous P72R SNP who harbored somatic mutations in TP53, we observed a selection bias against missense TP53 mutants encoding the P72 SNP (P = 1.64 x 10-13). Exogenously expressed hotspot p53 mutants with the P72 SNP were negatively selected in cancer cells. Gene expression analyses showed the enrichment of p53 pathway genes and inflammatory genes in cancer cells transduced with mutants encoding P72 SNP. Immune gene signature is enriched in patients harboring missense TP53 mutations with homozygous P72 SNP. These patients have improved overall survival as compared with those with the R72 SNP (P = .04). CONCLUSION: This is the largest study demonstrating a selection against the P72 SNP. Missense p53 mutants with the P72 SNP retain partial wild-type tumor-suppressive functions, which may explain the selection bias against P72 SNP across cancer types. Ovarian cancer patients with the P72 SNP have a better prognosis than with the R72 SNP. Our study describes a previously unknown role through which the rs1042522 SNP modifies tumor suppressor activities of mutant p53 in patients.

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.

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.

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.

Proteomic analysis of transitional cell carcinoma-like variant of tubo-ovarian high-grade serous carcinoma.

The current World Health Organization classification does not distinguish transitional cell carcinoma of the ovary (TCC) from conventional tubo-ovarian high-grade serous carcinoma (HGSC), despite evidence suggesting improved prognosis for patients with TCC; instead, it is considered a morphologic variant of HGSC. The immunohistochemical (IHC) markers applied to date do not distinguish between TCC and HGSC. Therefore, we sought to compare the proteomic profiles of TCC and conventional HGSC to identify proteins enriched in TCC. Prognostic biomarkers in HGSC have proven to be elusive, and our aim was to identify biomarkers of TCC as a way of reliably and reproducibly identifying patients with a favorable prognosis and better response to chemotherapy compared with those with conventional HGSC. Quantitative global proteome analysis was performed on archival material of 12 cases of TCC and 16 cases of HGSC using SP3 (single-pot, solid phase-enhanced, sample preparation)-Clinical Tissue Proteomics, a recently described protocol for full-proteome analysis from formalin-fixed paraffin-embedded tissues. We identified 430 proteins that were significantly enriched in TCC over HGSC. Unsupervised co-clustering perfectly distinguished TCC from HGSC based on protein expression. Pathway analysis showed that proteins associated with cell death, necrosis, and apoptosis were highly expressed in TCCs, whereas proteins associated with DNA homologous recombination, cell mitosis, proliferation and survival, and cell cycle progression pathways had reduced expression. From the proteomic analysis, three potential biomarkers for TCC were identified, claudin-4 (CLDN4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), and minichromosome maintenance protein 7 (MCM7), and tested by IHC analysis on tissue microarrays. In agreement with the proteomic analysis, IHC expression of those proteins was stronger in TCC than in HGSC (p < 0.0001). Using global proteomic analysis, we are able to distinguish TCC from conventional HGSC. Follow-up studies will be necessary to confirm that these molecular and morphologic differences are clinically significant.

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.

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.

Expression of L1 retrotransposon open reading frame protein 1 in gynecologic cancers.

LINE-1 (L1) retrotransposons are mobile genetic elements capable of "copy-and-pasting" their own sequences into random genomic loci, and one of the proteins it uses to achieve mobility is LINE-1 open reading frame 1 protein (L1ORF1p). L1ORF1p expression is found across many epithelial cancers, including small cohorts of ovarian and endometrial cancers, and is highly expressed in cancers with mutant p53 expressions. Here we aimed to gain insights into L1ORF1p expression levels within specific histotypes of ovarian cancers: high-grade serous (n = 585), low-grade serous (n = 26), clear cell (n = 132), endometrioid (n = 148), and mucinous (n = 32) ovarian cancers, as well as endometrial cancers (n = 607) using tissue microarray (TMA's). We demonstrated that L1ORF1p expression is associated with advanced stage and serous histotype in gynecological cancers. Like previous studies, we found a higher proportion of L1ORF1p expression in cases with aberrant p53 expression. We evaluated the expression of L1ORF1p in serous tubal intraepithelial carcinomas (STICs) (n = 6) and p53 signature lesions (n = 2) in fallopian tubes. Three STIC cases displayed aberrant p53 overexpression with corresponding L1ORF1p expression in the same tissues, but such correlation was not seen in the two p53 signature lesions, suggesting that L1 protein may be expressed after dysplastic transformation. The remaining three STIC cases have TP53 nonsense mutations with absent p53 expression but a strong and clear L1ORF1p expression within the STIC lesions. While L1ORF1p may not be prognostic in gynecological cancers, it may be useful clinically as a diagnostic IHC marker for p53 null STIC lesions and this warrants further investigation.

DICER1 and FOXL2 Mutation Status Correlates With Clinicopathologic Features in Ovarian Sertoli-Leydig Cell Tumors.

Sertoli-Leydig cell tumors (SLCTs) are rare ovarian sex cord-stromal neoplasms. The only known recurrent genetic abnormality is DICER1 mutation, with rare mutations reported in FOXL2. We set out to establish a molecular classifier using DICER1 and FOXL2 somatic mutation status and clinicopathologic features in 42 SLCTs. Five tumors (12%) were well differentiated, 31 (74%) moderately differentiated, and 6 (14%) poorly differentiated. Eight (19%) had heterologous elements, and 2 (5%) showed retiform differentiation; all 10 were moderately differentiated. DICER1 RNase IIIb domain mutations were identified in 18/41 (44%; 17 moderately, 1 poorly differentiated), including all cases with retiform or heterologous elements. FOXL2 c.402C>G (p.C134W) mutation was identified in 8/42 (19%) tumors (5 moderately, 3 poorly differentiated). DICER1 and FOXL2 mutations were mutually exclusive. Median age for the cohort was 47 years (range, 15 to 90 y). Patients with DICER1 mutations were younger (median, 24.5 y; range, 15 to 62 y) than patients with FOXL2 mutation (median, 79.5 y; range, 51 to 90 y) (P<0.0001). Nine of 10 tumors with retiform or heterologous elements occurred in premenopausal patients (median, 26.5 y; range, 15 to 57 y). Patients with tumors that were wild type for DICER1 and FOXL2 (15/42, 37%) had an intermediate age (median, 51 y; range, 17 to 74 y). All tumors were FOXL2 positive by immunohistochemistry. Patients with FOXL2 mutation trended toward presenting more often with abnormal bleeding (P=0.13); DICER1-mutant patients trended toward having more androgenic symptoms (P=0.22). Our data suggest at least 3 molecular subtypes of SLCT with distinct clinicopathologic features: DICER1 mutant (younger, more androgenic symptoms, moderately/poorly differentiated, retiform or heterologous elements), FOXL2 mutant (postmenopausal, abnormal bleeding, moderately/poorly differentiated, no retiform or heterologous elements), and DICER1/FOXL2 wild type (intermediate age, no retiform or heterologous elements, including all well-differentiated tumors).

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.