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We report a case of a cystic ovarian neoplasm in a 76-yr-old female composed of 2 distinct and intimately associated components: a macrocystic adult granulosa cell tumor (AGCT) and a serous borderline tumor. The granulosa cell nature of the tumor was confirmed with positive immunohistochemical staining for inhibin, calretinin, and WT1, while the neoplastic nature of the granulosa cell proliferation was supported by the presence of a point mutation of the FOXL2 gene. A review of 19 previously reported mixed AGCT and epithelial neoplasms of the ovary is included. Of the eight mixed AGCT and epithelial tumors, including our case, that were tested for FOXL2 mutation, 4 of the 5 mutation-positive cases were notable for demonstrating a macroscopically visible nodule or mass of AGCT at the time of gross examination, while 2 of the 3 mutation-negative cases lacked a mass-producing granulosa cell component. This feature by itself may be sufficient to predict the true neoplastic nature of the granulosa cell proliferation. This is the first reported case of a composite neoplastic AGCT and serous borderline tumor. We also discuss the current histogenetic models for these rare mixed AGCT and epithelial tumors., (Copyright © 2022 by the International Society of Gynecological Pathologists.)

Ovarian serous borderline tumors (SBTs) harboring the BRAFV600E mutation are associated with decreased risk of progression to low-grade serous carcinoma, and often prominently feature tumor cells with abundant eosinophilic cytoplasm. Since eosinophilic cells (ECs) may be a marker of the underlying genetic driver, we proposed morphologic criteria and evaluated the interobserver reproducibility for assessing this histologic feature. Following the completion of an online training module, representative tumor slides from 40 SBTs ( BRAFV600E -mutated, n=18, BRAF -wildtype, n=22) were independently reviewed by 5 pathologists. For each case, reviewers provided a semiquantitative assessment of the extent of ECs (0: absent, 1: <10%, 2: 10%-50%, or 3: >50%, of tumor area). Interobserver reproducibility for estimating the extent of ECs was moderate (κ=0.41). Applying a cut-off score of ≥2, the median sensitivity and specificity for predicting BRAFV600E mutation were 67% and 95%, respectively. With a cut-off score of ≥1, median sensitivity and specificity were 100% and 82%, respectively. Morphologic mimics of ECs, including tumor cells with tufting or hobnail change and detached cell clusters in micropapillary SBTs, were possible contributing factors for discordant interobserver interpretations. BRAFV600E immunohistochemistry showed diffuse staining in BRAF -mutated tumors, including those with few ECs. In conclusion, the finding of extensive ECs in SBT is highly specific for BRAFV600E mutation. However, in some BRAF -mutated SBTs, ECs may be focal and/or difficult to distinguish from other tumor cells with overlapping cytologic features. The morphologic finding of definitive ECs, even when scarce, should therefore prompt consideration for BRAFV 600E mutation testing., Competing Interests: M.H.C. has received an honorarium from Roche, outside of the submitted work. The remaining authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Background: Over the years, the detection rate of pancreatic cystic neoplasms (PCNs) has significantly increased; however, the differential diagnosis and identification of high-risk PCNs remain challenging. We sought to investigate whether chromosomal instability (CIN) features in cell-free DNA in the cystic fluid of PCNs could help to identify high-risk PCNs., Methods: Pancreatic cystic fluid samples from 102 patients with PCNs were intraoperatively collected for detection of CIN using an ultrasensitive chromosomal aneuploidy detector. Clinical and imaging data were retrospectively collected, and statistical analysis was performed to assess the potential role of CIN in clinical practice., Results: CIN was investigated in a total of 100 patients. Sixteen of 26 serous cystic cystadenomas (SCAs) harbored deletions of chr3p and/or chr6p, whereas low rates of CIN were detected in mucinous cystic neoplasms. Most malignant PCNs presented with more than one type of CIN; amplification of chr1q and chr8q found in nine and seven of 11 malignant PCNs (81.8% and 63.6%), respectively, could aid in distinguishing high-risk IPMNs from low-risk ones, with a higher sensitivity than imaging. A combination of the mural nodule imaging feature and amplification of chr1q and chr8q achieved a sensitivity of 70.0% and a specificity of 82.4% in identifying high-risk IPMNs., Conclusions: Our work revealed the distinct CIN signature of different types of PCNs. Deletions of chr3p and chr6p defined a subtype of SCAs. Gains of chr1q and chr8q were associated with insidious malignant PCNs and helped identify high-risk IPMNs., (© 2023. Society of Surgical Oncology.)

Oncogenic activation of the mitogen-activated protein kinase (MAPK) pathway due to KRAS or BRAF gain-of-function mutation is frequently found in ovarian serous borderline tumor (SBT) and their extraovarian implants. We investigated mutational status of KRAS and BRAF of the primary ovarian SBTs that had a high stage presentation in correlation with clinical outcome. Among 39 consecutive primary SBTs with either invasive implants (20 cases) or non-invasive implants (19 cases), KRAS and BRAF mutational analysis was informative in 34 cases. Sixteen cases (47%) harbored a KRAS mutation, while 5 cases (15%) had a BRAF V600E mutation. High-stage disease (IIIC) was seen in 31% (5/16) of patients with a KRAS mutation and 39% (7/18) of patients without a KRAS mutation (p = 0.64). KRAS mutations were present in 9/16 (56%) tumors with invasive implants/LGSC versus 7/18 (39%) tumors with non-invasive implants (p = 0.31). BRAF mutation was seen in 5 cases with non-invasive implants. Tumor recurrence was seen in 31% (5/16) of patients with a KRAS mutation, compared to 6% (1/18) of patients without a KRAS mutation (p = 0.04). A KRAS mutation predicted an adverse disease-free survival (31% survival at 160 months) compared to those with wild-type KRAS (94% at 160 months; log-rank test, p = 0.037; HR 4.47). In conclusion, KRAS mutation in primary ovarian SBTs is significantly associated with a worse disease-free survival, independent of the high tumor stage or histological subtypes of extraovarian implant. KRAS mutation testing of primary ovarian SBT may servce as a useful biomarker for tumor recurrence., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The development of cross-sectional imaging techniques has enhanced the detection of pancreatic cystic lesions (PCLs). PCLs are found in approximately 2% of the general population, often as incidentally detected lesions on computed tomography or MRI during the evaluation of other medical conditions. Broadly, PCLs are classified as mucinous or nonmucinous. Mucinous PCLs include mucinous cystic neoplasms and intraductal papillary mucinous neoplasms. Nonmucinous PCLs include pseudocysts, serous cystadenomas, solid pseudopapillary neoplasms, and cystic pancreatic neuroendocrine tumors, as well as cystic acinar cell carcinoma, cystic degeneration of pancreatic ductal adenocarcinoma, lymphoepithelial cyst, and others., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objective: To analyze the role of cancer stem cells (CSC) in ovarian carcinogenesis through the identification of CD133 expression in the normal ovary (NO), serous cystadenoma (SC), borderline serous tumour (BST), low-grade serous carcinoma (LGSC), and high-grade serous carcinoma (HGSC)., Materials and Methods: A total of 48 tissue samples contain 5 NO, 10 SC, 5 BST, 8 LGSC, and 20 HGSC were stained with anti-CD133 antibody by immunohistochemical protocol. The difference in the H-score of CD133 expression between groups and their relationship to age, histomorphology, and localization was analyzed., Results: CD133 expression varied among tumor groups, with clinicopathologic parameters showing diverse associations (age p = 0.773; histomorphology p = 0.001; and localization p = 0.026). The comparison of CD133 H-scores differed significantly between each group (p = 0.0031), in which precursor and malignant lesions possessed more robust CD133 expression., Conclusion: The presence of CD133 cellular expression and localization in different types of serous ovarian tumours suggests that these markers are involved in ovarian tumorigenesis.

High-grade serous ovarian cancer (HGSOC) is characterized by TP53 mutation and somatic copy number alterations (SCNAs). Here we show that the oncogenic transcription factor EVI1 (ecotropic viral integration site-1) is amplified and overexpressed up to 30% of 1640 HGSOC cases in The Cancer Genome Atlas (TCGA). Functionally, EVI1 promotes proliferation/invasion in vitro and tumor growth of xenograft model in vivo. Importantly, we discover that EVI1 regulates estrogen signaling by directly activating ESR1 (estrogen receptor 1) transcription determined by the ChIP and luciferase assay. Interestingly, EVI1 and ESR1 share common regulatory targets as indicated by the analysis of ChIP-Seq data. EVI1 and ESR1 collaborate in the regulation of some estrogen receptor-regulated genes. Furthermore, EVI1 drives tumor aggressiveness partially by regulating estrogen signaling. Estrogen enhances the proliferation, invasion and xenograft growth of ovarian cancer cells. Importantly, estrogen can rescue the inhibition of proliferation, invasion and xenograft growth induced by silencing EVI1. These findings suggest that EVI1 functions as a novel regulator of the estrogen signaling network in ovarian cancer., (Copyright © 2021. Published by Elsevier B.V.)

Fusion genes are structural chromosomal rearrangements resulting in the exchange of DNA sequences between genes. This results in the formation of a new combined gene. They have been implicated in carcinogenesis in a number of different cancers, though they have been understudied in high grade serous ovarian cancer. This study used high throughput tools to compare the transcriptome of high grade serous ovarian cancer and normal fallopian tubes in the interest of identifying unique fusion transcripts within each group. Indeed, we found that there were significantly more fusion transcripts in the cancer samples relative to the normal fallopian tubes. Following this, the role of fusion transcripts in chemo-response and overall survival was investigated. This led to the identification of fusion transcripts significantly associated with overall survival. Validation was performed with different analytical platforms and different algorithms to find fusion transcripts.

Ovarian cancer is the fifth leading cause of cancer-related mortality in women. Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme and there is growing evidence to suggest that it plays an important role in cancer progression. This is the first study to examine the expression of NNMT in serous ovarian cystadenomas, serous borderline tumours, low grade serous carcinomas (LGSC) and high grade serous carcinomas (HGSC) and investigate the potential independent association of NNMT expression with survival. Tissue samples were analysed immunohistochemically for NNMT expression. The stromal NNMT score was significantly higher in HGSC compared to serous cystadenomas and serous borderline tumours ( p  < .001, p  < .043, respectively). The mean stromal NNMT score of patients with HGSC was significantly higher than patients with LGSC ( p  = .043). Patients with low expression of NNMT had a significantly higher mean recurrence-free survival than patients with high expression ( p  = .036). NNMT may support tumour progression in ovarian cancer by promoting desmoplastic stromal tumour reaction. NNMT overexpression may be associated with poor prognosis and can be a therapeutic target in ovarian cancer.IMPACT STATEMENT What is already known on this subject? Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that is overexpressed in many malignancies. Its overexpression was shown to lead to histone hypomethylation, which in turn can decrease and increase the expression of tumour suppressor proteins and onco-proteins, respectively. NNMT was also shown to play a role in epithelial-to-mesenchymal transition, which is critical in tumour progression and the stromal tumour reaction. The stromal tumour reaction was recently targeted with promising therapeutic results in ovarian cancer. What do the results of this study add? The expression of NNMT in various ovarian neoplasms including serous cystadenomas, borderline tumours and serous carcinomas has not been studied and independently associated with poor survival, previously. This study suggests that NNMT is progressively overexpressed in the stroma of ovarian neoplasms from benign cysts to HGSCs. NNMT overexpression appears to be independently associated with poor survival in ovarian cancer. What are the implications of these findings for clinical practice and/or further research? The implications of these findings are that NNMT may play an important role in the stromal tumour reaction, and therefore its overexpression may contribute to poor survival. NNMT overexpression may be an important target of ovarian cancer therapy.

Background: Recent studies have revealed thousands of A-to-I RNA editing events in primates. These events are closely related to the occurrence and development of multiple cancers, but the origination and general functions of these events in ovarian cancer remain incompletely understood., Objective: To further the determination of molecular mechanisms of ovarian cancer from the perspective of RNA editing., Methods: Here, we used the SNP-free RNA editing Identification Toolkit (SPRINT) to detect RNA editing sites. These editing sites were then annotated, and related functional analysis was performed., Results: In this study, about 1.7 million RES were detected in each sample, and 98% of these sites were due to A-to-G editing and were mainly distributed in non-coding regions. More than 1,000 A-- to-G RES were detected in CDS regions, and nearly 700 could lead to amino acid changes. Our results also showed that editing in the 3'UTR regions could influence miRNA-target binding. We predicted the network of changed miRNA-mRNA interaction caused by the A-to-I RNA editing sites. We also screened the differential RNA editing sites between ovarian cancer and adjacent normal tissues. We then performed GO and KEGG pathway enrichment analysis on the genes that contained these differential RNA editing sites. Finally, we identified the potential dysregulated RNA editing events in ovarian cancer samples., Conclusion: This study systematically identified and analyzed RNA editing events in ovarian cancer and laid a foundation to explore the regulatory mechanism of RNA editing and its function in ovarian cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Purpose: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features., Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting., Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations., Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271 ., (©2020 American Association for Cancer Research.)

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis 1 . To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells 2 . Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape 3-5 and provides a resource for the development of novel therapeutic approaches.

Background: The prevalence of incidental pancreatic cystic neoplasms (PCNs) has increased dramatically with advancements in cross-sectional imaging. Diagnostic imaging is limited in differentiating between benign and malignant PCNs. The aim of this review is to provide an overview of biomarkers that can be used to distinguish PCNs., Methods: A review of the literature on molecular diagnosis of cystic neoplasms of the pancreas was performed., Results: Pancreatic cysts can be categorized into inflammatory and non-inflammatory lesions. Inflammatory cysts include pancreatic pseudocysts. Noninflammatory lesions include both mucinous and non-mucinous lesions. Mucinous lesions include intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm. Non-mucinous lesions include serous cystadenoma and solid-pseudopapillary tumor of the pancreas. Imaging, cyst aspiration, and histologic findings, as well as carcinoembryonic antigen and amylase are commonly used to distinguish between cyst types. However, molecular techniques to detect differences in genetic mutations, protein expression, glycoproteomics, and metabolomic profiling are important developments in distinguishing between cyst types., Discussion: Nomograms incorporating common clinical, laboratory, and imaging findings have been developed in a better effort to predict malignant IPMN. The incorporation of top molecular biomarker candidates to nomograms may improve the predictive ability of current models to more accurately diagnose malignant PCNs.

Noninvasive ovarian low-grade serous tumors [atypical proliferative serous tumor (APST)/serous borderline tumor] appear to progress to invasive low-grade serous carcinoma (LGSC) at a low but regular rate. The underlying biology of this phenomenon is unknown. We studied 18 patients with 30 ovarian tumors (12 bilateral), including APST, noninvasive LGSC and invasive LGSC, who also had low-grade serous carcinomatosis. Tumors were evaluated for microinvasion (usual eosinophilic cell type), microinvasive carcinoma (<5 mm invasion of micropapillary nests), and overt carcinoma (≥5 mm invasion of micropapillary nests). Tumors were evaluated based on the original numerical order of sections under the hypothetical scenarios in which sampling was stopped at 1 section/cm and 2 sections/cm. Sampling based on 1 section/cm of greatest tumor dimension identified invasion of any type in 21 tumors (70%). Among these 21 tumors, 10 had microinvasive carcinoma, and 11 overt carcinoma. Sampling based on 2 sections/cm identified microinvasive carcinoma in 9 tumors and overt carcinoma in 14 tumors. With increased sampling from 1 to 2 sections/cm, the diagnosis in 3 tumors would have changed from microinvasive carcinoma to overt carcinoma, and in an additional 2 tumors from APST to APST with microinvasive carcinoma. Sampling based on >2 sections/cm changed the diagnosis in 1 additional case of APST with microinvasive carcinoma to overt carcinoma. These findings support that undetected (unsampled) occult invasion in the primary ovarian tumors is a likely explanation for some cases of apparent progression of noninvasive low-grade serous ovarian tumors to invasive LGSC. To minimize undetected occult invasion, consideration of sampling noninvasive low-grade ovarian serous tumors with at least 2 sections/cm of maximum tumor diameter may be warranted. The eosinophilic cell type of microinvasion, or microinvasive carcinoma, regardless of size, should prompt further sampling to identify overt carcinoma. The eosinophilic type of microinvasion was never seen alone in this cohort and by itself may be biologically insignificant.

Background: Invasive mammary carcinomas that spontaneously develop in female cats are associated with high mortality, and resemble the most aggressive human breast cancers, especially triple-negative breast cancer (TNBC). Transcriptome studies showed that TNBCs are a heterogeneous group that includes a potentially hormone-dependent subtype named luminal-AR. Some authors proposed an immunohistochemical definition of the luminal-AR subtype, which is not only positive for Androgen Receptor (AR), but also either positive for the transcription factor Forkhead box A1 (FOXA1), or negative for basal markers. The objectives of this study were to describe AR and FOXA1 expressions in feline mammary carcinomas (FMCs), their prognostic value, and if their coexpression could define a "luminal-AR" subtype of triple-negative mammary carcinomas in cats., Methods: In a previously described retrospective cohort of 180 female cats with FMCs, with a 2-year follow-up post-mastectomy, we assessed AR, FOXA1, ER, PR, Ki-67, HER2, and CK14 expressions by automated immunohistochemistry., Results: Of the 180 FMCs, 57 (32%) were luminal; i.e., ER and/or PR positive, and 123 (68%) were triple-negative (ER-, PR- and HER2-) FMCs. AR overexpression (found in 33 cases/180, 18%) and FOXA1 index ≥1% (64/180, 36%) were associated with a longer disease-free interval, overall survival, and cancer-specific survival in cats with FMC. Analysis of AR, FOXA1 and CK14 coexpression in triple-negative FMCs showed that AR+ triple-negative FMCs were heterogeneous: there existed an AR+ FOXA1+ CK14- subgroup (n = 7) associated with a better cancer-specific survival by multivariate survival analysis (HR = 0.26, 95% CI: 0.07-0.89, p = 0.03) compared to AR+ FOXA1-CK14+ triple-negative FMCs (n = 46) (HR = 1.00), independently of the pathologic tumor size and pathologic nodal stage. The non-basal-like subtype of triple-negative FMCs that coexpresses AR and FOXA1 (the AR+ FOXA1+ CK14- subgroup) could represent the equivalent of the luminal-AR subgroup of human triple-negative breast cancer., Conclusions: We identified an AR+ FOXA1+ CK14- subgroup of triple-negative FMCs that might correspond to the luminal-AR subgroup of human triple-negative breast cancers. Cats with FMC may be interesting spontaneous animal models to investigate new strategies targeting the androgen receptor, especially in the aggressive subtype of AR+ basal-like triple-negative mammary carcinomas with loss of FOXA1 expression (the AR+ FOXA1-CK14+ subgroup).

Large-scale genomic studies have detailed the molecular landscape of tumors from patients with high-grade serous ovarian cancers (HGSC) who underwent primary debulking surgery and correlated the identified subgroups to survival. In recent years, there is increased use of neoadjuvant chemotherapy (NACT) for patients with HGSC and while abundant data exist for patients who underwent primary debulking, little data are available on the cancer cells remaining after NACT that could lead to recurrences. We aimed to analyze gene expression profiles of NACT-treated HGSC tumor samples, and correlate them to treatment response and outcome. Tumor samples were collected from patients with stage III or IV HGSC (NACT cohort, N = 57) at the time of surgery and diagnosis (biopsy samples N = 8). Tumor content was validated by histologic examination and bioinformatics. Gene expression analysis was performed using a tailored NanoString-based assay, while sequencing was performed using MiSeq. A cross-validated survival classifier revealed patient clusters with either a "Better" or "Worse" prognostic outcome. The association with overall survival remained significant after controlling for clinical variables, and differential gene expression, gene set enrichment analyses, and the appropriate survival models were used to assess the associations between alterations in gene expression in cancer cells remaining after NACT and outcome. Pathway-based analysis of the differentially expressed genes revealed comparatively high levels of cell cycle and DNA repair gene expression in the poor outcome group. IMPLICATIONS: Our work suggests mRNA expression patterns in key genes following NACT may reflect response to treatment and outcome in patient with HGSC., (©2019 American Association for Cancer Research.)

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex) 6 + (Man) 3 (GlcNAc) 2 , and the complex neutral structure, (Hex) 2 (HexNAc) 2 (Deoxyhexose) 1 + (Man) 3 (GlcNAc) 2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although risk factors have been established for the development of serous carcinoma after a diagnosis of serous borderline tumor (SBT), comprising atypical proliferative serous tumor (APST) (ie, conventional SBT) and noninvasive low-grade serous carcinoma (niLGSC) (ie, micropapillary SBT), subsequent invasive carcinoma still occurs in a subset of women who are not at increased risk. Whether subsequent serous carcinoma in women with a prior SBT represents malignant progression/recurrence or an independent primary tumor is unclear, and the combined clinicopathologic and molecular features of SBTs and their subsequent carcinomas have not been fully characterized. In this study, we analyzed a cohort of 42 women initially diagnosed with SBT who subsequently developed serous carcinoma of a total of 1025 cases of ovarian SBT from a nationwide population-based cohort. Review of the diagnostic slides was performed from this subset of SBTs and matched metachronous invasive serous carcinomas (39 low grade, 3 high grade). DNA was extracted from tissue blocks available for 41 cases (both SBT and carcinoma, n=36; SBT only, n=3; carcinoma only, n=2). Samples were subjected to digital droplet PCR to analyze mutation hotspots in KRAS (codon 12) and BRAF (V600E), which are frequently found in low-grade serous tumors. Eighty-one percent of SBTs (34/42) were APST, and 19% (8/42) were niLGSC. Forty percent of cases (17/42) were FIGO stage I, the majority of which were APST (14/17; 82%). The median time to development of carcinoma was 9 years (range, 0.6 to 25 y). Mutations in SBTs were distributed as follows: 5/39 (13%) BRAF mutant, 22/39 (56%) KRAS mutant, and 12/39 (31%) wild-type for both genes. There was a significant relationship between SBT gene mutation and histologic type, with BRAF mutations occurring exclusively in APST and a higher frequency of niLGSC among SBTs wild-type for BRAF and KRAS (P=0.01). The diffuse presence of tumor cells with abundant eosinophilic cytoplasm was significantly associated with the BRAF mutation (P=0.001). Mutational analyses of matched SBT/carcinoma pairs revealed concordant profiles in 33/36 (92%) cases, of which 19 (53%) were KRAS mutant, 4 (11%) were BRAF mutant, and 10 (28%) were wild type for both genes. The 3 discordant cases consisted of a wild-type niLGSC with a subsequent BRAF-mutant invasive LGSC, a KRAS-mutant APST with a KRAS-mutant LGSC, and a BRAF-mutant APST with subsequent development of a KRAS-mutant high-grade serous carcinoma. In conclusion, some women with SBTs can subsequently develop serous carcinoma, occasionally over 10 years later. Most subsequent carcinomas are low grade, but a small subset can be high grade. The type of gene mutation in SBT correlates with various histologic features. While most cases of serous carcinoma developing after a diagnosis of SBT probably represent tumor progression, a minority are independent primary tumors, presumably arising from endosalpingiosis.

Objectives: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND)., Methods: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs., Results: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs (TBX15, BMP3) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86-0.99), 0.71 (0.57-0.85), and 0.72 (0.60-0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77-0.94, P = 0.2)., Discussion: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.

Aims: Low-grade serous carcinomas (LGSCs) and their precursors serous borderline tumours (SBTs) characteristically harbour mutations in BRAF, KRAS or NRAS but rarely in TP53, whereas high-grade serous carcinomas (HGSCs) are characterised by frequent TP53 mutations but rare BRAF, KRAS or NRAS mutations. In a small subset of cases, LGSCs and/or SBTs develop into high-grade tumours, including HGSCs and poorly differentiated carcinomas (PDCs). Here, we sought to define the repertoire of somatic genetic alterations in low-grade serous tumours and synchronous or metachronous high-grade adnexal carcinomas., Methods and Results: DNA extracted from five SBTs/LGSCs and synchronous or metachronous HGSCs/PDCs and matched normal tissue was subjected to massively parallel sequencing targeting all exons and selected non-coding regions of 341 cancer-related genes. The low-grade and high-grade tumours from a given case were related, and shared mutations and copy number alterations. Progression from low-grade to high-grade lesions was observed, and involved the acquisition of additional mutations and/or copy number alterations, or shifts from subclonal to clonal mutations. Only two (an HGSC and a PDC) of the five high-grade tumours investigated harboured TP53 mutations, whereas NRAS and KRAS hotspot mutations were seen in two HGSCs and one HGSC, respectively., Conclusions: Our results suggest that progression from SBT to HGSC may take place in a subset of cases, and that at least some of the rare HGSCs lacking TP53 mutations may be derived from a low-grade serous precursor., (© 2018 John Wiley & Sons Ltd.)

Purpose: The ovarian cancer data set from The Cancer Genome Atlas integrates genomic and proteomic data with clinical annotations based on chart abstractions. We aimed to develop an algorithm to create a matching, more accessible clinical data set cataloging time to treatment failure (TTF) of sequential lines of treatment in patients with serous ovarian cancers., Materials and Methods: The master data set of 587 patients with serous ovarian cancer was condensed into a more homogeneous and clinically relevant population comprised of high-risk patients with both grade 3 cancers and stage III or IV disease, resulting in a subgroup of 450 patients. We quantified the TTF of different lines of therapy as well as different therapeutic combinations by extrapolating from the time of starting one therapy to the time of starting a subsequent therapy., Results: The overall survival (OS) of patients was highly related to platinum sensitivity status, with median OS times of 56.6, 27.0, and 11.6 months in patients who had platinum-sensitive, -resistant, or -refractory disease, respectively. In high-risk patients, the median TTFs were 14.8, 10.2, 5.7, and 4.1 months with the first, second, third, and fourth lines of chemotherapy, respectively. Patients with stable disease after first-line therapy had similar OS outcomes as patients with partial remissions (34.4 v 33.7 months, respectively)., Conclusion: This new data set enhances the clinical annotation by providing exploitable chemotherapy benefit data that can now be paired with genomic and proteomic data within The Cancer Genome Atlas data. The major determinant of OS in this study was platinum sensitivity status. TTF decreased with each successive line of therapy. However, patients who achieved only stable disease with first-line therapy had OS similar to those with partial remission.

Cystic neoplasms in the pancreas are encountered frequently on imaging, often detected incidentally during evaluation for other conditions. They can have a variety of clinical and imaging presentations, and similarly, wide-ranging prognostic and treatment implications. In the majority, imaging helps in diagnosis of pancreatic cystic neoplasms (PCNs) and guides management decisions. But, a significant minority of the PCNs remain indeterminate. There have been multiple recent advances in biomarkers and molecular genetics which will likely prove helpful in risk stratification of PCNs. Several prominent national and international societies, as well as consensus groups have put forth recommendations to help guide management of PCNs. The purpose of this article is to discuss the role of imaging in evaluation of PCNs, review the recent advances in molecular genetics and pancreatic cyst fluid analysis, and analyze the pros and cons of major evidence-based and consensus guidelines for management of PCNs.

In contrast to non-invasive extraovarian implants, invasive implants of ovarian serous borderline tumor/atypical proliferative serous tumor are associated with adverse outcome and have been reclassified as low-grade serous carcinoma. Mutations of KRAS and/or BRAF have been reported in up to 50% of serous borderline tumor/atypical proliferative serous tumor. We investigated KRAS and BRAF mutation frequencies in the two types of implants of serous borderline tumor/atypical proliferative serous tumor in correlation with clinical outcome. Forty-two implants of serous borderline tumor from 39 patients were included (invasive implants/low-grade serous carcinoma, n=20; non-invasive implants, n=22). KRAS mutation was found in 12 of 20 invasive implants (60%) and 3 of 22 non-invasive implants (14%). BRAF V600E mutation was found in 1 of 22 non-invasive implants (5%) and none in invasive implants (0%). Invasive implants were more frequently associated with higher stage disease. Nine of 14 patients (64%) with KRAS mutation were found to have stage IIIC disease, while 5 of 24 patients (20%) without the mutation had stage IIIC disease. Patients with invasive implants had higher recurrence rate compared to those with non-invasive implants (60 vs 14 %, P=0.0003, log-rank test) and worse disease-specific survival (P=0.0008, log-rank test). Regardless of the histological subtypes, patients with KRAS mutation positive implants had significantly higher recurrence rate than those without the mutation (71 vs 21%, P=0.0021, log-rank test) and an unfavorable disease-specific survival (P=0.0104, log-rank test). In conclusion, compared to those with non-invasive implants, patients with invasive implants present with higher stage of the disease, higher recurrence rate and worse survival. KRAS mutation, but not BRAF V600E mutation, is significantly associated with invasive implants of serous borderline tumor. Regardless of the histological subtypes of the implants, KRAS mutation is a significant prognostic indicator for high risk of tumor recurrence and worse disease-specific survival.

With the current epidemic of diagnosed pancreatic cystic neoplasms on the rise, a substantial amount of work has been done to unravel their biology, thus leading to implications on clinical decision making. Recent genetic profiling of resected human specimens has identified alterations in signaling pathways involving KRAS and GNAS signaling as early events in the pathogenesis of intraductal pancreatic mucinous neoplasms. Progressively, mutations in genes such as TP53, SMAD4, RNF43, and others are thought to characterize invasive and advanced lesions. The role of inflammation in fueling the growth and transformation of these cysts has also begun to be studied with greater interest. A number of promising clinical studies have attempted to integrate these genetic insights into classifying these cysts and treating patients. We have reviewed existing literature on similar lines besides commenting on some useful animal models that recapitulate molecular and phenotypic progression of these cysts.

We report the 402C-G FOXL2 mutation status in 1 epithelial ovarian lesion in a 38-yr-old woman showing stromal proliferations that were morphologically indistinguishable from adult granulosa cell tumor (AGCT). The lesion was a serous borderline tumor. The AGCT-like components were distributed within the septa and cyst walls. FOXL2 mutation was absent. The combination of an epithelial neoplasm and AGCT-like areas is rare but described. The AGCT-like components are likely to be tumor-like proliferations but not truly neoplastic AGCT. FOXL2 mutation testing may be useful in confirming an AGCT-like component.

Epithelial ovarian tumors are a group of morphologically and genetically heterogeneous neoplasms. Based on differences in clinical phenotype and genetic background, ovarian neoplasms are classified as low-grade and high-grade tumor. Borderline ovarian tumors represent approximately 10%-20% of all epithelial ovarian masses. Various histological subtypes of ovarian malignancies differ in terms of their risk factor profiles, precursor lesions, clinical course, patterns of spread, molecular genetics, response to conventional chemotherapy, and prognosis. The most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous borderline tumors, as well as in mucinous cancers, are mutations in BRAF and KRAS genes. The most commonly observed BRAF mutation is substitution of glutamic acid for valine in codon 600 (V600E) in exon 15. The primary aim of this study was to determine whether fully integrated, real-time polymerase chain reaction-based Idylla™ system may be useful in determination of BRAF gene mutation status in codon 600 in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 42 patients with histopathologically verified ovarian masses, who were operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland). Based on histopathological examination of surgical specimens, 35 lesions were classified as low-grade ovarian carcinomas, and 7 as borderline ovarian tumors. Specimens with expression of BRAF V600E (VE1) protein were tested for mutations in codon 600 of the BRAF gene, using an automated molecular diagnostics platform Idylla™. Cytoplasmic immunoexpression of BRAF V600E (VE1) protein was found in three specimens: serous superficial papilloma, serous papillary cystadenoma of borderline malignancy, and partially proliferative serous cystadenoma. All specimens with the expression of BRAF V600E (VE1) protein were tested positively for BRAF V600E/E2/D mutation. No statistically significant relationship (p > 0.05) was found between the presence of BRAF V600E mutation and the probability of 5-year survival. BRAF mutation testing with a rapid, fully integrated molecular diagnostics system Idylla™ may be also a powerful prognostic tool in subjects with newly diagnosed serous borderline tumors, identifying a subset of patients who are unlikely to progress.

Alterations in p16 protein expression have been reported to be associated with tumor development and progression. However, p16 expression status in the peritumoral stroma has been rarely investigated. We investigated the stromal p16 expression in ovarian neoplasms using immunohistochemistry, and differences in the expression status depending on the degree of malignancy and histological type were analyzed. This study included 24, 21, and 46 cases of benign, borderline, and malignant ovarian lesions, respectively, of which 29, 25, and 32 cases were serous, mucinous, and endometriosis-associated lesions. Most benign lesions showed negative or weak expression, whereas borderline lesions showed focal, moderate expression. Malignant lesions showed markedly elevated stromal p16 expression compared with benign or borderline lesions. There were significant differences in stromal p16 expression between benign and borderline lesions (P < 0.001) and between borderline and malignant lesions (P < 0.001). These significances remained when analysis was performed based on lesion classification as serous, mucinous, and endometriosis-associated. In contrast, differences in stromal p16 expression among the histological types were not significant. Stromal p16 expression in ovarian neoplasms was absent or weak in benign and focal, moderate in borderline lesions, whereas malignant lesions exhibited diffuse, moderate-to-strong p16 immunoreactivity. Our observations suggest that stromal p16 expression is involved in the development of ovarian carcinoma. Further studies are necessary to confirm our preliminary results.

Background and Aims: The American Gastroenterological Association (AGA) recently reported evidence-based guidelines for the management of asymptomatic neoplastic pancreatic cysts. These guidelines advocate a higher threshold for surgical resection than prior guidelines and imaging surveillance for a considerable number of patients with pancreatic cysts. The aims of this study were to assess the accuracy of the AGA guidelines in detecting advanced neoplasia and present an alternative approach to pancreatic cysts., Methods: The study population consisted of 225 patients who underwent EUS-guided FNA for pancreatic cysts between January 2014 and May 2015. For each patient, clinical findings, EUS features, cytopathology results, carcinoembryonic antigen analysis, and molecular testing of pancreatic cyst fluid were reviewed. Molecular testing included the assessment of hotspot mutations and deletions for KRAS, GNAS, VHL, TP53, PIK3CA, and PTEN., Results: Diagnostic pathology results were available for 41 patients (18%), with 13 (6%) harboring advanced neoplasia. Among these cases, the AGA guidelines identified advanced neoplasia with 62% sensitivity, 79% specificity, 57% positive predictive value, and 82% negative predictive value. Moreover, the AGA guidelines missed 45% of intraductal papillary mucinous neoplasms with adenocarcinoma or high-grade dysplasia. For cases without confirmatory pathology, 27 of 184 patients (15%) with serous cystadenomas (SCAs) based on EUS findings and/or VHL alterations would continue magnetic resonance imaging (MRI) surveillance. In comparison, a novel algorithmic pathway using molecular testing of pancreatic cyst fluid detected advanced neoplasias with 100% sensitivity, 90% specificity, 79% positive predictive value, and 100% negative predictive value., Conclusions: The AGA guidelines were inaccurate in detecting pancreatic cysts with advanced neoplasia. Furthermore, because the AGA guidelines manage all neoplastic cysts similarly, patients with SCAs will continue to undergo unnecessary MRI surveillance. The results of an alternative approach with integrative molecular testing are encouraging but require further validation., (Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Objective: Uterine serous carcinomas (USC) harbor simultaneous HER2 (ERBB2) over-expression and gain of function mutations in PIK3CA. These concurrent alterations may uncouple single agent anti-HER2 therapeutic efficacy making inhibition of the mammalian target of rapamycin (mTOR) a promising option to heighten anti-tumor response., Methods: Both in vitro and in vivo experiments were conducted to assess proliferation, cell death and anti-tumor activity of ridaforolimus, lapatinib and combination lapatinib, trastuzumab (L/T) and ridaforolimus. With institutional approval, NOD/SCID mice bearing xenografts of non-immortalized, HER2 gene amplified cell lines (ARK1, ARK2) with and without PIK3CA gene mutations were divided into four arm cohorts. Ridaforolimus was administered alone and in combination with L/T. Tumor volumes were assessed and posttreatment analysis was performed., Results: We observed dose dependent in vitro abrogation of downstream target proteins including phospho-AKT and phospho-S6. In both in vivo models, single agent ridaforolimus impaired xenograft tumor growth. Combination ridaforolimus and L/T, however, further improved the observed anti-tumor activity only in the ARK1 model with the PIK3CA gene mutation (E542K). The addition of mTOR inhibition to dual HER2 blockade added no additional anti-tumor effects in the ARK2 xenografts. Western blot and immunohistochemical analysis of downstream pathway alterations following in vivo treatment revealed dual HER2 blockade with ridaforolimus was necessary to induce apoptosis, decrease proliferation and abrogate phospho-S6 protein expression in the PIK3CA mutated model., Conclusions: These pilot data suggest that PIK3CA gene mutation may be an effective biomarker for selecting those HER2 over-expressing USC tumors most likely to benefit from mTOR inhibition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ovarian serous borderline tumors (SBTs) being a precursor of low-grade serous carcinomas are morphologically characterized by noninvasive growth and low-grade cytology. On the other hand, many pathologists regard cytologically high-grade, noninvasive (HG-noninv) ovarian serous tumors resembling SBTs in low magnification as conventional high-grade serous carcinomas (HGSCs) by personal experiences. Nonetheless, there are no established molecular characteristic of such tumors. In this study, therefore, we attempted to provide the molecular evidence. We selected 37 ovarian serous tumors that exhibited a cytologically HG-noninv growth pattern, including 36 tumors that coexisted with conventional invasive HGSC components (HG-inv) and a single tumor exclusively composed of pure HG-noninv. Histologically, all HG-noninv showed many mitotic figures, and serous tubal intraepithelial carcinomas were identified in 3 tumors with HG-noninv. Immunohistochemically, most HG-noninv showed aberrant p53 expression, frequent IMP3 positivity, p16 overexpression, a high MIB-1 labeling index, and infrequent PAX2. By molecular analysis, the pure HG-noninv and 13 HGSCs with HG-noninv showed TP53 mutations, but KRAS/BRAF mutations were not detected in any of them. In 1 tumor, we detected an identical TP53 mutation in both HG-noninv and HG-inv components by using laser capture microdissection. These immunohistochemical and molecular features of HG-noninv were similar to those of conventional invasive HGSCs but different from those of SBTs. In conclusion, our results showed that a cytologically HG-noninv growth pattern simulating an SBT is a morphological spectrum of HGSC, but not a true SBT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tumours of ovarian-epithelial type of the testis, including serous borderline tumours, represent very rare entities. They are identical to the surface epithelial tumours of the ovary and have been reported in patients from 14 to 68 years of age. We describe two cases of a 46- and a 39-year old man with incidental findings of intratesticular masses of the left respectively right testis. Under the assumption of a malignant testicular tumour the patients were subjected to inguinal orchiectomy. Histologically, the tumours were identical to their ovarian counterparts: They showed a cystic configuration with a fibrous wall and irregular papillary structures lined by partially multistratified columnar cells and areas of hobnail cells. Furthermore, there was mild cytological atypia with a proliferative activity of below 5% as proved by Ki67 staining; mitoses could not be detected. Immunohistochemically, the tumour cells displayed expression of pan-cytokeratin AE3, progesterone receptor, Wilms' tumour protein (WT1), and PAX8 (Paired box gene 8). Estrogen receptor was expressed in one case. Octamer-binding transcription factor-4 (OCT4), calretinin, thrombomodulin, and D2-40 were not expressed. Mutation testing of BRAF revealed a BRAF V600E mutation in one case, while testing for KRAS mutations proved to be negative in both. The BRAF mutated tumour showed strong cytosolic and membranous positivity for B-Raf also on immunohistochemical analysis. Comparative genomic hybridization of one case could not reveal any chromosomal aberrations.

Recent studies have highlighted the role of long non-coding RNAs (lncRNAs) in carcinogenesis and have suggested that genes of this class might be used as biomarkers in cancer. However, whether lncRNAs are involved in serous ovarian cancer (SOC) remains largely unknown. In the present study, we focused on lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) and investigated its expression pattern, clinical significance, and biological function in SOC. We found that ANRIL levels were elevated in SOC tissues compared with normal controls and were highly correlated with advanced FIGO stage, high histological grade, lymph node metastasis, and poor prognosis. Multivariate analysis further revealed that ANRIL is an independent prognostic factor for predicting overall survival of SOC patients. In vitro, we compared differential ANRIL levels between SOC parental cell lines (SK-OV-3, HO8910) and highly metastatic sublines (SK-OV-3.ip1, HO8910-PM). Notably, ANRIL was highly expressed in both SK-OV-3.ip1 cells and HO8910-PM cells. SiRNA-mediated ANRIL silencing in these cells impaired cell migration and invasion. Based on the metastasis-related mRNA microarray analysis and subsequent western blotting confirmation, we found that MET and MMP3 are key downstream genes of ANRIL involved in SOC cell migration/invasion. Together, our data suggest that lncRNA ANRIL plays an important role in SOC invasion/metastasis and could represent a novel biomarker for predicting poor survival as well a promising therapeutic target.

Serous borderline tumor also known as atypical proliferative serous tumor (APST) is the precursor of ovarian low-grade serous carcinoma (LGSC). In this study, we correlated the morphologic and immunohistochemical phenotypes of 71 APSTs and 18 LGSCs with the mutational status of KRAS and BRAF, the most common molecular genetic changes in these neoplasms. A subset of cells characterized by abundant eosinophilic cytoplasm (EC), discrete cell borders, and bland nuclei was identified in all (100%) 25 BRAF-mutated APSTs but in only 5 (10%) of 46 APSTs without BRAF mutations (P<0.0001). Among the 18 LGSCs, EC cells were found in only 2, and both contained BRAF mutations. The EC cells were present admixed with cuboidal and columnar cells lining the papillae and appeared to be budding from the surface, resulting in individual cells and clusters of detached cells "floating" above the papillae. Immunohistochemistry showed that the EC cells always expressed p16, a senescence-associated marker, and had a significantly lower Ki-67 labeling index than adjacent cuboidal and columnar cells (P=0.02). In vitro studies supported the interpretation that these cells were undergoing senescence, as the same morphologic features could be reproduced in cultured epithelial cells by ectopic expression of BRAF(V600E). Senescence was further established by markers such as SA-β-gal staining, expression of p16 and p21, and reduction in DNA synthesis. In conclusion, this study sheds light on the pathogenesis of this unique group of ovarian tumors by showing that BRAF mutation is associated with cellular senescence and the presence of a specific cell type characterized by abundant EC. This "oncogene-induced senescence" phenotype may represent a mechanism that impedes progression of APSTs to LGSC.

Objective: Transcriptional profiling of epithelial ovarian cancer has revealed molecular subtypes correlating to biological and clinical features. We aimed to determine gene expression differences between malignant, benign and borderline serous ovarian tumors, and investigate similarities with the well-established intrinsic molecular subtypes of breast cancer., Methods: Global gene expression profiling using Illumina's HT12 Bead Arrays was applied to 59 fresh-frozen serous ovarian malignant, benign and borderline tumors. Nearest centroid classification was performed applying previously published gene profiles for the ovarian and breast cancer subtypes. Correlations to gene expression modules representing key biological breast cancer features were also sought. Validation was performed using an independent, publicly available dataset., Results: 5,944 genes were significantly differentially expressed between benign and malignant serous ovarian tumors, with cell cycle processes enriched in the malignant subgroup. Borderline tumors were split between the two clusters. Significant correlations between the malignant serous tumors and the highly aggressive ovarian cancer signatures, and the basal-like breast cancer subtype were found. The benign and borderline serous tumors together were significantly correlated to the normal-like breast cancer subtype and the ovarian cancer signature derived from borderline tumors. The borderline tumors in the study dataset, in addition, also correlated significantly to the luminal A breast cancer subtype. These findings remained when analyzed in an independent dataset, supporting links between the molecular subtypes of ovarian cancer and breast cancer beyond those recently acknowledged., Conclusions: These data link the transcriptional profiles of serous ovarian cancer to the intrinsic molecular subtypes of breast cancer, in line with the shared clinical and molecular features between high-grade serous ovarian cancer and basal-like breast cancer, and suggest that biomarkers and targeted therapies may overlap between these tumor subsets. The link between benign and borderline ovarian cancer and luminal breast cancer may indicate endocrine responsiveness in a subset of ovarian cancers.

Purpose: Management guidelines for pancreatic intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (MCN) are based on the assumption that mucinous cysts can be accurately distinguished from other pancreatic cystic lesions. Previous studies using surgical material have identified recurrent mutations in GNAS and KRAS in pancreatic mucinous neoplasms. Yet, the diagnostic utility of testing for both genes in pancreatic cyst fluid obtained by endoscopic ultrasound-fine-needle aspiration (EUS-FNA) remains unclear., Experimental Design: GNAS and KRAS testing was performed on EUS-FNA pancreatic cyst fluid from 91 pancreatic cysts: 41 IPMNs, 9 IPMNs with adenocarcinoma, 16 MCNs, 10 cystic pancreatic neuroendocrine tumors (PanNET), 9 serous cystadenomas (SCA), 3 retention cysts, 2 pseudocysts, and 1 lymphoepithelial cyst., Results: Mutations in GNAS were detected in 16 (39%) IPMNs and 2 (22%) IPMNs with adenocarcinoma. KRAS mutations were identified in 28 (68%) IPMNs, 7 (78%) IPMNs with adenocarcinoma, and 1 (6%) MCN. Mutations in either gene were present in 34 (83%) IPMNs, 8 (89%) IPMNs with adenocarcinoma, and 1 (6%) MCN. No mutations were found in cystic PanNETs, SCAs, retention cysts, pseudocysts, and a lymphoepithelial cyst. GNAS and KRAS mutations had 100% specificity [95% confidence interval (CI), 0.83-1.00] but 65% sensitivity (95% CI, 0.52-0.76) for mucinous differentiation. Among IPMNs, mutations in either gene had 98% specificity (95% CI, 0.86-1.00) and 84% sensitivity (95% CI, 0.70-0.92)., Conclusions: The combination of GNAS and KRAS testing was highly specific and sensitive for IPMNs; however, the lack of sensitivity for MCNs highlights the need for additional markers to improve the detection of pancreatic mucinous neoplasms., (©2014 American Association for Cancer Research.)

Boolean implications (if-then rules) provide a conceptually simple, uniform and highly scalable way to find associations between pairs of random variables. In this paper, we propose to use Boolean implications to find relationships between variables of different data types (mutation, copy number alteration, DNA methylation and gene expression) from the glioblastoma (GBM) and ovarian serous cystadenoma (OV) data sets from The Cancer Genome Atlas (TCGA). We find hundreds of thousands of Boolean implications from these data sets. A direct comparison of the relationships found by Boolean implications and those found by commonly used methods for mining associations show that existing methods would miss relationships found by Boolean implications. Furthermore, many relationships exposed by Boolean implications reflect important aspects of cancer biology. Examples of our findings include cis relationships between copy number alteration, DNA methylation and expression of genes, a new hierarchy of mutations and recurrent copy number alterations, loss-of-heterozygosity of well-known tumor suppressors, and the hypermethylation phenotype associated with IDH1 mutations in GBM. The Boolean implication results used in the paper can be accessed at http://crookneck.stanford.edu/microarray/TCGANetworks/.

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissue(high)/sera(low)-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic 'drop and restoration' alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

Complete androgen insensitivity syndrome is an extremely infrequent disease. The patients exhibit female phenotype because of insensitivity to the androgen receptor and may develop tumors, especially in their undescended gonads. We report a case of bilateral Sertoli cell adenoma in gonads with unilateral serous cystadenoma, in an elderly phenotypic woman with primary amenorrhea. We also provide radiological and pathological studies.

Objective: This study assesses TRAIL-R2 (TNF-related apoptosis-inducing ligand receptor 2) and BCL2 (B cell CLL/lymphoma 2) expression as well as CpG island methylation within the TRAIL-R2 promoter in ovarian serous tumors and primary and metastatic serous EOC (epithelial ovarian cancer)., Methods: RNA and DNA were obtained from women with normal ovarian tissues (n = 18), ovarian serous cystadenoma tumors (n = 11) and serous EOC (n = 16) using Trizol®. Quantitative PCR was performed to quantify the relative levels of TRAIL-R2 and BCL2. The methylation frequency of the TRAIL-R3 promoter was assessed using a methylation-specific PCR assay after DNA bisulfite conversion. Differences between the groups were evaluated using the χ (2), Mann-Whitney U or Kruskal-Wallis tests, as indicated., Results: We identified TRAIL-R2 and BCL2 mRNA expressed in all ovarian tumor groups, and there were significant differences between the groups. Both genes had low expression levels in ovarian serous cystadenoma and primary EOC tumors when compared with metastatic EOC. Methylation of the TRAIL-R2 promoter was frequently observed in all groups; however, there were no statistically significant associations., Conclusions: Primary EOC is associated with lower TRAIL-R2 and BCL2 expression levels, while metastatic EOC is associated with higher expression of these genes. Promoter DNA methylation was not related to this finding, suggesting there are other mechanisms involved in transcriptional control.

Aims: To look for FOXL2 mutation in rare ovarian epithelial lesions showing stromal components with morphological features of adult granulosa cell tumour (AGCT)., Methods and Results: We report the 402C→G FOXL2 mutation status in five epithelial ovarian lesions in women aged 45-77 years showing stromal proliferations that were morphologically indistinguishable from AGCT. The lesions were mucinous cystadenoma, mixed epithelial cystadenoma, endometriotic cyst, mucinous borderline tumour (intestinal type), and mucinous carcinoma. In one case, the AGCT component formed a discrete nodule, and in the others it was distributed within the septa and cyst walls. FOXL2 mutation was present in two cases and absent in three cases. One mutation-positive case showed an AGCT nodule abutting a mucinous borderline tumour, interpreted as a collision tumour. The other positive case had an AGCT component within the septa of a mucinous carcinoma, and both components are likely to be neoplastic. In the three cases without FOXL2 mutation, the stromal component most likely represents a non-neoplastic AGCT-like proliferation., Conclusions: Areas typical of AGCT are rarely associated with epithelial ovarian lesions. These are heterogeneous and likely to be truly neoplastic in only a subset of cases. FOXL2 mutation testing may be useful in confirming a true neoplastic AGCT component., (© 2013 John Wiley & Sons Ltd.)

Kindlin-2 has been known to promote most cancer progression through regulation of multiple signaling pathways. However, a novel tumor suppressive role of Kindlin-2 was identified in serous epithelial ovarian cancer progression, which sharply contrasts to the tumor promoting roles for Kindlin-2 in most other cancers. While we demonstrated that Kindlin-2 was highly expressed in control tissues, a drastic low expression of Kindlin-2 was found in the tumor tissues of serous epithelial ovarian cancer, especially in the high-grade serous epithelial ovarian cancer. Importantly, Kindlin-2 inhibited serous epithelial ovarian cancer cell peritoneal dissemination in a mouse model. For clinical relevance, low Kindlin-2 expression correlated with higher tumor grade and older patients. Intriguingly, decreased Kindlin-2 expression predicts poor overall and progression-free survivals in serous epithelial ovarian cancer patients. Mechanistically, Kindlin-2 induced a mesenchymal to epithelial transition in serous epithelial ovarian cancer cells, at least in part, by up-regulation of estrogen receptor α which was recruited to the promoter of E-cadherin and thereby enhanced the transcription of E-cadherin. Collectively, we concluded that inadequate Kindlin-2 is an independent risk factor for serous epithelial ovarian cancer patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Borderline ovarian tumors show heterogeneity in clinical behavior. Most have excellent prognosis, although a small percentage show recurrence or progressive disease, usually to low-grade serous carcinoma. The aim of this study was to understand the molecular relationship between these entities and identify potential markers of tumor progression and therapeutic targets. We studied gene expression using Affymetrix HGU133plus2 GeneChip microarrays in 3 low-grade serous carcinomas, 13 serous borderline tumors and 8 serous cystadenomas. An independent data set of 18 serous borderline tumors and 3 low-grade serous carcinomas was used for validation. Unsupervised clustering revealed clear separation of benign and malignant tumors, whereas borderline tumors showed two distinct groups, one clustering with benign and the other with malignant tumors. The segregation into benign- and malignant-like borderline molecular subtypes was reproducible on applying the same analysis to an independent publicly available data set. We identified 50 genes that separate borderline tumors into their subgroups. Functional enrichment analysis of genes that separate borderline tumors to the two subgroups highlights a cell adhesion signature for the malignant-like subset, with Claudins particularly prominent. This is the first report of molecular subtypes of borderline tumors based on gene expression profiling. Our results provide the basis for identification of biomarkers for the malignant potential of borderline ovarian tumor and potential therapeutic targets for low-grade serous carcinoma.

Objective: To examine single nucleotide polymorphism (SNPs) in MUC16 (CA125) and MUC1 (CA15.3) in relation to ovarian cancer risk and survival., Methods: We genotyped germline variants of MUC16 (rs2547065, rs1559168, rs12984471, rs2121133) and MUC1 (rs2070803, rs4072037, rs1045253) using samples collected from 758 ovarian cancer cases and 788 controls enrolled in the New England Case-Control Study between 2003 and 2008. We calculated age-adjusted odds ratios (OR) and 95% confidence intervals (CIs) for disease risk using unconditional and polytomous logistic regression and hazard ratios (HR) for survival using Cox proportional hazard ratios. In a subset of cases, we compared log-normalized CA125 values by genotype using generalized linear models., Results: Cases homozygous for the variant allele of MUC16 SNP, rs12984471, had poorer overall survival (log-rank p = 0.03) and higher CA125 levels, especially cases over age 65 (p = 0.01). For MUC1 SNP, rs4072037, women homozygous for the G variant had a non-significantly decreased risk for serous invasive types but elevated risk for serous borderline tumors, mucinous borderline and invasive tumors, and endometrioid tumors. Women with the variant allele of MUC16 SNP, rs2547065, especially those who were homozygous had an elevated risk for ovarian cancer; but this association was not confirmed in an independent dataset., Conclusion: This targeted screen of seven polymorphisms of MUC16 and MUC1 genes failed to identify and confirm effects on ovarian cancer risk overall. However, there may be effects of MUC16 rs12984471 on survival and MUC1 rs4072037 on risk for histologic types of ovarian cancer other than invasive serous. Further study is warranted.

Despite impressive research efforts, the biology of epithelial ovarian cancer (EOC) remains poorly understood and alterations in the expression of CASPASE-8 contribute to a worse tumor prognosis. This study assesses the methylation of the CpG island within the CASPASE-8 promoter and CASPASE-8 gene expression both in cystadenoma tumors and in primary and metastatic EOC. DNA and RNA were obtained from women with normal ovarian tissues (n=18), ovarian serous cystadenoma tumors (n=11) and EOC (n=16) using Trizol(®). The methylation frequency of the CpG island in the CASPASE-8 promoter was assessed using the methylation-specific PCR assay after DNA bisulfite conversion. Quantitative PCR was performed to quantify the relative levels of CASPASE-8 in each sample. The differences between samples with each group were evaluated using the Mann-Whitney U and Kruskal-Wallis tests as indicated. Hemimethylation of the CASPASE-8 promoter was found in 11.8% of the normal ovary samples, 20% of the cystadenoma tumors and 20% of the metastatic EOC, while methylation of the CASPASE-8 promoter was absent in the EOC primary tissues (P=0.047). An increased CASPASE-8 expression level was observed in all tumor groups. Significant differences were observed in the CASPASE-8 expression levels when compared with all ovarian tumor groups (P=0.0278). Promoter DNA methylation did not associate with expression levels of CASPASE-8, suggesting the presence of other mechanisms in relation to gene expression control in EOC; thus providing a better understanding of this complex disease., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Epithelial ovarian cancer is one of the most lethal gynecological malignant tumors despite improvement of the treatment. Recent molecular studies show that ovarian cancer is a heterogeneous disease which is reflected by different histologic types. These subtypes differ from their origin, pathogenesis and molecular alterations and can be divided in two major groups. The type I cancer (low grade) evolves from precursor lesions in a step-wise process. In contrast, the type II cancer (high grade) grows rapidly without any identifiable precursors. Among all subtypes is heterogeneity in the biological behavior which has implications in patient prognosis and treatment especially for individualized therapies in the future.

Context: Von Hippel Lindau disease is a rare autosomal dominantly inherited multisystem disorder characterized by development of benign and malignant tumors. The abdominal manifestation of the syndrome are protean. Magnetic resonance plays an important role in identification of abdominal abnormalities and follow-up of lesions., Objective: To describe magnetic resonance imaging findings and patterns of pancreatic and other principal abdominal manifestations in a series of von Hippel-Lindau (VHL) disease patients and to review literature., Methods: We retrospectively reviewed abdominal magnetic resonance studies performed in 23 patients (10 males, 13 females) diagnosed of VHL., Results: In all examined patients abdominal involvement was present. The pancreatic imaging findings detected were: unilocular cystic lesions (6/23: 26.1%); serous cystadenomas (11/23: 47.8%), including diffuse lesions (8/23: 34.8%); solid neuroendocrine tumors (8/23: 34.8%); cystic neuroendocrine tumors (1/23: 4.3%). The renal findings detected were: simple renal cysts (18/23: 78.3%); complex renal cysts (13/23: 56.5%), including benign lesions (10/23: 43.5%) and malignant lesions (3/23: 13.0%); renal carcinomas (11/23: 47.8%) and 5 of these (45.5%) were multiple and bilateral. Five patients (21.7%) presented pheochromocytoma (4 of these were bilateral; 80.0%) and 1 patient (4.3%) presented cystadenoma of the epididymis., Conclusions: In VHL disease patients, magnetic resonance imaging plays an essential role in the identification of pancreatic and other abdominal lesions, in their follow-up, in the screening of asymptomatic gene carriers, and in their long-term surveillance.

To investigate the associations among the number of polyploid giant cancer cells (PGCCs) and vasculogenic mimicry (VM), EZH2 expression, and serous ovarian tumor grade, a total of 80 paraffin-embedded serous ovarian tumor samples including 21 cases of primary carcinoma and their metastatic tumors, 26 cases of primary carcinoma without metastasis, and 12 cases of serous borderline cystadenoma were analyzed. PGCCs and VM were detected in human serous ovarian tumor. The metastatic foci of ovarian carcinoma had the highest number of PGCCs and VM. The number of PGCCs and VM increased with the grade of ovarian carcinomas. PGCCs generated erythrocytes via budding and together they formed VM. Tumor cells and cancer-associated fibroblasts were positive for EZH2 immunohistochemical staining. The tumor cells and cancer associated fibroblasts in the metastatic foci had the highest staining index of EZH2 staining. Both tumor cells and cancer-associated fibroblasts express EZH2 which then contributes to the malignant grade of serous ovarian tumor.

BRAF and KRAS mutations in ovarian serous borderline tumours (OSBTs) and ovarian low-grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or whether those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumour samples from 23 recurrent LGSC patients with a known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for five patients, and either OSBTs or LGSCs were available for another 18 patients. Tumour cells from paraffin-embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumours that appeared to have wild-type KRAS by conventional PCR-Sanger sequencing were further analysed by full COLD (co-amplification at lower denaturation temperature)-PCR and deep sequencing. Full COLD-PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR-Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in ten patients. Full COLD-PCR deep sequencing detected low-abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in seven OSBT samples and six LGSC samples. Surprisingly, patients with the KRAS G12V mutation have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumour cells with or without detectable KRAS mutations., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Although high-grade serous ovarian cancer (OVC) is the most lethal gynecologic malignancy in women, little is known about the regulatory mechanisms in the cellular processes that lead to this cancer. Recently, accumulated lines of evidence have shown that the interplay between transcription factors (TFs) and microRNAs (miRNAs) is critical in cellular regulation during tumorigenesis. A comprehensive investigation of TFs and miRNAs, and their target genes, may provide a deeper understanding of the regulatory mechanisms in the pathology of OVC. In this study, we have integrated three complementary algorithms into a framework, aiming to infer the regulation by miRNAs and TFs in conjunction with gene expression profiles. We demonstrated the utility of our framework by inferring 67 OVC-specific regulatory feed-forward loops (FFL) initiated by miRNAs or TFs in high-grade serous OVC. By analyzing these regulatory behaviors, we found that all the 67 FFLs are consistent in their regulatory effects on genes that are jointly targeted by miRNAs and TFs. Remarkably, we unveiled an unbalanced distribution of FFLs with different oncogenic effects. In total, 31 of the 67 coherent FFLs were mainly initiated by oncogenes. On the contrary, only 4 of the FFLs were initiated by tumor suppressor genes. These overwhelmingly observed oncogenic genes were further detected in a sub-network with 32 FFLs centered by miRNA let-7b and TF TCF7L1 to regulate cell differentiation. Closer inspection of 32 FFLs revealed that 75% of the miRNAs reportedly play functional roles in cell differentiation, especially when enriched in epithelial-mesenchymal transitions. This study provides a comprehensive pathophysiological overview of recurring coherent circuits in OVC that are co-regulated by miRNAs and TFs. The prevalence of oncogenic coherent FFLs in serous OVC suggests that oncogene-driven regulatory motifs could cooperatively act upon critical cellular processes such as cell differentiation in a highly efficient and consistent manner.