Divergent Malignant Melanocytic Differentiation in Ovarian Endometrioid Adenocarcinoma With Aberrant ß-Catenin Expression: A Case Expanding the Histologic Spectrum of ß-Catenin Activated Gynecologic Neoplasia.

Divergent differentiation in gynecologic carcinomas encompasses a broad range of lineages, including mesenchymal, germ cell, high-grade neuroendocrine, neuroectodermal, and cutaneous adnexal differentiation. Here we present a case of ovarian endometrioid adenocarcinoma with divergent malignant melanocytic differentiation (MMeD). The background ovarian endometrioid adenocarcinoma showed focally aberrant ß-catenin expression and histologic patterns associated with ß-catenin activation, including spindled elements and corded and hyalinized foci. The areas with MMeD had both spindled and epithelioid morphology, diffusely aberrant ß-catenin expression, expression of melanocytic markers (an HMB45/Mart-1 cocktail, MITF, and S100), and no staining for myogenic markers (SMA and desmin) or epithelial markers (cytokeratins and E-cadherin). INI1, BRG1, PMS2, and MSH6 were retained, and p53 showed a wild-type expression pattern. No areas with definitive carcinosarcomatous differentiation were identified despite extensive sampling. While a single case of gynecologic carcinosarcoma with a serous epithelial component and a small focus on malignant melanoma has been reported in the English literature, the current case represents what is, to the best of our knowledge, the first case of MMeD arising in the context of a ß-catenin activated endometrioid adenocarcinoma. Pathogenetic and differential diagnostic considerations are discussed.

Immunohistochemistry as an adjunct for challenging histological patterns of borderline Brenner tumors: An illustrative study of 4 cases.

Borderline Brenner tumors (BBT) have a range of morphology that shows considerable overlap with that of malignant Brenner tumors (MBT). In particular, two histological patterns of BBT can be particularly challenging: 1) BBT with intraepithelial carcinoma (BBT-IEC) and 2) BBT with a small nested pattern (BBT-SNP). BBT-IEC is characterized by a tumor with the low-power non-infiltrative silhouette of a conventional BBT, but with increased cytological atypia and mitotic activity similar to that of MBT. Conversely, BBT-SNP is characterized by a complex proliferation of small tumor nests that closely resemble the infiltrative growth pattern of MBT, but without the obligate cytologic atypia and mitotic activity of MBT. We suggest that the combination of p16, p53 and Ki-67 may be helpful in distinguishing these 2 patterns of BBT from both conventional BBT and from MBT. While both conventional BBT and BBT-IEC show a null pattern of p16 expression, our case of BBT-IEC showed aberrant p53 overexpression, albeit with a maturation pattern similar to that described for TP53 mutant mucinous ovarian carcinoma and differentiated vulvar intraepithelial neoplasia (dVIN). Similarly, while BBT-SNP shows an infiltrative-like growth pattern similar to that of MBT, our case also showed a wild-type pattern of p53 expression and a Ki-67 proliferative index similar to areas with conventional BBT histology. In conclusion, in our small case series, we show that the use of immunohistochemistry for p53 and Ki-67 may help to distinguish challenging patterns of BBT from MBT. Further studies are needed to validate this finding in a larger case cohort.

HER2 Expression in Endometrial Cancers Diagnosed as Clear Cell Carcinoma.

There is increasing evidence that many endometrial cancers (EC) diagnosed as clear cell carcinoma (CCC) have substantial overlap with both serous carcinoma (SC) and endometrioid carcinoma (EmC), not only in terms of morphology and immunophenotype but also by molecular characterization. Now with use of HER2-based therapy in SC, a CCC diagnosis in serous-like tumors has the potential to exclude patients from receiving beneficial therapy. To assess HER2 in CCC in relation to other characteristics, a tissue microarray of archived CCC, EmC, and SC was stained for HER2 alongside a battery of immunostains used in EC. Cases with equivocal HER2 IHC were also assessed by in situ hybridization. HER2 status was assessed in 229 cases (23 CCC, 74 SC, 132 EmC). HER2 was positive in 48% of cases diagnosed as CCC, 19% of SC, and 0% of EmC. Rigorous morphologic and immunophenotypic review by 5 gynecologic pathologists revealed diagnostic disagreement in 8/11 HER2+ cases diagnosed as CCC, with SC as the other major diagnostic consideration. All HER2+ (n=25) cases were MMR-intact and most HER2+ EC had aberrant p53 staining (22/25, 88%); the 3 cases with a wild type pattern for p53 (12%) were all negative for ER. Based on these findings, patients with a diagnosis of CCC should be included in future clinical trials of HER2-targeted therapy. Moreover, given the diagnostic difficulty surrounding CCC, immunohistochemistry-based algorithms that include aberrant p53 and/or the absence of ER expression may provide a more objective means of establishing eligibility criteria than is currently possible using traditional histologic classification.

Fatal adrenal crisis due to Addison's disease arising in the context of autoimmune polyglandular syndrome type 1.

The autoimmune polyglandular syndromes (APS) are rare immune-mediated endocrinopathies causing destruction of multiple endocrine and non-endocrine organs. Involvement of adrenal glands associated with any type of APS results in Addison's disease. While patients with Addison's disease often suffer from symptoms of neuroglycopenia, lethal hypotension and hypoglycemia are uncommon. Here, we report a fatal case of APS type 1 with hypotension and profound hypoglycemia in a 24-year-old man who was found unconsciousness at home and progressively evolved into pulseless electrical activity. Although his condition was initially considered to be possibly due to drug toxicity, subsequent drug screening tests failed to detect alcohol or any other substances. Emergent medical evaluation revealed severe hypotension (51/30 mm/Hg) and profound hypoglycemia (blood glucose of 20-30 mg/dl). Despite vigorous supportive care, the patient died following 3 days of respiratory dependency due to irreversible anoxic brain injury. Postmortem examination revealed severely atrophic adrenal glands with lymphocytic infiltration. Subsequent review of the patient's medical history and correlation with autopsy findings confirmed the presence of multiple organ involvement, consistent with APS type 1. This case serves as a reminder for forensic pathologists that death from an acute adrenal (Addisonian) crisis, while uncommon, should remain a differential diagnostic consideration. Furthermore, it underscores the fact that Addison's disease can occur as part of a constellation of autoimmune manifestations within the context of an underlying polyglandular syndrome, such as APS type 1.

Genetic alterations of triple negative breast cancer by targeted next-generation sequencing and correlation with tumor morphology.

Triple negative breast cancer represents a heterogeneous group of breast carcinomas, both at the histologic and genetic level. Although recent molecular studies have comprehensively characterized the genetic landscape of these tumors, few have integrated a detailed histologic examination into the analysis. In this study, we defined the genetic alterations in 39 triple negative breast cancers using a high-depth targeted massively parallel sequencing assay and correlated the findings with a detailed morphologic analysis. We obtained representative frozen tissue of primary triple negative breast cancers from patients treated at our institution between 2002 and 2010. We characterized tumors according to their histologic subtype and morphologic features. DNA was extracted from paired frozen primary tumor and normal tissue samples and was subjected to a targeted massively parallel sequencing platform comprising 229 cancer-associated genes common across all experiments. The average number of non-synonymous mutations was 3 (range 0-10) per case. The most frequent somatic alterations were mutations in TP53 (74%) and PIK3CA (10%) and MYC amplifications (26%). Triple negative breast cancers with apocrine differentiation less frequently harbored TP53 mutations (25%) and MYC gains (0%), and displayed a high mutation frequency in PIK3CA and other PI3K signaling pathway-related genes (75%). Using a targeted massively parallel sequencing platform, we identified the key somatic genetic alterations previously reported in triple negative breast cancers. Furthermore, our findings show that triple negative breast cancers with apocrine differentiation constitute a distinct subset, characterized by a high frequency of PI3K pathway alterations similar to luminal subtypes of breast cancer.

Mesonephric-like adenocarcinoma of the ovary with squamoid morular metaplasia, aberrant ß-catenin expression, and concurrent FGFR2 and CTNNB1 mutations: a case report.

In general, endometrioid-defining features such as squamoid morular metaplasia are not thought to be associated with mesonephric adenocarcinoma (MA) and mesonephric-like adenocarcinoma (MLA). Here, we report a case of FGFR2-mutated ovarian MLA with squamoid morular metaplasia accompanied by aberrant nuclear and cytoplasmic ß-catenin expression and CTNNB1 mutation. Histologically, the tumor showed classical MLA histology, including well-formed glands with intraluminal eosinophilic secretions and cells with papillary thyroid carcinoma-like nuclei. Squamoid morular metaplasia was intimately associated with the tumor. Glandular epithelial elements, including those immediately associated with the squamoid morules, were negative for ER, but positive for both GATA3 and PAX8; aberrant ß-catenin expression was limited to the squamoid morules. This case illustrates the ability of mesonephric neoplasia to exhibit histological features previously thought to be restricted to an endometrioid phenotype.

Adenosquamous carcinoma in situ of the anus: a case series.

While human papillomavirus (HPV)-associated adenocarcinoma of the anus resembling endocervical adenocarcinoma has recently been described, anal adenocarcinoma in situ/AIS has not. Moreover, to the best of our knowledge, truly biphasic anal adenosquamous carcinoma in situ (ADSQ-IS) is essentially non-existent in the literature. Here, we report four cases of anal ADSQ-IS including one with associated invasive adenosquamous carcinoma. Histologically, all cases of ADSQ-IS showed a basal/peripheral population of stratified, p40-positive dysplastic squamous cells adjacent to luminal, columnar, p40-negative dysplastic glandular cells, bearing a striking resemblance to the normal anal transitional epithelium. Both lesional components were diffusely and strongly positive for p16 and positive for high-risk HPV by RNA in situ hybridization. These cases expand the morphological spectrum of high-risk HPV-associated pre-invasive lesions and underscore the plasticity of HPV-associated neoplasia.

A combined, supraclavicular, infraclavicular, transaxillary, and posterior subscapular approaches for en bloc resection of giant myxofibrosarcoma.

Background: Myxofibrosarcoma (MFS) is a rare and locally infiltrative tumor that commonly occurs in extremities in older adults; however, truncal and head and neck cases have been reported. They are characterized by multinodular growth, incomplete fibrous septa, and myxoid stroma. Surgical resection is the mainstay of treatment. Case Description: The authors report a case of a combined, supraclavicular, infraclavicular, transaxillary, and posterior subscapular approaches for resection of giant MFS. Conclusion: The anatomical complexity and rarity of tumors involving the brachial plexus impose many challenges onto surgeons performing surgical resections. Treatment choices and surgical outcomes rely heavily on meticulous multidisciplinary planning, anatomical knowledge, careful dissection, and extent of resection. This case is unique in utilizing four different approaches to the brachial plexus to resect one tumor.

Prominent decidualization following progestin treatment for endometrial hyperplasia and carcinoma as a mimic of large residual tumor: A cautionary tale.

OBJECTIVE: Progestin-based therapy is common for patients with endometrial neoplasia who desire fertility preservation, but some patients ultimately require surgery. Intraoperative assessment, which can use gross lesion size, may impact the extent of surgery performed. We sought to characterize the extent to which grossly identified lesions in the setting of progestin therapy correspond to microscopic findings. METHODS: Thirteen hysterectomy specimens with progestin-treated atypical hyperplasia or endometrioid carcinoma were identified. Clinicopathologic factors were collected by chart review. Slides were assessed for the extent to which decidualized stroma (DS) comprised grossly identified lesions and comparisons were drawn with tumor size, age, and menopausal status. RESULTS: Mass lesions were described in 11 cases with a median of 4.5 cm (range 1-8.2) and the 2 cases without discrete masses had diffuse thickening. Two patients had only focal residual hyperplasia despite having mass lesions (7 & 2.2 cm). DS was more prominent in premenopausal patients (median 65%, range 10-90%) than in postmenopausal patients (median 18%, range 10-40%; p = 0.06). The distribution of DS throughout mass lesions was variable. CONCLUSIONS: Large mass lesions following progestin therapy may histologically consist of DS with little to no residual neoplastic disease, such that perceived tumor size does not necessarily reflect extensive residual disease, especially in pre-menopausal patients. Intraoperative gross assessment alone may lead to unnecessary lymphadenectomy and/or oophorectomy, but this can potentially be prevented by using frozen section.

Ovarian Cells Have Increased Proliferation in Response to Heparin-Binding Epidermal Growth Factor as Collagen Density Increases.

It is well known that during ovarian cancer progression, the omentum transforms from a thin lacy organ to a thick tougher tissue. However, the mechanisms regulating this transformation and the implications of the altered microenvironment on ovarian cancer progression remain unclear. To address these questions, the global and local concentrations of collagen I were determined for normal and metastatic human omentum. Collagen I was increased 5.3-fold in omenta from ovarian cancer patients and localized to areas of activated fibroblasts rather than regions with a high density of cancer cells. Transforming growth factor beta 1 (TGFß1) was detected in ascites from ovarian cancer patients (4 ng/mL), suggesting a potential role for TGFß1 in the observed increase in collagen. Treatment with TGFß1 induced fibroblast activation, proliferation, and collagen deposition in mouse omental explants and an in vitro model with human omental fibroblasts. Finally, the impact of increased collagen I on ovarian cancer cells was determined by examining proliferation on collagen I gels formulated to mimic normal and cancerous omenta. While collagen density alone had no impact on proliferation, a synergistic effect was observed with collagen density and heparin-binding epidermal growth factor treatment. These results suggest that TGFß1 induces collagen deposition from the resident fibroblasts in the omentum and that this altered microenvironment impacts cancer cell response to growth factors found in ascites. Impact statement Using quantitative analysis of patient samples, in vitro models of the metastatic ovarian cancer microenvironment were designed with pathologically relevant collagen densities and growth factor concentrations. Studies in these models support a mechanism where transforming growth factor ß1 in the ascites fluid induces omental fibroblast proliferation, activation, and deposition of collagen I, which then impacts tumor cell proliferation in response to additional ascites growth factors such as heparin-binding epidermal growth factor. This approach can be used to dissect mechanisms involved in microenvironmental modeling in multiple disease applications.

Scaffold stiffness influences breast cancer cell invasion via EGFR-linked Mena upregulation and matrix remodeling.

Clinically, increased breast tumor stiffness is associated with metastasis and poorer outcomes. Yet, in vitro studies of tumor cells in 3D scaffolds have found decreased invasion in stiffer environments. To resolve this apparent contradiction, MDA-MB-231 breast tumor spheroids were embedded in 'low' (2 kPa) and 'high' (12 kPa) stiffness 3D hydrogels comprised of methacrylated gelatin/collagen I, a material that allows for physiologically-relevant changes in stiffness while matrix density is held constant. Cells in high stiffness materials exhibited delayed invasion, but more abundant actin-enriched protrusions, compared to those in low stiffness. We find that cells in high stiffness had increased expression of Mena, an invadopodia protein associated with metastasis in breast cancer, as a result of EGFR and PLCγ1 activation. As invadopodia promote invasion through matrix remodeling, we examined matrix organization and determined that spheroids in high stiffness displayed a large fibronectin halo. Interestingly, this halo did not result from increased fibronectin production, but rather from Mena/α5 integrin dependent organization. In high stiffness environments, FN1 knockout inhibited invasion while addition of exogenous cellular fibronectin lessened the invasion delay. Analysis of fibronectin isoforms demonstrated that EDA-fibronectin promoted invasion and that clinical invasive breast cancer specimens displayed elevated EDA-fibronectin. Combined, our data support a mechanism by which breast cancer cells respond to stiffness and render the environment conducive to invasion. More broadly, these findings provide important insight on the roles of matrix stiffness, composition, and organization in promoting tumor invasion.

RENIN-SECRETING OVARIAN ADULT GRANULOSA CELL TUMOR IN A PATIENT WITH NEW-ONSET HYPERTENSION AND HYPOKALEMIA.

OBJECTIVE: To describe a case of a renin-secreting ovarian tumor, which is a rare cause of hyperreninemia leading to secondary hypertension and hypokalemia. METHODS: We present the first case of a patient with a molecularly confirmed renin-secreting ovarian adult granulosa cell tumor. RESULTS: A 31-year-old female presented with hypertension and amenorrhea. She was found to have spontaneous hypokalemia. Computed tomography scan showed a pelvic mass which was resected and found to be a renin-secreting ovarian adult granulosa cell tumor. The hypertension and hypokalemia resolved after the mass was resected. CONCLUSION: Renin-secreting ovarian adult granulosa cell tumors are rare causes of hyperreninemia leading to hypertension and hypokalemia.

Alternatively activated macrophage-derived secretome stimulates ovarian cancer spheroid spreading through a JAK2/STAT3 pathway.

High-grade serous ovarian cancer (HGSOC) metastasizes when tumor spheroids detach from the primary tumor and re-attach throughout the peritoneal cavity. Once the cancer cells have implanted in these new sites, the development of metastatic lesions is dependent on the disaggregation of cancer cells from the spheroids and subsequent expansion across the collagenous extracellular matrix (ECM). As HGSOC progresses an increase in alternatively activated macrophages (AAMs) in the surrounding ascites fluid has been observed and AAMs have been shown to enhance tumor invasion and growth in a wide range of cancers. We hypothesized that soluble factors from AAMs in the peritoneal microenvironment promote the disaggregation of ovarian cancer spheroids across the underlying ECM. We determined that co-culture with AAMs significantly increased HGSOC spheroid spreading across a collagen matrix. Multivariate modeling identified AAM-derived factors that correlated with enhanced spread of HGSOC spheroids and experimental validation showed that each individual cell line responded to a distinct AAM-derived factor (FLT3L, leptin, or HB-EGF). Despite this ligand-level heterogeneity, we determined that the AAM-derived factors utilized a common signaling pathway to induce spheroid spreading: JAK2/STAT3 activation followed by MMP-9 mediated spreading. Furthermore, immunostaining demonstrated that FLT3, LEPR, EGFR, and pSTAT3 were upregulated in metastases in HGSOC patients, with substantial patient-to-patient heterogeneity. These results suggest that inhibiting individual soluble factors will not inhibit AAM-induced effects across a broad group of patients; instead, the downstream JAK2/STAT3/MMP-9 pathway should be examined as potential therapeutic targets to slow metastasis in ovarian cancer.

The Extracellular Matrix of Ovarian Cortical Inclusion Cysts Modulates Invasion of Fallopian Tube Epithelial Cells.

A growing body of research supports the idea that the fallopian tube epithelium (FTE) is the precursor for most high-grade serous ovarian canacers (HGSOC) but that the ovary plays a critical role in tumor metastasis. Cortical inclusion cysts (CICs) in the ovarian cortex have been hypothesized to create a niche environment that plays a role in HGSOC progression. Through histological analysis of pathology samples from human ovaries, we determined that collagen I and III were elevated near CICs and that the collagen fibers in this dense region were oriented parallel to the cyst boundary. Using this information from human samples as design parameters, we engineered an in vitro model that recreates the size, shape, and extracellular matrix (ECM) properties of CICs. We found that FTE cells within our model underwent robust invasion that was responsive to stimulation with follicular fluid, while ovarian surface epithelial (OSE) cells, the native cells of the ovary, were not invasive. We provide experimental evidence to support a role of the extracellular matrix in modulating FTE cell invasion, as decreased collagen I concentration or the addition of collagen III to the matrix surrounding FTE cells increased FTE cell invasion. Taken together, we show that an in vitro model of CICs informed by the analysis of human tissue can act as an important tool for understanding FTE cell interactions with their environment.

Alternatively-Activated Macrophages Upregulate Mesothelial Expression of P-Selectin to Enhance Adhesion of Ovarian Cancer Cells.

Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a coculture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAM) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1ß activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this de novo P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling underflow. C57/BL6 mice treated with MIP-1ß exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion ex vivo and ID8 adhesion in vivo Analysis of samples from patients with HGSOC confirmed increased MIP-1ß and P-selectin, suggesting that this novel multicellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti-CCR5 and P-selectin therapies developed for other indications.Significance: This study reports novel insights on the peritoneal dissemination occurring during progression of ovarian cancer and has potential for therapeutic intervention.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg Cancer Res; 78(13); 3560-73. ©2018 AACR.

Criteria for Risk Stratification of Vulvar and Vaginal Smooth Muscle Tumors: An Evaluation of 71 Cases Comparing Proposed Classification Systems.

Accurate risk stratification of smooth muscle tumors (SMTs) is essential for appropriate patient management. Yet, the rarity of SMTs of the vagina and vulva makes development of a prognostically meaningful classification system challenging. While 2 classification methods for vulvar SMTs and 1 for vaginal SMTs have been proposed, it is our experience that many pathologists tend to apply criteria for uterine SMTs when evaluating vulvovaginal tumors. We retrospectively reviewed a large cohort of vulvovaginal SMTs with clinical follow-up and evaluated which method most accurately classified tumors according to patient outcome. A total of 71 tumors, 53 vaginal (75%) and 18 vulvar (25%), from 71 patients were identified. All tumors were centrally examined for degree of cytologic atypia, morphology (spindled, epithelioid, myxoid), mitotic index per 10 high power fields, atypical mitotic figures, tumor cell necrosis, ischemic necrosis, tumor interface (circumscribed or infiltrative) and margin status. Clinical features were recorded for each patient. Follow-up was available for 63 patients (89%), and ranged from 1 to 234 months (median: 64 mo). While site-specific and uterine criteria showed equally excellent sensitivity in classifying smooth muscle neoplasms as leiomyosarcoma according to patient outcome, uterine criteria showed improved specificity relatively to site-specific methods in classifying tumors as nonsarcoma according to patient outcome. We recommend that uterine SMT criteria and nomenclature be adopted for evaluation and classification of vulvovaginal SMTs.