Oncogenic BRAF and KRAS mutations in endosalpingiosis.

Endosalpingiosis, a microscopic lesion composed of ectopic Fallopian tube epithelium, frequently involves the peritoneum and lymph nodes in patients with ovarian serous borderline tumour or low-grade serous carcinoma, but its pathogenic significance remains unclear. Using laser-capture microdissection and droplet digital PCR, we investigated whether endosalpingiosis harbours the driver mutations in BRAF and KRAS that characterise ovarian low-grade serous neoplasms. Somatic mutations were detected in 14 (33%) of 43 endosalpingiotic lesions analysed. Of 21 women with endosalpingiosis associated with a synchronous or metachronous ovarian low-grade serous tumour, mutations were identified in endosalpingiotic lesions from 11 (52%) women, with most cases (10/11, 91%) demonstrating identical mutations in both tumour and endosalpingiosis. In contrast, of 13 cases of endosalpingiosis not associated with an ovarian tumour, only one harboured a KRAS mutation. The proliferative activity as assessed by Ki-67 immunohistochemistry was lower in endosalpingiosis than in low-grade serous tumours, and endosalpingiosis with either a BRAF or KRAS mutation had a significantly lower Ki-67 index than those without. Ectopic expression of KRASG12V in Fallopian tube epithelial cells led to ERK phosphorylation, p21 induction, growth arrest and cellular senescence. In conclusion, we demonstrate that endosalpingiosis represents an interesting example of cancer driver mutations in deceptively normal-appearing cells, which may be prone to neoplastic transformation upon bypass of endogenous oncosuppressive mechanisms. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Frequent somatic mutations of the telomerase reverse transcriptase promoter in ovarian clear cell carcinoma but not in other major types of gynaecological malignancy.

Up-regulated expression of telomerase reverse transcriptase (TERT) and subsequent maintenance of telomere length are essential in tumour development. Recent studies have implicated somatic gain-of-function mutations at the TERT promoter as one of the mechanisms that promote transcriptional activation of TERT; however, it remains unclear whether this genetic abnormality is prevalent in gynaecological neoplasms. We performed mutational analysis in a total of 525 gynaecological cancers, and correlated TERT promoter mutations with clinicopathological features. With the exception of ovarian clear cell carcinomas, in which mutations were found in 37 (15.9%) of 233 cases, the majority of gynaecological malignancies were wild-type. TERT promoter mutation does not appear to be an early event during oncogenesis, as it was not detected in the contiguous endometriosis associated with ovarian clear cell carcinoma. Ovarian clear cell carcinoma cell lines with TERT promoter mutations exhibited higher TERT mRNA expression than those with wild-type sequences (p = 0.0238). TERT promoter mutation tended to be mutually exclusive with loss of ARID1A protein expression (p = 4.4 × 10(-9) ) and PIK3CA mutation (p = 0.0019) in ovarian clear cell carcinomas. No associations with disease-specific survival were observed for ovarian clear cell carcinoma. The above results, in conjunction with our previous report showing longer telomeres in ovarian clear cell carcinomas relative to other types of ovarian cancer, suggests that aberrations in telomere biology may play an important role in the pathogenesis of ovarian clear cell carcinoma.

Insights into cancer metastasis from a clinicopathologic perspective: Epithelial-Mesenchymal Transition is not a necessary step.

Epithelial-mesenchymal transition (EMT) has been implicated as the critical event initiating cancer invasion and metastasis. After disseminating through the circulation, the malignant cells have been proposed to undergo subsequent mesenchymal-epithelial transition (MET) to form secondary tumors. However, strong evidence from human tumor specimens for this paradigm is lacking. In carcinomas, cancers derived from epithelial tissues, epithelial morphology and gene expression are always retained to some degree. While mesenchymal transdifferentiation may be involved in the pathogenesis of carcinosarcomas, even in these neoplasms, as well as in germ cell tumors capable of multilineage differentiation, the mesenchymal phenotype does not facilitate metastatic progression. Indeed, most cancers invade and travel through lymphatic and blood vessels via cohesive epithelial migration, rather than going through the EMT-MET sequence. EMT gene expression is also consistently associated with high histologic grade and while the transcription factors, Snail, Slug and Twist have traditionally been thought of as inducers of EMT, under certain conditions, they also mediate dedifferentiation and maintenance of the stem cell state. In various malignancies, including basal-like breast cancer and colorectal cancer, the genetically unstable, undifferentiated phenotype predicts early metastatic spread and poor prognosis. This article discusses some of the controversies surrounding differentiation and metastasis from a clinicopathologic perspective and presents evidence that the epithelial phenotype is maintained throughout the process of cancer metastasis.

Identifying Lynch syndrome in patients with ovarian carcinoma: the significance of tumor subtype.

Up to 15% of ovarian cancers are etiologically linked with hereditary susceptibility. Within this group, germline mutations in mismatch repair (MMR) genes, known otherwise as Lynch syndrome (LS), account for the majority of cases that are not associated with mutations in BRCA1 or BRCA2. Clinical schemas specific for gynecologic cancers have been developed to identify patients with LS; however, many of the recommendations are poorly defined. Few case series of germline-confirmed LS-associated ovarian cancers have been reported, limited by small sample size and often lacking central pathology review. Much insight has been gained from studies of unselected cohorts, using immunohistochemical assessment of MMR protein expression or microsatellite instability analysis. In spite of contradictory results, likely reflective of differences in study design, sample size and methodology, a recurring observation is the overrepresentation of "endometriosis-associated tumors," namely, endometrioid and clear cell subtypes, in the group of ovarian tumors with MMR deficiency. In this review, we summarize the clinical and histomorphologic features of LS-associated/MMR-deficient ovarian epithelial cancers and recommend that reflex testing be performed on the basis of tumor subtype.

Mixed Ovarian Neoplasms With Gastrointestinal-type Mucinous and Mullerian Epithelial Components: A Rare Group of Tumors Demonstrating the Phenotypic Plasticity of the Mullerian Epithelial Cell.

Primary mucinous ovarian neoplasms, gastrointestinal-type (GI-type), are composed of mucin-producing tumor cells resembling intestinal goblet cells or gastric foveolar epithelium. In contrast to seromucinous tumors, which exhibit endocervical-type mucinous differentiation and are thought to be derived from endometriosis, the cell/tissue-of-origin of most GI-type mucinous ovarian tumors is unknown. We identified 8 GI-type mucinous ovarian tumors (cystadenomas, n=4; borderline tumor/carcinoma, n=4) with spatially distinct areas that showed morphologic features of Mullerian-type epithelial differentiation (ciliated cells or endometrioid-type glands). Immunohistochemistry for cell lineage markers and Alcian blue (pH 2.5)/Periodic Acid-Schiff staining were performed. Morphologically distinct components were isolated by microdissection, from which extracted DNA was analyzed by targeted next-generation sequencing. In all cases, immunohistochemistry demonstrated mucin-producing cells to be positive for at least one GI marker (CK20 or CDX2), while areas with morphologic features of Mullerian differentiation were positive for PAX8, ER and/or PR, and lacked expression of CK20 and CDX2; CK7 was strongly and diffusely positive in all tumor cells. Tumor cells with a gastric-type phenotype produced neutral mucin, while acidic mucin was present within intestinal-type goblet cells. Targeted sequencing revealed ARID1A mutations in all mixed borderline tumors/carcinomas (n=4); other recurrent genetic alterations included KRAS (n=2) and TP53 mutations (n=2). Shared mutations were present in paired Mullerian and GI-type mucinous tumor components in 4 mixed borderline tumors/carcinomas, with more shared mutations between components than private mutations specific to each component. All mixed borderline tumors/carcinomas were associated with endometriosis (n=3) or Mullerian inclusion cysts (n=1); mutation or loss of ARID1A expression was seen in these putative precursor lesions in 2 cases. Hence, ovarian neoplasms composed of clonally related GI-type mucinous and Mullerian-type epithelial components harbor ARID1A mutations and are frequently associated with endometriosis. The existence of a Mullerian stem/progenitor cell with the capacity to differentiate toward cell lineages within the GI-tract may be involved in the pathogenesis of at least a subset of GI-type mucinous ovarian neoplasms.

Clinicopathologic and Molecular Features of Paired Cases of Metachronous Ovarian Serous Borderline Tumor and Subsequent Serous Carcinoma.

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.

Correlation of PD-L1 Expression with Tumor Mutation Burden and Gene Signatures for Prognosis in Early-Stage Squamous Cell Lung Carcinoma.

OBJECTIVES: Anti-programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD-1/PD-L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti-PD-1/PD-L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti-PD-1/PD-L1 immunotherapy-related biomarkers in early-stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early-stage SqCLC, providing data for identifying the potential role for patients with anti-PD-1/PD-L1 treatment in early-stage SqCLC. METHODS: A total of 255 specimens from patients with early-stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD-L1 protein expression by immunohistochemistry was evaluated by using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T-effector and interferon gamma (IFN-γ) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. RESULTS: The prevalence of PD-L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD-L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells. PD-L1 protein expression on tumor-infiltrating immune cells was correlated with the T-effector and IFN-γ gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. CONCLUSIONS: Evaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the cohort with early-stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

Histopathologic Features of Prognostic Significance in High-Grade Osteosarcoma.

Context .- In osteosarcoma treated with neoadjuvant chemotherapy the extent of tumor necrosis on resection is considered an indicator of treatment response, and this has been shown to correlate with survival in most but not all studies. Objective .- To identify additional histologic variables of prognostic significance in high-grade osteosarcoma. Design .- Slides of pretreatment biopsy and primary postneoadjuvant chemotherapy resections from 165 patients with high-grade osteosarcoma were reviewed. Univariate (Kaplan-Meier) and multivariate (Cox regression) analyses were performed to identify clinical and histomorphologic attributes associated with overall survival. Results .- Univariate analyses confirmed the prognostic significance of metastatic status on presentation, primary tumor size, anatomic site, and histologic subtype. Additionally, the identification of lymphovascular invasion, 10% or more residual viable tumor, and 10 or more mitoses per 10 high-powered fields assessed in posttreatment resections were associated with poor survival, retaining significance in multivariate analyses. Based on results from multivariate analysis, we developed a prognostic index incorporating primary tumor size and site, and significant histologic features assessed on resection (ie, lymphovascular invasion status, mitotic rate, and extent of viable tumor). This scoring system segregates patients into 3 risk categories with significant differences in overall survival and retained significance in an independent validation set of 42 cases. Conclusions .- The integration of clinical and microscopic features improves prognostication of patients with osteosarcoma.

In-depth molecular profiling of the biphasic components of uterine carcinosarcomas.

Uterine carcinosarcoma is a clinically aggressive malignancy composed of a mix of carcinomatous and sarcomatous elements. We performed targeted next-generation sequencing of 27 uterine cancer and sarcoma genes together with immunohistochemical analyses of selected proteins in 30 uterine carcinosarcomas. This included 13 cases in which the distinct carcinoma and sarcoma components were sequenced separately and 10 cases where the metastatic tumours were analysed in addition to the primary tumours. We identified non-synonymous somatic mutations in 90% of the cases, with 27 of 30 cases (90%) harbouring TP53 alterations. The PI3K pathway was the most commonly mutated signalling pathway with mutations identified in PIK3CA, PTEN, PIK3R1, and/or PIK3R2 in two-thirds of the cases. Mutations in FBXW7, PPP2R1A, ARID1A and KRAS were demonstrated in a minority of cases. In cases where the carcinomatous and sarcomatous components were separately analysed, most of the mutations identified were present in both components, indicating a common origin for the two components. Furthermore, the same TP53 alterations and/or PI3K pathway mutations seen in the primary tumours were also identified in the metastatic sites. Overall, carcinosarcomas exhibited heterogeneous molecular features that resemble the heterogeneity seen in endometrial carcinomas, with some showing endometrioid carcinoma-like and others showing serous carcinoma-like mutation profiles. While patients with serous-like tumours presented more frequently with advanced-stage disease compared to patients with endometrioid-like tumours, there was no statistical difference in outcome between the two groups. Our results provide insights into the oncogenesis of uterine carcinosarcoma and identify targetable mutations that represent early oncogenic events. The findings of the different molecular types of uterine carcinosarcoma that parallel the different molecular types in endometrial carcinoma may have future treatment implications with targeted therapies.

The histomorphology of Lynch syndrome-associated ovarian carcinomas: toward a subtype-specific screening strategy.

Women with Lynch syndrome (LS) are at increased risk for the development of epithelial ovarian cancer (OC). Analogous to previous studies on BRCA1/2 mutation carriers, there is evidence to suggest a histotype-specific association in LS-associated OCs (LS-OC). Whereas the diagnosis of high-grade serous carcinoma is an indication for BRCA1/2 germline testing, in contrast, there are no screening guidelines in place for triaging OC patients for LS testing based on histotype. We performed a centralized pathology review of tumor subtype on 20 germline mutation-confirmed LS-OCs, on the basis of morphologic assessment of hematoxylin and eosin-stained slides, with confirmation by immunohistochemistry when necessary. Results from mismatch-repair immunohistochemistry (MMR-IHC) and microsatellite instability (MSI) phenotype status were documented, and detailed pedigrees were analyzed to determine whether previously proposed clinical criteria would have selected these patients for genetic testing. Review of pathology revealed all LS-OCs to be either pure endometrioid carcinoma (14 cases), mixed carcinoma with an endometrioid component (4 cases), or clear cell carcinoma (2 cases). No high-grade or low-grade serous carcinomas or mucinous carcinomas of intestinal type were identified. Tumor-infiltrating lymphocytes were prominent (≥40 per 10 high-powered fields) in 2 cases only. With the exception of 1 case, all tumors tested for MMR-IHC or MSI had an MMR-deficient phenotype. Within this cohort, 50%, 55%, 65%, and 85% of patients would have been selected for genetic workup by Amsterdam II, revised Bethesda Guidelines, SGO 10% to 25%, and SGO 5% to 10% criteria, respectively, with <60% of index or sentinel cases detected by any of these schemas. To further support a subtype-driven screening strategy, MMR-IHC reflex testing was performed on all consecutive non-serous OCs diagnosed at 1 academic hospital over a 2-year period; MMR deficiency was identified in 10/48 (21%) cases, all with endometrioid or clear cell histology. We conclude that there is a strong association between endometrioid and clear cell ovarian carcinomas and hereditary predisposition due to MMR gene mutation. These findings have implications for the role of tumor subtype in screening patients with OC for further genetic testing and support reflex MMR-IHC and/or MSI testing for newly diagnosed cases of endometrioid or clear cell ovarian carcinoma.

Follicular epithelial dysplasia of the thyroid: morphological and immunohistochemical characterization of a putative preneoplastic lesion to papillary thyroid carcinoma in chronic lymphocytic thyroiditis.

In chronic lymphocytic thyroiditis (CLT), the follicular epithelial cells display cytological atypia resembling papillary thyroid carcinoma (PTC), and epidemiological studies have suggested an increased risk of PTC in patients with this condition. While reactive atypia is observed diffusely in CLT-affected thyroid parenchyma, it is not unusual to find microscopic foci morphologically distinct from the surrounding parenchyma, exhibiting more pronounced cytological and architectural atypia. These small atypical lesions, which we term "follicular epithelial dysplasia" (FED), are particularly prominent in cases of severe CLT, yet lack invasive growth, papillary architecture, or intranuclear pseudoinclusions. To gain further insight into their biological significance, we constructed a tissue microarray of 70 cases of CLT, comprised of morphologically normal thyroid, thyroid with reactive atypia, FED, follicular nodular disease (nodular hyperplasia or follicular adenoma), and PTC. Immunohistochemical staining was performed for a marker panel including PTC (HBME-1, cytokeratin 19, galectin-3, and cyclin-D1) as well as TTF-1, thyroglobulin, and p63. Slides were digitally scanned and immunohistochemical staining evaluated using automated image analysis software. FED lesions were positive for TTF-1 and thyroglobulin (50/50, 100 %), though some (13/50, 26 %) also expressed p63. Similar to PTC, strong diffuse staining was observed for HBME-1 (43/50, 86 %), cytokeratin 19 (48/50, 96 %), galectin-3 (20/50, 40 %) and cyclin-D1 (38/50, 76 %). In contrast, normal thyroid, reactive atypia, and follicular nodular disease were negative, or at most, exhibited focal weak staining for HBME-1, cytokeratin 19, and galectin-3. The results of this study demonstrate the presence of atypical microscopic lesions in CLT with an immunohistochemical profile similar to PTC, supporting the concept of a premalignant lesion preceding PTC, arising in the context of severe chronic inflammation.

Case report: Adrenal LH/hCG receptor overexpression and gene amplification causing pregnancy-induced Cushing's syndrome.

Transient pregnancy-induced Cushing's syndrome (CS) is extremely rare, with only several cases reported in the literature. Ectopic LH/hCG-receptors (LHCGR) in the adrenal gland have been suggested to be involved in the pathogenesis of this condition. We report the clinical, molecular, and genetic features of a patient with pregnancy-induced CS. A 29-year-old female patient developed CS during multiple pregnancies, leading to repeated miscarriage. Signs and symptoms of hypercortisolism resolved soon after delivery or abortion, only to recur in subsequent pregnancies. In the non-pregnant state, hCG stimulation testing resulted in elevated cortisol levels. Serum cortisol was not suppressible with dexamethasone. The adrenals exhibited bilateral adrenal cortical nodular hyperplasia. Quantitative RT-PCR revealed a 2-fold increase in LHCGR and progesterone receptor mRNA expression and decreased estrogen receptor-beta expression in the patient's adrenal tissue relative to normal adrenals. Higher intensity of immunostaining for LHCGR was observed, particularly within the nodular lesions, compared to controls. Quantitative PCR revealed a LHCGR-to-beta-actin ratio of 1.5 in genomic DNA from adrenal and peripheral leukocytes, suggesting the presence of a germline duplication of the LHCGR gene. LHCGR overexpression resulting from germline gene duplication may be a potential pathogenic mechanism underlying this case of pregnancy-induced CS.

Antioxidant vitamins reduce acute meal-induced memory deficits in adults with type 2 diabetes.

Memory impairment is observed in adults with type 2 diabetes mellitus (T2DM), with further acute deficits after meal ingestion. This study explored whether postprandial oxidative stress was a contributor to these meal-induced memory deficits. Sixteen adults with T2DM (mean age, 63.5 +/- 2.1 years) who were not regularly taking high-dose antioxidant supplements were fed a high-fat meal, the same test meal with vitamins C (1000 mg) and E (800 IU) tablets, or water on 3 separate occasions. After meal ingestion, a battery of cognitive tests were administered, which included measures of delayed verbal memory, assessed at 60 and 105 minutes after meal ingestion. Relative to water consumption, the high-fat meal resulted in poorer performance at 105 minutes postingestion on measures of delayed verbal recall (word list and paragraph recall) and working memory (Digit-Span Forward). Coconsumption of antioxidant vitamins and high-fat meal prevented this meal-induced deficit such that performance on these tasks was indistinguishable from that after water intake. At the same time point, a small but significant improvement on the word-naming and color-naming components of Stroop was observed after meal ingestion, relative to water, irrespective of whether antioxidants were consumed, demonstrating the specificity of meal-induced impairments to memory function. Executive function, assessed by Trails Parts A and B, was not influenced by meal or antioxidant ingestion. In adults with T2DM, coconsumption of antioxidant vitamins minimizes meal-induced memory impairment, implicating oxidative stress as a potential contributor to these decrements.