Mesonephric-like adenocarcinoma of the female genital tract: novel observations and detailed molecular characterisation of mixed tumours and mesonephric-like carcinosarcomas.

AIMS: To report novel observations in five mesonephric-like adenocarcinomas (MLAs) of the female genital tract. METHODS AND RESULTS: We report two endometrial MLAs in association with endometrioid carcinoma and atypical hyperplasia and three (one endometrial, two ovarian) cases with a sarcomatoid component (mesonephric-like carcinosarcoma). Pathogenic KRAS mutations, which are characteristic of MLA, were identified in all cases although interestingly, in one of the mixed carcinomas, this was confined to the endometrioid component. The concurrent MLA, endometrioid carcinoma and atypical hyperplasia components in one case harboured identical EGFR, PTEN and CCNE1 mutations, suggesting that the atypical hyperplasia gave rise to a Müllerian carcinoma with both endometrioid and mesonephric-like components. The carcinosarcomas all contained a component of MLA and a sarcomatous component with chondroid elements. In the ovarian carcinosarcomas, the coexisting epithelial and sarcomatous components shared some mutations including KRAS and CREBBP, suggesting that they are clonally related. Furthermore, in one case CREBBP and KRAS mutations detected in the MLA and sarcomatous components were also detected in an associated undifferentiated carcinoma component, suggesting that it was clonally related to the MLA and sarcomatous components. CONCLUSIONS: Our observations provide additional evidence that MLAs have a Müllerian origin and characterise mesonephric-like carcinosarcomas in which chondroid elements appear to be characteristic. In reporting these findings, we provide recommendations for distinction between a mesonephric-like carcinosarcoma and a MLA with a spindle cell component.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high-grade carcinoma: a targeted next-generation sequencing study.

AIMS: Our understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low-grade endometrial cancer (DEC-LG). However, cases of UC arising in the setting of high-grade EC (DEC-HG) have been noted in the literature. Our knowledge of the genomics of DEC-HG is limited. To characterise the molecular landscape of DEC-HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC-HG and four DEC-LG. METHODS AND RESULTS: DEC-HG and DEC-LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC-HG and 4/4 (100%) DEC-LG, while SMARCA4 mutations were present in 4/7 (57%) DEC-HG and in 1/4 (25%) DEC-LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC-HG and DEC-LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC-HG and in 2/4 (50%) DEC-LG, while mutation-pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC-HG and none of the DEC-LG. MLH1 mutations were observed in 1/7 (14%) DEC-HG and 1/4 (25%) DEC-LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC-HG, but neither was associated with corresponding loss of protein expression. CONCLUSION: The findings support expanding the definition of DEC to include DEC-HG, a previously under-recognised phenomenon with genomic similarities to DEC-LG.

Budget impact analysis of molecular subtype profiling in endometrial cancer.

OBJECTIVE: This study evaluated the costs associated with four approaches to classifying endometrial cancer (EC), including histomorphological, histomorphological with ancillary immunohistochemical assays, histomolecular and selective molecular classification. METHODS: Direct costs were determined per EC sample from the hospital's perspective. A budget impact analysis and sensitivity analysis were conducted to estimate the mean, minimum and maximum costs per sample and annual institutional costs in adjusted 2022 Canadian dollars. A provincial cost forecast was projected based on expected 2022 EC biopsies. RESULTS: In 2018, our institution performed 190 EC biopsies. The mean cost per biopsy was $158 ($156-$212) for histomorphological classification, $384 ($360-$514) for histomorphological classification with immunohistochemistry and $1297 ($1265-1833) for histomolecular classification. Total annual institutional cost for histomorphological classification was $29,980 and $72,950 with immunohistochemistry. For histomolecular classification, the first year cost was $246,521, accounting for initial educational learning curve, and $233,461 thereafter, assuming a consistent number of biopsies per year. Targeted implementation of histomolecular classification among high-grade, p53 abnormal and/or MMR-deficient ECs (56% of cases) cost $169,688 in the first year and $162,418 annually thereafter. With a projected 3400 EC biopsies in Ontario in 2022, histomorphological classification would annually cost $537,078 and $1,305,677 with immunohistochemistry. Histomolecular classification would cost $4,410,203 in the first year and $4,176,737 annually once established. Selective molecular classification would lead to a cost of $3,044,178 in the first year and $2,913,443 thereafter. CONCLUSIONS: The study highlights the need for informed decision-making when implementing molecular classification in clinical practice, given the substantial incremental healthcare costs associated with these approaches.

Lats1 and Lats2 are required for the maintenance of multipotency in the Müllerian duct mesenchyme.

WNT signaling plays essential roles in the development and function of the female reproductive tract. Although crosstalk with the Hippo pathway is a key regulator of WNT signaling, whether Hippo itself plays a role in female reproductive biology remains largely unknown. Here, we show that conditional deletion of the key Hippo kinases Lats1 and Lats2 in mouse Müllerian duct mesenchyme cells caused them to adopt the myofibroblast cell fate, resulting in profound reproductive tract developmental defects and sterility. Myofibroblast differentiation was attributed to increased YAP and TAZ expression (but not to altered WNT signaling), leading to the direct transcriptional upregulation of Ctgf and the activation of the myofibroblast genetic program. Müllerian duct mesenchyme cells also became myofibroblasts in male mutant embryos, which impeded the development of the male reproductive tract and resulted in cryptorchidism. The inactivation of Lats1/2 in differentiated uterine stromal cells in vitro did not compromise their ability to decidualize, suggesting that Hippo is dispensable during implantation. We conclude that Hippo signaling is required to suppress the myofibroblast genetic program and maintain multipotency in Müllerian mesenchyme cells.

Brief family history questionnaire to screen for Lynch syndrome in women with newly diagnosed non-serous, non-mucinous ovarian cancers.

OBJECTIVES: While ovarian cancer is the third most common Lynch syndrome-associated cancer in women, there is no established screening strategy to identify Lynch syndrome in this population. The objective of this study was to assess whether the 4-item brief Family History Questionnaire can be used as a screening tool to identify women with ovarian cancer at risk of Lynch syndrome. METHODS: In this prospective cohort study, participants with newly diagnosed non-serous, non-mucinous ovarian cancer completed the brief Family History Questionnaire, extended Family History Questionnaire, and had tumors assessed with immunohistochemistry for mismatch repair proteins, MLH1 methylation, and microsatellite instability testing. All underwent universal germline testing for Lynch syndrome. Performance characteristics were compared between the brief Family History Questionnaire, extended Family History Questionnaire, immunohistochemistry±MLH1 methylation, and microsatellite instability testing. RESULTS: Of 215 participants, 169 (79%) were evaluable with both the brief Family History Questionnaire and germline mutation status; 12 of these 169 were confirmed to have Lynch syndrome (7%). 10 of 12 patients (83%) with Lynch syndrome were correctly identified by the brief Family History Questionnaire, compared with 6 of 11 (55%) by the extended Family History Questionnaire, 11 of 13 (85%) by immunohistochemistry±MLH1 methylation, and 9 of 11 (82%) by microsatellite instability testing. The sensitivity, specificity, positive predictive values, and negative predictive values of the brief Family History Questionnaire were 83%, 65%, 15%, and 98%, respectively. A combined approach with immunohistochemistry and the brief Family History Questionnaire correctly identified all 12 patients with Lynch syndrome. The brief Family History Questionnaire was more sensitive than the extended Family History Questionnaire and took <10 min for each patient to complete. CONCLUSIONS: The brief Family History Questionnaire alone or combined with immunohistochemistry may serve as an adequate screening strategy, especially in centers without access to universal tumor testing.

Maximizing cancer prevention through genetic navigation for Lynch syndrome detection in women with newly diagnosed endometrial and nonserous/nonmucinous epithelial ovarian cancer.

BACKGROUND: Despite recommendations for reflex immunohistochemistry (IHC) for mismatch repair (MMR) proteins to identify Lynch syndrome (LS), the uptake of genetic assessment by those who meet referral criteria is low. The authors implemented a comprehensive genetic navigation program to increase the uptake of genetic testing for LS in patients with endometrial cancer (EC) or nonserous/nonmucinous ovarian cancer (OC). METHODS: Participants with newly diagnosed EC or OC were prospectively recruited from 3 cancer centers in Ontario, Canada. Family history questionnaires were used to assess LS-specific family history. Reflex IHC for MMR proteins was performed with the inclusion of clinical directives in pathology reports. A trained genetic navigator initiated a genetic referral on behalf of the treating physician and facilitated genetic referrals to the closest genetics center. RESULTS: A total of 841 participants (642 with EC, 172 with OC, and 27 with synchronous EC/OC) consented to the study; 194 (23%) were MMR-deficient by IHC. Overall, 170 women (20%) were eligible for a genetic assessment for LS: 35 on the basis of their family history alone, 24 on the basis of their family history and IHC, 82 on the basis of IHC alone, and 29 on the basis of clinical discretion. After adjustments for participants who died (n = 6), 149 of 164 patients (91%) completed a genetic assessment, and 111 were offered and completed genetic testing. Thirty-four women (4.0% of the total cohort and 30.6% of those with genetic testing) were diagnosed with LS: 5 with mutL homolog 1 (MLH1), 9 with mutS homolog 2 (MSH2), 15 with mutS homolog 6 (MSH6), and 5 with PMS2. CONCLUSIONS: The introduction of a navigated genetic program resulted in a high rate of genetic assessment (>90%) in patients with gynecologic cancer at risk for LS.

Understanding the clinical implication of mismatch repair deficiency in endometrioid endometrial cancer through a prospective study.

OBJECTIVES: Findings on impact of mismatch repair deficiency (MMRd) on patient outcomes in endometrial cancer (EC) have been inconsistent to date. The objective of this study was to compare the oncologic outcomes and recurrence patterns between MMRd and MMR-intact (MMRi) endometrioid EC (EEC). METHODS: Between 2015 and 2018, we prospectively recruited 492 EEC cases from three cancer centers in Ontario, Canada. Tumors were reflexively assessed for MMR protein expression by immunohistochemistry (IHC). Clinicopathological, survival and recurrence patterns were compared between MMRd and MMRi cases. RESULTS: Of 492 EEC, 348 were MMRi (71%) and 144 were MMRd (29%) with median follow-up of 16.8 months (0-69.6). MMRd tumors tended to be grade 2 or 3 (56% vs. 29%, p < 0.001), with propensity for lymphovascular space invasion (28% vs. 18%, p = 0.024), lymph node involvement (7% vs. 5%, p < 0.001) and received more adjuvant treatment (46% vs. 33%, p = 0.027). This group also had significantly lower 3-year recurrence-free survival (78% vs. 90%, p = 0.014) although there was no difference in OS (p = 0.603). MMRd cases were more likely to recur in retroperitoneal lymph nodes (p = 0.045). Upon subgroup analysis, MLH1 methylated tumors had the worst prognostic features and survival outcomes. CONCLUSIONS: MLH1 methylated EECs exhibit more aggressive features compared to other MMRd and MMRi EECs. This may indicate an inherent difference in tumor biology, suggesting the importance of individualized management based on EC molecular phenotype.

NTRK-rearranged Cervical Sarcoma: Expanding the Clinicopathologic Spectrum.

The NTRK genes (NTRK1, NTRK2, and NTRK3) encode for TrkA, TrkB, and TrkC, neurotrophic tyrosine receptor kinases which serve a variety of functions including in the regulation of pathways involved in carcinogenesis. A number of reports have described NTRK gene fusions in a variety of adult and pediatric tumor types from various organ systems including the central nervous system, thyroid gland, breast, and soft tissue. NTRK-rearranged uterine sarcomas are a recently described group of tumors which occur in both the uterine corpus and cervix, tend to morphologically resemble fibrosarcoma, and may behave aggressively, although data is limited given the newly recognized nature and thus relative rarity of these tumors. Herein, we present the case of a cervical sarcoma with SPECC1L-NTRK3 fusion (detected with Illumina RNA Fusion Panel), prospectively diagnosed at the time of cervical biopsy and subsequently treated with hysterectomy. The clinical presentation, radiologic findings, morphologic features, and immunohistochemical profile of this case will be reviewed and compared with the body of existing literature to date. Identification of NTRK-rearranged neoplasms is important as targeted therapy in the form of NTRK inhibitors has recently become widely available.

Performance characteristics of screening strategies to identify Lynch syndrome in women with ovarian cancer.

BACKGROUND: For women with ovarian cancer (OC), the optimal screening strategy to identify Lynch syndrome (LS) has not been determined. In the current study, the authors compared the performance characteristics of various strategies combining mismatch repair (MMR) immunohistochemistry (IHC), microsatellite instability testing (MSI), and family history for the detection of LS. METHODS: Women with nonserous and/or nonmucinous ovarian cancer were recruited prospectively from 3 cancer centers in Ontario, Canada. All underwent germline testing for LS and completed a family history assessment. Tumors were assessed using MMR IHC and MSI. The sensitivity, specificity, and positive and negative predictive values of screening strategies were compared with the gold standard of a germline result. RESULTS: Of 215 women, germline data were available for 189 (88%); 13 women (7%) had pathogenic germline variants with 7 women with mutS homolog 6 (MSH6); 3 women with mutL homolog 1 (MLH1); 2 women with PMS1 homolog 2, mismatch repair system component (PMS2); and 1 woman with mutS homolog 2 (MSH2). A total of 28 women had MMR-deficient tumors (13%); of these, 11 had pathogenic variants (39%). Sequential IHC (with MLH1 promoter methylation analysis on MLH1-deficient tumors) followed by MSI for nonmethylated and/or MMR-intact patients was the most sensitive (92.3%; 95% confidence interval, 64%-99.8%) and specific (97.7%; 95% confidence interval, 94.2%-99.4%) approach, missing 1 case of LS. IHC with MLH1 promoter methylation analysis missed 2 patients of LS. Family history was found to have the lowest sensitivity at 55%. CONCLUSIONS: Sequential IHC (with MLH1 promoter methylation analysis) followed by MSI was found to be most sensitive. However, IHC with MLH1 promoter methylation analysis also performed well and is likely more cost-effective and efficient in the clinical setting. The pretest probability of LS is high in patients with MMR deficiency and warrants universal screening for LS.

Histological grading of ovarian mucinous carcinoma - an outcome-based analysis of traditional and novel systems.

AIMS: Grading of primary ovarian mucinous carcinoma (OMC) is inconsistent among practices. The International Collaboration on Cancer Reporting recommends grading OMC using the International Federation of Gynecology and Obstetrics (FIGO) system for endometrial endometrioid carcinoma, when needed. The growth pattern (expansile versus infiltrative), a known prognostic variable in OMC, is not considered in any grading system. We herein analysed the prognostic value of various grading methods in a well-annotated cohort of OMC. METHODS AND RESULTS: Institutional OMCs underwent review and grading by the Silverberg and FIGO schemes and a novel system, growth-based grading (GBG), defined as G1 (expansile growth or infiltrative invasion in ≤10%) and G2 (infiltrative growth >10% of tumour). Of 46 OMCs included, 80% were FIGO stage I, 11% stage II and 9% stage III. On follow-up (mean = 52 months, range = 1-190), five patients (11%) had adverse events (three recurrences and four deaths). On univariate analysis, stage (P = 0.01, Cox proportional analysis), Silverberg grade (P = 0.01), GBG grade (P = 0.001) and percentage of infiltrative growth (P < 0.001), but not FIGO grade, correlated with disease-free survival. Log-rank analysis showed increased survival in patients with Silverberg grade 1 versus 2 (P < 0.001) and those with GBG G1 versus G2 (P < 0.001). None of the parameters evaluated was significant on multivariate analysis (restricted due to the low number of adverse events). CONCLUSIONS: Silverberg and the new GBG system appear to be prognostically significant in OMC. Pattern-based grading allows for a binary stratification into low- and high-grade categories, which may be more appropriate for patient risk stratification. Despite current practices and recommendations to utilise FIGO grading in OMC, our study shows no prognostic significance of this system and we advise against its use.

Undifferentiated endometrial carcinoma arising in the background of high-grade endometrial carcinoma - Expanding the definition of dedifferentiated endometrial carcinoma.

Dedifferentiated endometrial adenocarcinoma (DEC) is defined by the coexistence of undifferentiated carcinoma with low-grade (FIGO grade 1 or 2) endometrioid carcinoma. Few cases of DEC arising in the background of high-grade carcinoma (DEC-HG) have been reported, however, this phenomenon is poorly characterized. In this study we describe the morphologic, immunohistochemical and clinico-pathologic characteristics of DEC-HG. 18 DECs were diagnosed at our institution between 2008-2019, and in 11 (61%), the undifferentiated component was associated with high-grade carcinoma (8 endometrioid FIGO grade 3, 2 with ambiguous features, 1 serous). The remaining 7 (39%) represented DEC-LG (3 FIGO grade 1 and 4 FIGO grade 2). 7/11 (64%) patients with DEC-HG presented with advanced stage (FIGO stage III/IV), whereas most with DEC-LG (6/7, 86%) were stage I. On follow up, 2 patients in the DEC-HG group died of disease and 2 had progressive disease within 2 months of surgery. There was only one recurrence in the DEC-LG, 6 months post-surgery. The DEC component in both groups showed similar morphology and immunophenotype, with predominantly focal or complete loss of expression of pan-keratin, EMA, E-cadherin, CK8/18, PAX8 and ER. The DEC component in the DEC-HG group had wild-type p53 expression in 8/11 (73%) cases, loss of MLH1 and PMS2 in 6/11 (55%) and loss of SMARCA4 in 3/9 (33%). Although numbers are small, we show that DEC-HG is a previously under-recognized phenomenon, with morphologic and immunophenotypic similarities to DEC-LG, which supports expanding the definition of DEC to include DEC-HG. DEC-HG may be more aggressive than DEC-LG.

The prognostic role of horizontal and circumferential tumor extent in cervical cancer: Implications for the 2019 FIGO staging system.

OBJECTIVE: The FIGO 2019 update on cervical cancer staging removed horizontal tumor extent (HZTE) as a staging variable. Evidence is needed to substantiate this change. The prognostic significance of HZTE and a related variable, circumferential tumor extent (%CTE), is similarly unknown. We aimed to investigate the association of HZTE and %CTE with survival outcomes in cervical cancer patients. METHODS: We identified patients treated with primary surgery for stage I cervical cancer in a single institution during a 9-year period. HZTE and, when available, %CTE were obtained from pathology records. Cases were staged using 2019 FIGO staging. Correlations between HZTE, %CTE and FIGO stage with recurrence-free (RFS) and disease-specific survival (DSS) were determined using univariable and multivariable analyses. RESULTS: 285 patients were included with a median follow-up of 48 (range 7-123) months. HZTE was statistically associated with RFS and DSS on univariate and multivariate analysis. None of the 168 stage IA patients in our series had tumor recurrence or death during follow-up, including 42 with HZTE ≥7 mm. None of the patients with a tumor horizontal extent <7 mm experienced recurrence or death. %CTE correlated only with RFS on univariate analysis. 2019 FIGO stage did not independently correlate with RFS or DSS in our sample. CONCLUSIONS: HZTE is an independent predictor of survival in cervical carcinoma. In stage IA tumors, however, HZTE does not offer superior prognostic value, supporting the 2019 FIGO recommendations to remove this variable from staging in these cases. HZTE may be useful in larger tumors in which staging depends on maximum tumor size. %CTE is not an independent prognostic variable in cervical cancer, and we advise against its use.

Tumor site discordance in mismatch repair deficiency in synchronous endometrial and ovarian cancers.

OBJECTIVES: For synchronous endometrial and ovarian cancers, most centers rely on mismatch repair testing of the endometrial cancer to identify Lynch syndrome, and neglect the ovarian tumor site completely. We examined the mismatch repair immunohistochemistry and microsatellite instability results from the endometrium and ovary to assess discordance between the tumor sites and between tests. METHODS: 30 women with newly diagnosed synchronous endometrial and ovarian cancer were prospectively recruited from three cancer centers in Ontario, Canada. Both tumor sites were assessed for mismatch repair deficiency by immunohistochemistry and microsatellite instability test; discordance in results between tumor sites and discordance between test results at each site was examined. Cases with discordant results had tumors sequenced with a targeted panel in order to reconcile the findings. All women underwent mismatch repair gene germline testing. RESULTS: Of 30 patients, 11 (37%) were mismatch repair deficient or microsatellite instable at either tumor site, with 5 (17%) testing positive for Lynch syndrome. Mismatch repair immunohistochemistry expression was discordant between endometrial and ovarian tumor sites in 2 of 27 patients (7%) while microsatellite instability results were discordant in 2 of 25 patients (8%). Relying on immunohistochemistry or microsatellite instability alone on the endometrial tumor would have missed one and three cases of Lynch syndrome, respectively. One patient with Lynch syndrome with a PMS2 pathogenic variant was not detected by either immunohistochemistry or microsatellite instability testing. The rate of discordance between immunohistochemistry and microsatellite instability test was 3.8% in the ovary and 12% in the endometrium. CONCLUSIONS: There was discordance in immunohistochemistry and microsatellite instability results between tumor sites and between tests within each site. Endometrial tumor testing with mismatch repair immunohistochemistry performed well, but missed one case of Lynch syndrome. Given the high incidence of Lynch syndrome (17%), consideration may be given to germline testing in all patients with synchronous endometrial and ovarian cancers.

Myxoid smooth muscle neoplasia of the uterus: comprehensive analysis by next-generation sequencing and nucleic acid hybridization.

Uterine myxoid smooth muscle tumors, including myxoid leiomyosarcoma, are rare and their genomic profile has not been fully characterized. With the discovery of uterine sarcomas with ZC3H7B-BCOR fusion and BCOR internal tandem duplications, the differential diagnosis of myxoid smooth muscle lesions is expanding to include molecularly-defined tumors. Thus, we aimed to explore the genomic landscape of myxoid smooth muscle tumor using comprehensive tools. We performed whole exome next-generation sequencing and a pan-sarcoma RNA fusion assay in tumoral paraffin-embedded tissue from nine well-characterized uterine myxoid smooth muscle tumors (seven myxoid leiomyosarcomas and two myxoid smooth muscle tumors of unknown malignant potential). By immunohistochemistry, all tumors were strongly positive for smooth muscle markers and negative for BCOR staining; 4/6 expressed PLAG1. None of the tumors harbored known fusions including ZC3H7B-BCOR, TRPS1-PLAG1, and RAD51B-PLAG1. None harbored exon 15 BCOR internal tandem duplications; however, four tumors contained BCOR internal tandem duplications of unknown significance (mostly intronic). Mutational burden was low (median 3.8 mutations/megabase). DNA damage repair pathway gene mutations, including TP53 and BRCA2, were found. Copy number variation load, inferred from sequencing data, was variable with genomic indexes ranging from 2.2 to 74.7 (median 25.7), with higher indexes in myxoid leiomyosarcomas than myxoid smooth muscle tumors of unknown malignant potential. The absence of clear driver mutations suggests myxoid smooth muscle tumors to be genetically heterogeneous group of tumours and that other genetic (eg., undiscovered translocation) or epigenetic events drive the pathogenesis of uterine myxoid smooth muscle neoplasia.

Canadian Consensus-based and Evidence-based Guidelines for Benign Endometrial Pathology Reporting in Biopsy Material.

Standardized terminology has proven benefits in cancer reporting; in contrast, reporting of benign diagnoses in endometrial biopsy currently lacks such standardization. Unification and update on the lexicon can provide the structure and consistency needed for optimal patient care and quality assurance purposes. The Special Interest Group in Gynecologic Pathology of the Canadian Association of Pathologists-Association Canadienne des Pathologistes (CAP-ACP) embarked in an initiative to address the current need for consensus terminology in benign endometrial biopsy pathology reporting. Nine members of the Special Interest Group developed a guideline for structured diagnosis of benign endometrial pathology through critical appraisal of the available peer-reviewed literature and joint discussions. The first version of the document was circulated for feedback to a group of professionals in akin fields, the CAP-ACP Executive Committee and the CAP-ACP general membership. The final 1-page document included 17 diagnostic terms comprising the most common benign endometrial entities, as well as explanatory notes for pathologists. The proposed terminology was implemented in the practice of 5 pathologists from the group, who applied the guideline to all benign endometrial biopsies over a 2-wk period. A total of 212 benign endometrial biopsies were evaluated in this implementation step; the recommended terminology adequately covered the diagnosis in 203 cases (95.8%). A list of terminology for benign endometrial biopsy reporting, based on expert consensus and critical appraisal of the available literature, is presented. On the basis of our results of implementation at multiple centers, the proposed guideline can successfully cover the large majority of diagnostic scenarios. The document has the potential to positively impact patient care, promote quality assurance, and facilitate research initiatives aimed at improving histopathologic assessment of benign endometrium.

Nuclear ß-catenin localization and mutation of the CTNNB1 gene: a context-dependent association.

Although the majority of low-grade, early-stage endometrial cancer patients have good survival with surgery alone, patients who recur tend to do poorly. Identification of patients at high risk of recurrence who would benefit from adjuvant treatment or more extensive surgical staging would help optimize individualized care of endometrial cancer patients. CTNNB1 (encodes ß-catenin) mutations identify a subset of low-grade, early-stage endometrial cancer patients at high risk of recurrence. Mutation of CTNNB1 exon 3 is classically associated with translocation of the ß-catenin protein from the membrane to the nucleus and activation of Wnt/ß-catenin signaling. Given the clinical utility of identifying endometrial carcinomas with CTNNB1 mutation, the purpose of this study was to determine if immunohistochemistry could act as a surrogate for CTNNB1 gene sequencing. Next-generation sequencing was performed on 345 endometrial carcinomas. Immunohistochemical localization of ß-catenin was determined for 53/63 CTNNB1 exon 3 mutant tumors for which tissue was available and a subset of wild-type tumors. Nuclear localization of ß-catenin had 100% specificity in distinguishing CTNNB1 mutant from wild type, but sensitivity was lower (84.9%). Nearly half of CTNNB1 mutant cases had only 5-10% of tumor cells with ß-catenin nuclear localization. The concordance between pathologists blinded to mutation status in assessing nuclear localization was 100%. The extent of ß-catenin nuclear localization was not associated with specific CTNNB1 gene mutation, tumor grade, presence of non-endometrioid component, or specific concurrent gene mutations in the tumor. For comparison, nuclear localization of ß-catenin was more diffuse in desmoid fibromatosis, a tumor also associated with CTNNB1 mutation. Thus, nuclear localization of ß-catenin assessed by immunohistochemistry does not detect all endometrial cancers with CTNNB1 gene mutation. The extent of nuclear localization may be tumor type dependent. For endometrial cancer, immunohistochemistry could be an initial screen, with CTNNB1 sequencing employed when nuclear localization of ß-catenin is absent.

Cervical Adenocarcinoma: A Comparison of the Reproducibility of the World Health Organization 2003 and 2014 Classifications.

OBJECTIVE: The aim of the study was to compare the reproducibility of malignant glandular tumors of the uterine cervix classified per World Health Organization (WHO) 2003 and 2014. MATERIALS AND METHODS: Two pathologists reviewed 228 cases composed of adenocarcinoma in situ and 22 adenocarcinoma histotypes and selected 405 representative hematoxylin and eosin slides, which were digitally scanned. Six other pathologists (3 gynecological and 3 anatomical) independently reviewed and classified the images per both WHO classifications. One year later, they classified a random sample of 25 cases. Inter- (inter-OR) and intra-observer (intra-OR) reproducibility of the 6 pathologists and separately for gynecological compared with anatomical pathologists was tested using κ statistics. RESULTS: Both classifications were collapsed into 6 categories as benign, adenocarcinoma in situ, and mucinous, endometrioid, rare, and adenosquamous-miscellaneous carcinomas. WHO 2014 had an additional category: endocervical adenocarcinoma, usual type. Inter-observer κ values were more reliable than the intra-OR results based on 95% CIs. The average inter-OR κ values with both classifications were moderate between the 6 pathologists and between the 3 anatomical pathologists. In contrast, they were substantial between the 3 gynecological pathologists. With both classifications, the average intra-OR κ values of the 6 pathologists and both pathologist groups trended toward substantial. CONCLUSIONS: Reproducibility among 6 pathologists is unaffected by changes in the WHO 2014 classification and averages moderate between different and trends toward substantial between the same pathologist. Reproducibility between different pathologists can improve to substantial when they have expertise in gynecological pathology.

Genomic abnormalities in invasive endocervical adenocarcinoma correlate with pattern of invasion: biologic and clinical implications.

The pattern-based classification system for HPV-related endocervical adenocarcinoma, which classifies tumors based on the destructiveness of stromal invasion, is predictive of the risk of nodal metastases and adverse outcome. Previous studies have demonstrated clinically important molecular alterations in endocervical adenocarcinoma, including KRAS and PIK3CA mutations; however, correlation between the molecular landscape and pathological variables including pattern of invasion has not been thoroughly explored. In this study, 20 endocervical adenocarcinomas were classified using the pattern-based classification system and were subjected to targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) that surveys hotspot regions of 50 oncogenes and tumor suppressor genes. Single-nucleotide polymorphisms were correlated with clinical and pathologic variables including pattern of invasion. Five (25%), six (30%), and nine (45%) cases were classified as patterns A, B, and C respectively. Lymph node metastases, advanced stage at presentation and mortality from disease were exclusively seen in destructively invasive tumors (patterns B or C). Prevalent mutations in the cohort involved PIK3CA (30%), KRAS (30%), MET (15%), and RB1 (10%). Most (94%) relevant genomic alterations were present in destructively invasive tumors with PIK3CA, KRAS, and RB1 mutations seen exclusively in pattern B or C subgroups. KRAS mutations correlated with advanced stage at presentation (FIGO stage II or higher). Our findings indicate that the pattern of stromal invasion correlates with genomic abnormalities detected by next-generation sequencing, suggesting that tumors without destructive growth (pattern A) are biologically distinct from those with destructive invasion (patterns B and C), and that pattern B endocervical adenocarcinoma is more closely related to its pattern C counterpart. The pattern-based classification may be used as a triage tool when considering molecular testing for prognostic or therapeutic purposes.

Pattern-based classification of invasive endocervical adenocarcinoma, depth of invasion measurement and distinction from adenocarcinoma in situ: interobserver variation among gynecologic pathologists.

A pattern-based classification for invasive endocervical adenocarcinoma has been proposed as predictive of the risk of nodal metastases. We aimed to determine the reproducibility of such classification in the context of common diagnostic challenges: distinction between in situ and invasive adenocarcinoma and depth of invasion measurement. Nine gynecologic pathologists independently reviewed 96 cases of endocervical adenocarcinoma (two slides per case). They diagnosed each case as in situ or invasive carcinoma classifying the latter following the pattern-based classification as pattern A (non-destructive), B (focally destructive) or C (diffusely destructive). Depth of invasion, when applicable, was measured (mm). Overall, diagnostic reproducibility of pattern diagnosis was good (κ=0.65). Perfect agreement (9/9 reviewers) was seen in only 11 cases (11%), all destructively invasive (10 pattern C and 1 pattern B). In all, ≥5/9 reviewer concordance was achieved in 82/96 cases (85%). Distinction between in situ and invasive carcinoma, regardless of the pattern, showed only slight agreement (κ=0.37). Likewise, distinction restricted to in situ versus pattern A was poor (κ=0.23). Distinction between non-destructive (in situ+pattern A) and destructive (patterns B+C) carcinoma showed significantly higher agreement (κ=0.62). Estimation of depth of invasion showed excellent reproducibility (ICC=0.82). However, different measurements resulting in differing FIGO stages were common (from at least 1 reviewer in 79% cases). On the basis of interobserver agreement, the pattern-based classification is best at diagnosing destructive invasion, which carries a risk for nodal metastases. Agreement in diagnosing in situ versus invasive carcinoma, including pattern A, was poor. Given the nil risk of nodal spread in in situ and pattern A lesions, the term 'endocervical adenocarcinoma with non-destructive growth' can be considered when the distinction is difficult, after excluding destructive invasion. Depth of invasion measurement was highly reproducible among pathologists; thus, the pattern-based approach can complement, but should not replace, the depth of invasion metric.