Search

Vulvar squamous cell carcinomas and their precursors are currently classified by the World Health Organization based on their association with high-risk human papillomavirus (HPV). HPV independent lesions often harbor driver alterations in TP53, usually seen in the setting of chronic vulvar inflammation. However, a group of pre-invasive vulvar squamous lesions is independent from both HPV and mutant TP53. The lesions described within this category feature marked acanthosis, verruciform growth and altered squamous maturation, and over the last two decades several studies have added to their characterization. They have a documented association with verrucous carcinoma and conventional squamous cell carcinoma of the vulva, suggesting a precursor role. They also harbor recurrent genomic alterations in several oncogenes, mainly PIK3CAand HRAS, indicating a neoplastic nature. In this review, we provide a historical perspective and a comprehensive description of these lesions. We also offer an appraisal of the terminology used over the years, going from Vulvar Acanthosis with Altered Differentiationand Verruciform Lichen Simplex Chronicusto Differentiated Exophytic Vulvar Intraepithelial Lesionand Vulvar Aberrant Maturation, the latter term having been recently proposed by the International Society for the Study of Vulvovaginal Diseases. In line with the recognition of these lesions by the 2020 World Health Organization Classification of Tumours as a neoplastic precursor, we herein propose the term HPV-independent, p53-wild-type verruciform acanthotic Vulvar Intraepithelial Neoplasia(HPVi(p53wt) vaVIN), which better conveys not only the pathology but also the neoplastic nature and the biologic risk inherent to these uncommon and challenging lesions. We outline strict morphologic and immunohistochemical criteria for its diagnosis and distinction from mimickers. Immunohistochemistry for p16 and p53 should be performed routinely in the diagnostic work-up of these lesions, and the morphologic alternative term vaVIN should be reserved for instances in which p16/HPV/p53 status is unknown. We also discuss management considerations and the need to further explore precursors within and beyond the spectrum of verruciform acanthotic vulvar intraepithelial neoplasia.

Vulvar squamous cell carcinomas and their precursors are currently classified by the World Health Organization based on their association with high-risk human papillomavirus (HPV). HPV independent lesions often harbor driver alterations in TP53, usually seen in the setting of chronic vulvar inflammation. However, a group of pre-invasive vulvar squamous lesions is independent from both HPV and mutant TP53. The lesions described within this category feature marked acanthosis, verruciform growth and altered squamous maturation, and over the last two decades several studies have added to their characterization. They have a documented association with verrucous carcinoma and conventional squamous cell carcinoma of the vulva, suggesting a precursor role. They also harbor recurrent genomic alterations in several oncogenes, mainly PIK3CAand HRAS, indicating a neoplastic nature. In this review, we provide a historical perspective and a comprehensive description of these lesions. We also offer an appraisal of the terminology used over the years, going from Vulvar Acanthosis with Altered Differentiationand Verruciform Lichen Simplex Chronicusto Differentiated Exophytic Vulvar Intraepithelial Lesionand Vulvar Aberrant Maturation, the latter term having been recently proposed by the International Society for the Study of Vulvovaginal Diseases. In line with the recognition of these lesions by the 2020 World Health Organization Classification of Tumours as a neoplastic precursor, we herein propose the term HPV-independent, p53-wild-type verruciform acanthotic Vulvar Intraepithelial Neoplasia(HPVi(p53wt) vaVIN), which better conveys not only the pathology but also the neoplastic nature and the biologic risk inherent to these uncommon and challenging lesions. We outline strict morphologic and immunohistochemical criteria for its diagnosis and distinction from mimickers. Immunohistochemistry for p16 and p53 should be performed routinely in the diagnostic work-up of these lesions, and the morphologic alternative term vaVIN should be reserved for instances in which p16/HPV/p53 status is unknown. We also discuss management considerations and the need to further explore precursors within and beyond the spectrum of verruciform acanthotic vulvar intraepithelial neoplasia.

Uterine leiomyosarcoma is the most common uterine mesenchymal malignancy. The majority present at stage I, and clinical outcomes vary widely. However, no widely accepted risk stratification system for stage I uterine leiomyosarcoma is currently available. We studied 17 routinely evaluated clinicopathologic parameters in 203 stage I uterine leiomyosarcoma from three institutions to generate a novel risk stratification model for these tumors. Mitoses >25 per 2.4 mm2(10 high-power fields), atypical mitoses, coagulative necrosis, lymphovascular invasion, and serosal abutment were significantly associated with disease-free and disease-specific survival in univariate and multivariate analyses. These prognostic parameters were each scored as binary (“yes” or “no”) variables and fitted to a single optimized algebraic risk model:

Uterine leiomyosarcoma is the most common uterine mesenchymal malignancy. The majority present at stage I, and clinical outcomes vary widely. However, no widely accepted risk stratification system for stage I uterine leiomyosarcoma is currently available. We studied 17 routinely evaluated clinicopathologic parameters in 203 stage I uterine leiomyosarcoma from three institutions to generate a novel risk stratification model for these tumors. Mitoses >25 per 2.4 mm2(10 high-power fields), atypical mitoses, coagulative necrosis, lymphovascular invasion, and serosal abutment were significantly associated with disease-free and disease-specific survival in univariate and multivariate analyses. These prognostic parameters were each scored as binary (“yes” or “no”) variables and fitted to a single optimized algebraic risk model:

A subset of clinically benign uterine polyps shows atypical morphologic features worrisome for, but not diagnostic of, Mullerian adenosarcoma. We report clinicopathologic data for 63 polyps from 58 women with atypical morphologic features including abnormal architecture, abnormal periglandular stroma, stromal atypia, and mitoses >2 per 10 hpf. Four (11%) of 36 women with follow-up tissue sampling had residual/recurrent atypical polyp. Twelve (27%) of 44 women underwent hysterectomy subsequent to a diagnosis of atypical polyp. No patient developed adenosarcoma over median follow-up of 150 months. Twenty-one primary atypical polyps underwent molecular profiling. Five (24%) harbored chr 12q13-15 gain or amplification, 9/20 (45%) harbored chr 6q25.1 gain, and 7/21 (33%) had no significant copy number alterations. Gains of chr 1q, chr 8p12, and chr 10q11.21-23, amplifications of chr 12q24.12-13, chr 15p24.1-26.1, and chr 18q21.33, and loss of chr 7 and chr 11q21 were each seen in a single polyp. Mean tumor mutational burden was 3.1 (range, 0.76–8.365) mutations/Mb. Pathogenic point mutations were identified in 12/20 (60%) primary atypical polyps. We propose the term “atypical uterine polyps” for these lesions, which show biologic overlap with early Mullerian adenosarcoma but lack molecular alterations characteristic of clinically aggressive adenosarcoma and appear to follow a benign clinical course. Conservative management with close clinical follow-up and repeat sampling can be considered for these lesions, when clinically appropriate.

A subset of clinically benign uterine polyps shows atypical morphologic features worrisome for, but not diagnostic of, Mullerian adenosarcoma. We report clinicopathologic data for 63 polyps from 58 women with atypical morphologic features including abnormal architecture, abnormal periglandular stroma, stromal atypia, and mitoses >2 per 10 hpf. Four (11%) of 36 women with follow-up tissue sampling had residual/recurrent atypical polyp. Twelve (27%) of 44 women underwent hysterectomy subsequent to a diagnosis of atypical polyp. No patient developed adenosarcoma over median follow-up of 150 months. Twenty-one primary atypical polyps underwent molecular profiling. Five (24%) harbored chr 12q13-15 gain or amplification, 9/20 (45%) harbored chr 6q25.1 gain, and 7/21 (33%) had no significant copy number alterations. Gains of chr 1q, chr 8p12, and chr 10q11.21-23, amplifications of chr 12q24.12-13, chr 15p24.1-26.1, and chr 18q21.33, and loss of chr 7 and chr 11q21 were each seen in a single polyp. Mean tumor mutational burden was 3.1 (range, 0.76–8.365) mutations/Mb. Pathogenic point mutations were identified in 12/20 (60%) primary atypical polyps. We propose the term “atypical uterine polyps” for these lesions, which show biologic overlap with early Mullerian adenosarcoma but lack molecular alterations characteristic of clinically aggressive adenosarcoma and appear to follow a benign clinical course. Conservative management with close clinical follow-up and repeat sampling can be considered for these lesions, when clinically appropriate.

Desmoplastic small round cell tumor (DSRCT) is a high-grade round cell sarcoma that typically arises in the abdominopelvic cavity of young males, co-expresses keratins and desmin, and carries a pathognomonic EWSR1-WT1gene fusion. The EWSR1-WT1gene fusion is generally considered specific for DSRCT, although there are two reports of this fusion in tumors otherwise lacking features of DSRCT. We report three female genital tract tumors with EWSR1-WT1fusions but showing morphologic and immunohistochemical features incompatible with DSRCT. The tumors occurred in the uterine cervix, uterine corpus/ovaries, and vagina, respectively, of 46, 30, and 20-year-old women. Two tumors consisted of a sheet-like to fascicular proliferation of relatively uniform spindled to occasionally more epithelioid cells arrayed about thick-walled, hyalinized, and capillary-sized vessels, with distinctive areas of pseudovascular change, and absence of desmoplastic stroma. The third tumor resembled a monomorphic spindle cell sarcoma with necrosis. All had diffuse desmin and variable but more limited keratin expression, two of three expressed smooth muscle actin, and all were negative for h-caldesmon, CD10, estrogen receptor, myogenin, N-terminus WT-1, and S100 protein. One patient received neoadjuvant chemotherapy and radiation therapy followed by resection and is disease-free 42 months after diagnosis. Another patient was managed by resection only and is disease-free 9 months after initial diagnosis. The remaining patient recently underwent resection of multifocal pelvic disease. Comprehensive differential gene expression analysis on two tumors compared to two classic DSRCTs with known EWSR1-WT1fusions resulted in 1726 genes that were differentially expressed (log2 fold change >2 or < −2) and statistically significant (FDR < 5%). In combination with previous reports, our findings suggest pleiotropy of the EWSR1-WT1fusion is possible and not limited to DSRCT. Subsets of non-DSRCT EWSR1-WT1positive tumors may represent discrete entities, but further study is necessary.

Desmoplastic small round cell tumor (DSRCT) is a high-grade round cell sarcoma that typically arises in the abdominopelvic cavity of young males, co-expresses keratins and desmin, and carries a pathognomonic EWSR1-WT1gene fusion. The EWSR1-WT1gene fusion is generally considered specific for DSRCT, although there are two reports of this fusion in tumors otherwise lacking features of DSRCT. We report three female genital tract tumors with EWSR1-WT1fusions but showing morphologic and immunohistochemical features incompatible with DSRCT. The tumors occurred in the uterine cervix, uterine corpus/ovaries, and vagina, respectively, of 46, 30, and 20-year-old women. Two tumors consisted of a sheet-like to fascicular proliferation of relatively uniform spindled to occasionally more epithelioid cells arrayed about thick-walled, hyalinized, and capillary-sized vessels, with distinctive areas of pseudovascular change, and absence of desmoplastic stroma. The third tumor resembled a monomorphic spindle cell sarcoma with necrosis. All had diffuse desmin and variable but more limited keratin expression, two of three expressed smooth muscle actin, and all were negative for h-caldesmon, CD10, estrogen receptor, myogenin, N-terminus WT-1, and S100 protein. One patient received neoadjuvant chemotherapy and radiation therapy followed by resection and is disease-free 42 months after diagnosis. Another patient was managed by resection only and is disease-free 9 months after initial diagnosis. The remaining patient recently underwent resection of multifocal pelvic disease. Comprehensive differential gene expression analysis on two tumors compared to two classic DSRCTs with known EWSR1-WT1fusions resulted in 1726 genes that were differentially expressed (log2 fold change >2 or < −2) and statistically significant (FDR < 5%). In combination with previous reports, our findings suggest pleiotropy of the EWSR1-WT1fusion is possible and not limited to DSRCT. Subsets of non-DSRCT EWSR1-WT1positive tumors may represent discrete entities, but further study is necessary.

Uterine serous carcinoma is an aggressive subtype of endometrial cancer that accounts for fewer than 10% of endometrial carcinomas but is responsible for about half of deaths. A subset of cases has HER2 overexpression secondary to ERBB2gene amplification, and these patients may benefit from anti-HER2 therapies, such as trastuzumab. HER2 protein overexpression is currently assessed by immunohistochemistry (IHC) and ERBB2gene amplification by fluorescence in situ hybridization (FISH). Targeted next-generation sequencing (NGS) is increasingly used to routinely identify predictive and prognostic molecular abnormalities in endometrial carcinoma. To investigate the ability of a targeted NGS panel to detect ERBB2amplification, we identified cases of uterine serous carcinoma (n?=?93) and compared HER2 expression by IHC and copy number assessed by FISH with copy number status assessed by NGS. ERBB2copy number status using a combination of IHC and FISH was interpreted using the 2018 ASCO/CAP guidelines for breast carcinoma. ERBB2amplification by NGS was determined by the relative number of reads mapping to ERBB2in tumor DNA compared to control nonneoplastic DNA. Cases with copy number =6 were considered amplified and copy number <6 were non-amplified. By IHC, 70 specimens were classified as negative (0 or 1+), 19 were classified as equivocal (2+), and 4 were classified as positive (3+). Using combined IHC/FISH, ERBB2amplification was observed in 8 of 93 cases (9%). NGS identified the same 8 cases with copy number =6; all 85 others had copy number <6. In this series, NGS had 100% concordance with combined IHC/FISH in identifying ERBB2amplification. NGS is highly accurate in detecting ERBB2amplification in uterine serous carcinoma and provides an alternative to measurement by IHC and FISH.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

Uterine serous carcinoma is an aggressive subtype of endometrial cancer that accounts for fewer than 10% of endometrial carcinomas but is responsible for about half of deaths. A subset of cases has HER2 overexpression secondary to ERBB2gene amplification, and these patients may benefit from anti-HER2 therapies, such as trastuzumab. HER2 protein overexpression is currently assessed by immunohistochemistry (IHC) and ERBB2gene amplification by fluorescence in situ hybridization (FISH). Targeted next-generation sequencing (NGS) is increasingly used to routinely identify predictive and prognostic molecular abnormalities in endometrial carcinoma. To investigate the ability of a targeted NGS panel to detect ERBB2amplification, we identified cases of uterine serous carcinoma (n= 93) and compared HER2 expression by IHC and copy number assessed by FISH with copy number status assessed by NGS. ERBB2copy number status using a combination of IHC and FISH was interpreted using the 2018 ASCO/CAP guidelines for breast carcinoma. ERBB2amplification by NGS was determined by the relative number of reads mapping to ERBB2in tumor DNA compared to control nonneoplastic DNA. Cases with copy number ≥6 were considered amplified and copy number <6 were non-amplified. By IHC, 70 specimens were classified as negative (0 or 1+), 19 were classified as equivocal (2+), and 4 were classified as positive (3+). Using combined IHC/FISH, ERBB2amplification was observed in 8 of 93 cases (9%). NGS identified the same 8 cases with copy number ≥6; all 85 others had copy number <6. In this series, NGS had 100% concordance with combined IHC/FISH in identifying ERBB2amplification. NGS is highly accurate in detecting ERBB2amplification in uterine serous carcinoma and provides an alternative to measurement by IHC and FISH.

Mucinous ovarian tumors rarely harbor mural nodules, which have historically been classified as sarcoma-like, anaplastic carcinomatous, or sarcomatous on the basis of predominant morphologic features. The molecular relationship between mural nodules and associated mucinous ovarian tumors remains poorly characterized, as does the molecular pathogenesis of these mural nodules. Thus, we analyzed the morphological, immunohistochemical, and genetic features of 13 mucinous ovarian tumors and associated mural nodule(s). Three harbored sarcoma-like mural nodules and ten contained anaplastic carcinomatous nodules, including 1 tumor with spatially discrete anaplastic carcinomatous and sarcomatous nodules. Twelve of 13 cases showed genetic evidence of clonality between the mural nodule(s) and associated mucinous ovarian tumor, including all three tumors with sarcoma-like morphology. Mural nodules were genetically identical in the five cases in which there were multiple discrete mural nodules that were sequenced separately. MTAP and p53 immunohistochemistry confirmed the distribution of neoplastic cells in a subset of sarcoma-like and anaplastic carcinomatous nodules. No single recurrent genetic alteration was associated with mural nodule development. No recurrent genetic differences were identified between mural nodules with sarcoma-like, anaplastic carcinomatous, and sarcomatous morphology. Of 11 patients with clinical follow-up, three died of disease 3, 8, and 9 months after diagnosis, but no recurrent genetic events were associated with poor outcome. These molecular data suggest that sarcoma-like, anaplastic carcinomatous, and sarcomatous nodules represent a morphologic spectrum of clonal neoplasms arising in mucinous ovarian tumors rather than three discrete biological entities.

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-BCORfusion and BCORinternal 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 BCORinternal tandem duplications; however, four tumors contained BCORinternal tandem duplications of unknown significance (mostly intronic). Mutational burden was low (median 3.8 mutations/megabase). DNA damage repair pathway gene mutations, including TP53and 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.

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-BCORfusion and BCORinternal 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 BCORinternal tandem duplications; however, four tumors contained BCORinternal tandem duplications of unknown significance (mostly intronic). Mutational burden was low (median 3.8 mutations/megabase). DNA damage repair pathway gene mutations, including TP53and 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.

Small cell carcinoma of the ovary, hypercalcemic type is a rare, aggressive malignancy which usually occurs in young women and is characterized by mutations in SMARCA4, with few other alterations. We recently encountered uterine tumors with morphologic, immunohistochemical, and genetic similarities to small cell carcinoma of the ovary, hypercalcemic type. Herein we report the clinicopathologic and molecular features (using a targeted massively parallel sequencing [MPS] assay) of these tumors. The cases were diagnosed on cervical and endometrial biopsies (n= 2, 34, and 29 years) or hysterectomy and bilateral salpingo-oophorectomy (n= 3, 25, 33, and 58 years). The tumors were composed of sheets of large atypical epithelioid cells with prominent rhabdoid morphology, indistinguishable from the “large cell” variant of small cell carcinoma of the ovary, hypercalcemic type. In three cases, the ovaries were pathologically examined to exclude a primary ovarian malignancy. Immunohistochemically, four of four cases showed SMARCA4 loss, and were negative or only focally positive for keratins, EMA, and claudin-4. One of three cases was positive for WT-1. Targeted MPS was successfully performed on 4 of 5 tumors, and showed recurrent mutations in SMARCA4, with few other alterations. Of the cases diagnosed on hysterectomy, all had extensive lymphovascular invasion, extra-uterine spread, and marked infiltrative growth. These tumors were uniformly aggressive; all patients died of disease (median survival 7 months, range 1–43 months). We propose this entity be called “SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus)”, a term which describes both the tumor's underlying molecular abnormality and its morphology. Its unique clinicopathologic and molecular features differentiate it from other related malignancies, including undifferentiated endometrial carcinoma, small cell carcinoma of the ovary (hypercalcemic type), and epithelioid sarcoma. We review and discuss previously reported “rhabdoid tumors of the uterus;” while they are a heterogenous group of tumors, some of them are likely examples of this entity. Correctly identifying cases of SMARCA4-deficient uterine sarcoma from histologic mimics is important as it may have prognostic, predictive, and germline implications.

Small cell carcinoma of the ovary, hypercalcemic type is a rare, aggressive malignancy which usually occurs in young women and is characterized by mutations in SMARCA4, with few other alterations. We recently encountered uterine tumors with morphologic, immunohistochemical, and genetic similarities to small cell carcinoma of the ovary, hypercalcemic type. Herein we report the clinicopathologic and molecular features (using a targeted massively parallel sequencing [MPS] assay) of these tumors. The cases were diagnosed on cervical and endometrial biopsies (n= 2, 34, and 29 years) or hysterectomy and bilateral salpingo-oophorectomy (n= 3, 25, 33, and 58 years). The tumors were composed of sheets of large atypical epithelioid cells with prominent rhabdoid morphology, indistinguishable from the “large cell” variant of small cell carcinoma of the ovary, hypercalcemic type. In three cases, the ovaries were pathologically examined to exclude a primary ovarian malignancy. Immunohistochemically, four of four cases showed SMARCA4 loss, and were negative or only focally positive for keratins, EMA, and claudin-4. One of three cases was positive for WT-1. Targeted MPS was successfully performed on 4 of 5 tumors, and showed recurrent mutations in SMARCA4, with few other alterations. Of the cases diagnosed on hysterectomy, all had extensive lymphovascular invasion, extra-uterine spread, and marked infiltrative growth. These tumors were uniformly aggressive; all patients died of disease (median survival 7 months, range 1–43 months). We propose this entity be called “SMARCA4-deficient undifferentiated uterine sarcoma (malignant rhabdoid tumor of the uterus)”, a term which describes both the tumor’s underlying molecular abnormality and its morphology. Its unique clinicopathologic and molecular features differentiate it from other related malignancies, including undifferentiated endometrial carcinoma, small cell carcinoma of the ovary (hypercalcemic type), and epithelioid sarcoma. We review and discuss previously reported “rhabdoid tumors of the uterus;” while they are a heterogenous group of tumors, some of them are likely examples of this entity. Correctly identifying cases of SMARCA4-deficient uterine sarcoma from histologic mimics is important as it may have prognostic, predictive, and germline implications.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CAand ARID2, respectively. No TP53mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53and CDKN2Amutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CAmutations followed by a keratinizing carcinoma with mutations in both PIK3CAand TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CAmutant/TP53wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CAmutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term ‘differentiated exophytic vulvar intraepithelial lesion’ for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CAmutations to address this question.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term ‘differentiated exophytic vulvar intraepithelial lesion’ for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CA mutations to address this question.

Extrauterine high-grade serous carcinomas can exhibit various histologic patterns including (1) classic architecture that is papillary, micropapillary and infiltrative and (2) solid, endometrioid, and transitional (ie, SET) patterns. Although the SET pattern has been associated with germline BRCA mutations, potential molecular underpinnings have not been fully investigated. DNA was isolated from 174 carcinomas of the fallopian tube, ovary, or peritoneum. Targeted next-generation sequencing was performed and single-nucleotide and copy number variants were correlated with morphologic subtype. Overall, 79% of tumors were classified as high-grade serous carcinoma (n=138), and the most common mutations in high-grade serous carcinomas were TP53 (94%), BRCA1 (25%), BRCA2 (11%), and ATM (7%). Among chemotherapy-naive high-grade serous carcinomas, 40 cases exhibited classic morphology and 40 cases had non-classic morphology (SET or ambiguous features). Mutations in homologous recombination pathways were seen across all tumor histotypes. High-grade serous carcinomas with homologous recombination mutations were six times more likely to be associated with non-classic histology (P=0.002) and were significantly more likely to be platinum sensitive and have improved progression-free survival (PFS) (P=0.007 and P=0.004, respectively). In a multivariate analysis adjusted for age, homologous recombination mutation status and increased copy number variants were independently associated with improved PFS (P=0.008 and P=0.005, respectively). These findings underscore the potential significance of variant morphologic patterns and comprehensive genomic analysis in high-grade serous carcinomas with potential implications for pathogenesis, as well as response to targeted therapies.

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.

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 situand 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 situor 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 situand invasive carcinoma, regardless of the pattern, showed only slight agreement (κ=0.37). Likewise, distinction restricted to in situ versuspattern 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 versusinvasive carcinoma, including pattern A, was poor. Given the nil risk of nodal spread in in situand 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.

Extrauterine high-grade serous carcinomas can exhibit various histologic patterns including (1) classic architecture that is papillary, micropapillary and infiltrative and (2) solid, endometrioid, and transitional (ie, SET) patterns. Although the SET pattern has been associated with germline BRCAmutations, potential molecular underpinnings have not been fully investigated. DNA was isolated from 174 carcinomas of the fallopian tube, ovary, or peritoneum. Targeted next-generation sequencing was performed and single-nucleotide and copy number variants were correlated with morphologic subtype. Overall, 79% of tumors were classified as high-grade serous carcinoma (n=138), and the most common mutations in high-grade serous carcinomas were TP53(94%), BRCA1(25%), BRCA2(11%), and ATM(7%). Among chemotherapy-naive high-grade serous carcinomas, 40 cases exhibited classic morphology and 40 cases had non-classic morphology (SET or ambiguous features). Mutations in homologous recombination pathways were seen across all tumor histotypes. High-grade serous carcinomas with homologous recombination mutations were six times more likely to be associated with non-classic histology (P=0.002) and were significantly more likely to be platinum sensitive and have improved progression-free survival (PFS) (P=0.007 and P=0.004, respectively). In a multivariate analysis adjusted for age, homologous recombination mutation status and increased copy number variants were independently associated with improved PFS (P=0.008 and P=0.005, respectively). These findings underscore the potential significance of variant morphologic patterns and comprehensive genomic analysis in high-grade serous carcinomas with potential implications for pathogenesis, as well as response to targeted therapies.

Intravenous leiomyomatosis is an unusual smooth muscle neoplasm with quasi-malignant intravascular growth but a histologically banal appearance. Herein, we report expression and molecular cytogenetic analyses of a series of 12 intravenous leiomyomatosis cases to better understand the pathogenesis of intravenous leiomyomatosis. All cases were analyzed for the expression of HMGA2, MDM2, and CDK4 proteins by immunohistochemistry based on our previous finding of der(14)t(12;14)(q14.3;q24) in intravenous leiomyomatosis. Seven of 12 (58%) intravenous leiomyomatosis cases expressed HMGA2, and none expressed MDM2 or CDK4. Colocalization of hybridization signals for probes from the HMGA2locus (12q14.3) and from 14q24 by interphase fluorescence in situhybridization (FISH) was detected in a mean of 89.2% of nuclei in HMGA2-positive cases by immunohistochemistry, but in only 12.4% of nuclei in negative cases, indicating an association of HMGA2 expression and this chromosomal rearrangement (P=8.24 × 10−10). Four HMGA2-positive cases had greater than two HMGA2hybridization signals per cell. No cases showed loss of a hybridization signal by interphase FISH for the frequently deleted region of 7q22 in uterine leiomyomata. One intravenous leiomyomatosis case analyzed by array comparative genomic hybridization revealed complex copy number variations. Finally, expression profiling was performed on three intravenous leiomyomatosis cases. Interestingly, hierarchical cluster analysis of the expression profiles revealed segregation of the intravenous leiomyomatosis cases with leiomyosarcoma rather than with myometrium, uterine leiomyoma of the usual histological type, or plexiform leiomyoma. These findings suggest that intravenous leiomyomatosis cases share some molecular cytogenetic characteristics with uterine leiomyoma, and expression profiles similar to that of leiomyosarcoma cases, further supporting their intermediate, quasi-malignant behavior.

Intravenous leiomyomatosis is an unusual smooth muscle neoplasm with quasi-malignant intravascular growth but a histologically banal appearance. Herein, we report expression and molecular cytogenetic analyses of a series of 12 intravenous leiomyomatosis cases to better understand the pathogenesis of intravenous leiomyomatosis. All cases were analyzed for the expression of HMGA2, MDM2, and CDK4 proteins by immunohistochemistry based on our previous finding of der(14)t(12;14)(q14.3;q24) in intravenous leiomyomatosis. Seven of 12 (58%) intravenous leiomyomatosis cases expressed HMGA2, and none expressed MDM2 or CDK4. Colocalization of hybridization signals for probes from the HMGA2 locus (12q14.3) and from 14q24 by interphase fluorescence in situ hybridization (FISH) was detected in a mean of 89.2% of nuclei in HMGA2-positive cases by immunohistochemistry, but in only 12.4% of nuclei in negative cases, indicating an association of HMGA2 expression and this chromosomal rearrangement (P=8.24 × 10−10). Four HMGA2-positive cases had greater than two HMGA2 hybridization signals per cell. No cases showed loss of a hybridization signal by interphase FISH for the frequently deleted region of 7q22 in uterine leiomyomata. One intravenous leiomyomatosis case analyzed by array comparative genomic hybridization revealed complex copy number variations. Finally, expression profiling was performed on three intravenous leiomyomatosis cases. Interestingly, hierarchical cluster analysis of the expression profiles revealed segregation of the intravenous leiomyomatosis cases with leiomyosarcoma rather than with myometrium, uterine leiomyoma of the usual histological type, or plexiform leiomyoma. These findings suggest that intravenous leiomyomatosis cases share some molecular cytogenetic characteristics with uterine leiomyoma, and expression profiles similar to that of leiomyosarcoma cases, further supporting their intermediate, quasi-malignant behavior.

Lipoblastoma-like tumor (LLT) is a rare adipocytic neoplasm with a predilection for the vulva. Since 2002, <30 cases have been reported, characterizing it as an indolent tumor that may sometimes recur locally. Diagnosis can be challenging due to its rarity and morphologic overlap with other adipocytic tumors. Thus far, there are no specific molecular or immunohistochemical features to aid in the diagnosis of LLT. Recent case reports have described LLT arising at other sites, including the spermatic cord and gluteal region, suggesting wider anatomical distribution. We present a large series of LLT to further characterize its clinicopathologic and molecular features. Twenty-eight cases of LLT were retrieved from departmental and consult archives (including 8 from a prior series). The cohort comprised 28 patients (8 males, 20 females) with a median age of 28 years (range: 1-80 years). There were 17 primary LLT of the vulva. Other anatomical sites included the scrotum (n = 3), spermatic cord (n = 2), inguinal region (n = 2), limbs (n = 2), pelvis (n = 1), and retroperitoneum (n = 1). Median tumor size was 6.0 cm (range: 1.8-30.0 cm). The tumors had a lobulated architecture and were typically composed of adipocytes, lipoblasts, and spindle cells in a myxoid stroma with prominent thin-walled vessels. Using immunohistochemistry, a subset showed loss of Rb expression (12/23 of samples). Follow-up in 15 patients (median: 56 months) revealed 8 patients with local recurrence and 1 patient with metastases to the lung/pleura and breasts. Targeted DNA sequencing revealed a simple genomic profile with limited copy number alterations and low-mutational burden. No alterations in RB1were identified. The metastatic LLT showed concurrent pathogenic PIK3CAand MTORactivating mutations, both in the primary and in the lung/pleural metastasis; the latter also harbored TERTpromoter mutation. One tumor had a pathogenic TSC1mutation, and one tumor showed 2-copy deletion of CDKN2A, CDKN2B,and MTAPgenes. No biologically significant variants were identified in 8 tumors. No gene fusions were identified by RNA sequencing in 4 tumors successfully sequenced. This study expands the clinicopathologic spectrum of LLT, highlighting its wider anatomical distribution and potential for occasional metastasis. Molecularly, we identified activating mutations in the PI3K-MTOR signaling pathway in 2 tumors, which may contribute to exceptional aggressive behavior.

RAD51B-rearranged sarcomas are rare neoplasms that exhibit a heterogeneous morphology. To date, 6 cases have been reported all involving the uterus, including 4 perivascular epithelioid cell tumors (PEComas) and 2 leiomyosarcomas (LMS). In this study, we describe the morphologic, immunohistochemical, and molecular features of 8 additional sarcomas with RAD51B-rearrangement, including the first extrauterine example. All patients were females with a median age of 57 at presentation. Seven tumors originated in the uterus and one in the lower extremity soft tissue, with a median tumor size of 12 cm. Histologically, 4 tumors showed predominantly spindle cell morphology with eosinophilic fibrillary cytoplasm, with or without nuclear pleomorphism, while 2 pleomorphic epithelioid cells, featuring clear to eosinophilic, granular cytoplasm. Two neoplasms exhibited undifferentiated cytomorphology, including one with uniform small blue round cells. All tumors showed high grade cytologic atypia, high mitotic activity (median, 30/10 HPFs), while coagulative necrosis was seen in 6 cases and lymphovascular invasion in 2. By immunohistochemistry, 2 showed myoid and melanocytic markers in keeping with PEComa, while 4 cases were only positive for smooth muscle markers consistent with LMS (including 3 myxoid). The remaining 2 cases had a non-specific immunoprofile. Five cases tested by targeted RNA sequencing (Archer FusionPlex, Illumina TruSight) showed different fusion partners (HMGA2, PDDC1, CEP170). RAD51Brearrangements were identified by FISH in the remaining 3 cases. Targeted DNA sequencing in 2 cases were negative for TSCgene alterations. Clinical outcome available in 5 patients (median follow-up 19 months) revealed 3 local recurrences, 2 lung metastases, and 4 died of disease. Our results expand the spectrum of sarcomas with RAD51B-fusions, demonstrating variable clinical presentations, morphologic spectrum, and fusion partners. These tumors have a predilection for a uterine location, with either LMS, PEComa or undifferentiated phenotypes, and are associated with an aggressive clinical course.

Gestational endometrium can demonstrate a spectrum of atypical but benign changes. One such lesion is Localized Endometrial Proliferation of Pregnancy (LEPP), first described in a series of 11 cases. To understand its biologic and clinical importance, we explore the pathologic, immunophenotypic and molecular features of this entity. Nine cases of LEPP identified in a 15-year period were retrieved from departmental archives and reviewed. Immunohistochemistry and next generation sequencing using a comprehensive 446-gene panel were performed when material was available. Eight cases were identified in curettage specimens performed after first-trimester pregnancy loss, and one in the basal plate of a mature placenta. Mean patient age was 35 (range 27-41) years. Mean lesion size was 6.3 (range 2-12) mm. Architectural patterns, often coexisting in the same case, included cribriform (n=7), solid (n=5), villoglandular (n=2), papillary (n=2) and micropapillary (n=1). Cytologic atypia was mild in 7 cases and moderate in two. Mitotic activity was low (up to 3 per 2.4 mm2). All lesions were associated with neutrophils. Background Arias-Stella phenomenon was present in 4 cases. Immunohistochemistry was performed in 7 LEPP, all of which demonstrated wildtype p53, retained MSH6 and PMS2, membranous beta-catenin, and positive ER (mean 71%) and PR (mean 74%). All were negative for p40 except one case (focal weak positivity). PTEN was markedly reduced in background secretory glands in all cases; in 5/7, LEPP foci showed complete absence of PTEN expression. PIK3CApathogenic variants were identified in 4/4 cases sequenced; 3/4 had inactivating PTENmutations. Follow-up, available in 8 patients (mean length = 51 months, range 7-161), was conservative with observation only, and showed no persistence or adverse outcomes. LEPP is characterized by intraglandular cribriform/solid architecture, positive ER/PR, PTEN loss, and PIK3CA&PTENmutations. While our findings indicate that LEPP is of neoplastic nature, for now we advise against diagnosing LEPP as endometrial carcinoma or hyperplasia since LEPP has a particular clinico-pathologic context (concurrent gestation), distinct morphology (purely intraepithelial complex growth) and indolent outcome. Thus, it should be distinguished from endometrial intraepithelial neoplasia and carcinoma for which therapeutic interventions are indicated.

Mullerian clear cell carcinomas (CCC) are often aggressive, chemoresistant tumors. The prognostic significance of molecular subclassification of endometrioid carcinomas is well-established. However, less is known about the molecular landscape of CCC. The aim of this study was to better characterize the genetic landscape of a large cohort of CCC and correlate these findings with clinicopathologic features. CCC of the ovary (n=72), endometrium (n=24), and peritoneal/abdominal wall (n=5) were retrospectively identified. Tumors had undergone tumor-only targeted sequencing using a hybrid capture next-generation sequencing panel. Median tumor mutational burden (TMB) was 6.8 mutations/megabase (range 1.3-185, 21% were ≥10 mut/Mb). The most frequently mutated genes were ARID1A(48%), PIK3CA(45%), TP53(23%), and PTEN(10%). ERBB2amplification occurred in 4%. When classified according to the TCGA/ProMisE endometrial carcinoma molecular subgroups, 3 (3%) were POLE ultramutated, 5 (5%) were microsatellite unstable (MSI-H), 20 (20%) were TP53-mutant subgroup and 73 (72%) were no specific molecular profile (NSMP). Immunohistochemical expression of estrogen receptor, progesterone receptor, and programmed death-ligand 1 (PD-L1) were not associated with molecular subgroup. POLE and MSI-H tumors were characterized by an excellent prognosis, and the TP53-mutant subgroup had a worse disease-free survival than NSMP. NSMP tumors could be further substratified as high-risk NSMP if they lacked PIK3CA, PIK3R1, and ARID1Amutations, and/or harbored a TERTpromoter mutation. The overall TCGA and NSMP-specific stratifications were prognostic for both the entire cohort and the subset of stage 1 ovarian tumors. On multivariable analysis, stage, lymphovascular invasion, and TMB were prognostic for disease free survival, while advanced stage and TP53-mutant subgroup – but not a TP53mutation in isolation – were negative prognostic factors for overall survival. These data suggest that routine molecular profiling of Mullerian CCC may be warranted for both prognosis and identification of potential targeted treatment such as immunotherapy and anti-HER2 agents.

Uterine mesenchymal tumors continue to be a challenge to diagnose due to their non-specific clinical presentation, often non-distinctive gross appearance, varied (and many times overlapping) morphologic appearance, and unsuspected pitfalls in immunohistochemical expression. This review will focus on endometrial stromal tumors and those features that help in their distinction. In particular, a practical approach to the diagnosis of endometrial stromal neoplasia will be covered including recognition as a stromal process in a biopsy/curettage and distinction from a highly cellular leiomyoma. In addition, distinction of a stromal nodule from a low-grade endometrial stromal sarcoma (LGESS) and stromal sarcoma with limited infiltration in a hysterectomy specimen will be covered. The salient features that help distinguish a LGESS from a uterine tumor resembling ovarian sex-cord tumor as well as high-grade endometrial stromal sarcoma, the latter a tumor recently reintroduced in the WHO classification will also be discussed. Finally, a practical approach to the diagnosis of undifferentiated uterine sarcoma (UUS) will be presented.

Uterine mesenchymal tumors continue to be a challenge to diagnose due to their non-specific clinical presentation, often non-distinctive gross appearance, varied (and many times overlapping) morphologic appearance, and unsuspected pitfalls in immunohistochemical expression. This review will focus on endometrial stromal tumors and those features that help in their distinction. In particular, a practical approach to the diagnosis of endometrial stromal neoplasia will be covered including recognition as a stromal process in a biopsy/curettage and distinction from a highly cellular leiomyoma. In addition, distinction of a stromal nodule from a low-grade endometrial stromal sarcoma (LGESS) and stromal sarcoma with limited infiltration in a hysterectomy specimen will be covered. The salient features that help distinguish a LGESS from a uterine tumor resembling ovarian sex-cord tumor as well as high-grade endometrial stromal sarcoma, the latter a tumor recently reintroduced in the WHO classification will also be discussed. Finally, a practical approach to the diagnosis of undifferentiated uterine sarcoma (UUS) will be presented.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situhybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS(n=12) or NRAS(n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CAand PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRASmutations in 7% and 25% of cases, respectively. KRAS/NRASmutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Mesonephric carcinoma is a rare form of gynecologic cancer derived from mesonephric remnants usually located in the lateral wall of the uterine cervix. An analogous tumor occurs in the adnexa, female adnexal tumor of probable Wolffian origin. The pathogenesis and molecular events in mesonephric carcinoma are not known. The aim of this study was to examine the molecular alterations in mesonephric carcinoma to identify driver mutations and therapeutically targetable mutations. This study consisted of 19 tumors from 17 patients: 18 mesonephric carcinomas (15 primary tumors and three metastatic tumors) and 1 female adnexal tumor of probable Wolffian origin. In two patients, both primary and metastatic tumors were available. Genomic DNA was isolated and targeted next-generation sequencing was performed to detect mutations, copy number variations, and structural variants by surveying full exonic regions of 300 cancer genes and 113 selected intronic regions across 35 genes. Fluorescence in situ hybridization (FISH) for 1p and 1q was performed in two cases. Eighty-one percent (13/16) of mesonephric carcinomas had either a KRAS (n=12) or NRAS (n=1) mutation. Mutations in chromatin remodeling genes (ARID1A, ARID1B, or SMARCA4) were present in 62% of mesonephric carcinomas. All mesonephric carcinomas lacked mutations in PIK3CA and PTEN. The most common copy number alteration was 1q gain, found in 12 (75%) mesonephric carcinomas; this was confirmed by FISH in two cases. Mesonephric carcinoma is characterized by molecular alterations that differ from those of more common variants of cervical and endometrial adenocarcinoma, which harbor KRAS/NRAS mutations in 7% and 25% of cases, respectively. KRAS/NRAS mutations are common in mesonephric carcinoma and are often accompanied by gain of 1q and mutations in chromatin remodeling genes. Targeting inhibitors of the RAS/MAPK pathway may be useful in the treatment of mesonephric carcinoma.

Endometrial stromal sarcomas with the YWHAE-NUTM2A/B genetic fusion characteristically contain high-grade round to epithelioid cell component that is strongly and diffusely cyclin D1-positive and it may or may not show an associated low-grade fibroblastic/myxoid cell component. They are clinically more aggressive than endometrial stromal sarcomas with the JAZF1-SUZ12 genetic fusion and frequently demonstrate extrauterine extension at initial clinical presentation. In this setting, the tumor may be misdiagnosed as gastrointestinal stromal tumor. This study examines the expression of KIT and ANO1 in 14 YWHAE-NUTM2A/B tumors by immunohistochemistry. Staining localization was determined as membranous and/or cytoplasmic, and the staining intensity was assessed (negative, weak, moderate and strong). Of the 14 tumors, 6 contained only a high-grade round cell component, 2 only a low-grade fibroblastic component and 6 had both components in the slides evaluated. The high-grade round cell component displayed moderate to strong membranous/cytoplasmic KIT staining in all tumors (12 of 12). The low-grade fibroblastic cell component showed only weak cytoplasmic KIT staining in 3 of 8 tumors. In contrast, ANO1 was negative in all 14 neoplasms, irrespective of the component evaluated. Sanger sequencing analysis (exons 9, 11, 13 and 17) and Ampliseq Cancer Panel mutation screen (Ion Torrent) demonstrated no KIT mutations in three KIT-positive YWHAE-NUTM2A/B tumors. This study shows that the high-grade round cell component of YWHAE-NUTM2A/B endometrial stromal sarcoma consistently expresses KIT but lacks KIT hotspot mutations. KIT expression may represent a potential diagnostic pitfall in the evaluation of YWHAE-NUTM2A/B endometrial stromal sarcoma presenting with pelvic/abdominal mass, particularly in situations where its uterine origin is not definitive, and thus a panel of antibodies that includes ANO1 and cyclin D1 is necessary.

Endometrial stromal sarcomas with the YWHAE-NUTM2A/Bgenetic fusion characteristically contain high-grade round to epithelioid cell component that is strongly and diffusely cyclin D1-positive and it may or may not show an associated low-grade fibroblastic/myxoid cell component. They are clinically more aggressive than endometrial stromal sarcomas with the JAZF1-SUZ12genetic fusion and frequently demonstrate extrauterine extension at initial clinical presentation. In this setting, the tumor may be misdiagnosed as gastrointestinal stromal tumor. This study examines the expression of KIT and ANO1 in 14 YWHAE-NUTM2A/Btumors by immunohistochemistry. Staining localization was determined as membranous and/or cytoplasmic, and the staining intensity was assessed (negative, weak, moderate and strong). Of the 14 tumors, 6 contained only a high-grade round cell component, 2 only a low-grade fibroblastic component and 6 had both components in the slides evaluated. The high-grade round cell component displayed moderate to strong membranous/cytoplasmic KIT staining in all tumors (12 of 12). The low-grade fibroblastic cell component showed only weak cytoplasmic KIT staining in 3 of 8 tumors. In contrast, ANO1 was negative in all 14 neoplasms, irrespective of the component evaluated. Sanger sequencing analysis (exons 9, 11, 13 and 17) and Ampliseq Cancer Panel mutation screen (Ion Torrent) demonstrated no KITmutations in three KIT-positive YWHAE-NUTM2A/Btumors. This study shows that the high-grade round cell component of YWHAE-NUTM2A/Bendometrial stromal sarcoma consistently expresses KIT but lacks KIThotspot mutations. KIT expression may represent a potential diagnostic pitfall in the evaluation of YWHAE-NUTM2A/Bendometrial stromal sarcoma presenting with pelvic/abdominal mass, particularly in situations where its uterine origin is not definitive, and thus a panel of antibodies that includes ANO1 and cyclin D1 is necessary.

When classifying cellular uterine mesenchymal neoplasms, histological distinction of endometrial stromal from smooth muscle neoplasms can be difficult. The only widely established marker of endometrial stromal differentiation, CD10, has marginal specificity. We took a bioinformatics approach to identify more specific markers of endometrial stromal differentiation by searching the Human Protein Atlas, a public database of protein expression profiles. After screening the database using different methods, interferon-induced transmembrane protein 1 (IFITM1) was selected for further analysis. Immunohistochemistry for IFITM1 was performed using tissue sections from the selected cases of proliferative endometrium (22), secretory endometrium (6), inactive endometrium (19), adenomyosis (10), conventional leiomyoma (11), cellular leiomyoma (16), endometrial stromal nodule (2), low-grade endometrial stromal sarcoma (16), high-grade endometrial stromal sarcoma (2) and undifferentiated uterine sarcoma (2). Stained slides were scored in terms of intensity and distribution. Normal endometrial samples uniformly showed diffuse and strong IFITM1 staining. Endometrial stromal neoplasms, particularly low-grade endometrial stromal sarcoma, showed higher IFITM1 expression compared with smooth muscle neoplasms (P<0.0001). IFITM1 immunohistochemistry has high sensitivity and specificity, particularly in the distinction between low-grade endometrial stromal sarcoma and leiomyoma (81.2 and 86.7%, respectively). Our results indicate that IFITM1 is a sensitive and specific marker of endometrial stromal differentiation across the spectrum from proliferative endometrium to metastatic stromal sarcoma. IFITM1 is a potential valuable addition to immunohistochemical panels used in the diagnosis of cellular mesenchymal uterine tumors. Further studies with larger number of cases are necessary to corroborate this impression and determine the utility of IFITM1 in routine practice. This study is a clear example of how bioinformatics, particularly tools for mining genomic and proteomic databases, can enhance and accelerate biomarker development in diagnostic pathology.

When classifying cellular uterine mesenchymal neoplasms, histological distinction of endometrial stromal from smooth muscle neoplasms can be difficult. The only widely established marker of endometrial stromal differentiation, CD10, has marginal specificity. We took a bioinformatics approach to identify more specific markers of endometrial stromal differentiation by searching the Human Protein Atlas, a public database of protein expression profiles. After screening the database using different methods, interferon-induced transmembrane protein 1 (IFITM1) was selected for further analysis. Immunohistochemistry for IFITM1 was performed using tissue sections from the selected cases of proliferative endometrium (22), secretory endometrium (6), inactive endometrium (19), adenomyosis (10), conventional leiomyoma (11), cellular leiomyoma (16), endometrial stromal nodule (2), low-grade endometrial stromal sarcoma (16), high-grade endometrial stromal sarcoma (2) and undifferentiated uterine sarcoma (2). Stained slides were scored in terms of intensity and distribution. Normal endometrial samples uniformly showed diffuse and strong IFITM1 staining. Endometrial stromal neoplasms, particularly low-grade endometrial stromal sarcoma, showed higher IFITM1 expression compared with smooth muscle neoplasms (P<0.0001). IFITM1 immunohistochemistry has high sensitivity and specificity, particularly in the distinction between low-grade endometrial stromal sarcoma and leiomyoma (81.2 and 86.7%, respectively). Our results indicate that IFITM1 is a sensitive and specific marker of endometrial stromal differentiation across the spectrum from proliferative endometrium to metastatic stromal sarcoma. IFITM1 is a potential valuable addition to immunohistochemical panels used in the diagnosis of cellular mesenchymal uterine tumors. Further studies with larger number of cases are necessary to corroborate this impression and determine the utility of IFITM1 in routine practice. This study is a clear example of how bioinformatics, particularly tools for mining genomic and proteomic databases, can enhance and accelerate biomarker development in diagnostic pathology.

Leiomyoma and leiomyosarcoma share morphological features and smooth muscle differentiation, and both arise most frequently within the uterine corpus of middle-aged women. However, they are considered biologically unrelated tumors due to their disparate clinical, cytogenetic, and molecular features. MED12, the mediator complex subunit 12 gene, has been recently implicated as an oncogene in as many as 70% of sporadic uterine leiomyoma. In the present study, we show MED12 hotspot exon 2 mutations in extrauterine leiomyoma (3 of 19 cases) and in leiomyosarcoma (3 of 13 uterine cases). We also show that MED12 mutations are found in both primary and metastatic leiomyosarcoma. Immunoblotting studies demonstrated MED12 protein expression in 100% of leiomyomas (13) and leiomyosarcomas (20), irrespective of MED12 exon 2 mutation status or histological grade. These findings indicate that MED12 has oncogenic roles in a broad range of smooth muscle neoplasia, including tumors arising in extrauterine locations.

Leiomyoma and leiomyosarcoma share morphological features and smooth muscle differentiation, and both arise most frequently within the uterine corpus of middle-aged women. However, they are considered biologically unrelated tumors due to their disparate clinical, cytogenetic, and molecular features. MED12, the mediator complex subunit 12 gene, has been recently implicated as an oncogene in as many as 70% of sporadic uterine leiomyoma. In the present study, we show MED12hotspot exon 2 mutations in extrauterine leiomyoma (3 of 19 cases) and in leiomyosarcoma (3 of 13 uterine cases). We also show that MED12mutations are found in both primary and metastatic leiomyosarcoma. Immunoblotting studies demonstrated MED12 protein expression in 100% of leiomyomas (13) and leiomyosarcomas (20), irrespective of MED12exon 2 mutation status or histological grade. These findings indicate that MED12has oncogenic roles in a broad range of smooth muscle neoplasia, including tumors arising in extrauterine locations.

Premalignant endometrial lesions (endometrial intraepithelial neoplasia (EIN)) are clonal neoplasms that arise focally and can be diagnosed using specific criteria: (1) area of glands exceeds that of stroma (glands/stroma >1), (2) nuclear and/or cytoplasmic features of epithelial cells differ between architecturally abnormal glands and normal background glands, and (3) maximum linear dimension exceeds 1 mm. However, localized groups of crowded endometrial glands may be encountered that do not fulfill all of the criteria for EIN, are interpreted as ambiguous, and are reported as ‘focal gland crowding’. We conducted a retrospective study of gland crowding using a free-text index search for this term in our pathology files. The age of the patients, number of subsequent specimens, the duration, and the outcome of the follow-ups were recorded. Of the 71 579 consecutive gynecological pathology reports, 206 (0.3%) ‘gland crowding’ cases were identified, in which 69% (143/206) had follow-up sampling. Of these, 33 (23%) had an outcome diagnosis of EIN (27 cases; 19%) or carcinoma (6 cases; 4%). Included were 18 cases (55%) diagnosed within the first year and presumed concurrent, and an additional 15 (45%) discovered after 1 year and interpreted as a later phase of disease or new events. The term ‘crowded glands’ is a highly significant finding that carries a substantial risk of an outcome of EIN and occasionally malignancy. It underscores the importance of follow-up when some but not all of the criteria for EIN are encountered in the appropriate clinical setting.

Premalignant endometrial lesions (endometrial intraepithelial neoplasia (EIN)) are clonal neoplasms that arise focally and can be diagnosed using specific criteria: (1) area of glands exceeds that of stroma (glands/stroma >1), (2) nuclear and/or cytoplasmic features of epithelial cells differ between architecturally abnormal glands and normal background glands, and (3) maximum linear dimension exceeds 1 mm. However, localized groups of crowded endometrial glands may be encountered that do not fulfill all of the criteria for EIN, are interpreted as ambiguous, and are reported as ‘focal gland crowding’. We conducted a retrospective study of gland crowding using a free-text index search for this term in our pathology files. The age of the patients, number of subsequent specimens, the duration, and the outcome of the follow-ups were recorded. Of the 71 579 consecutive gynecological pathology reports, 206 (0.3%) ‘gland crowding’ cases were identified, in which 69% (143/206) had follow-up sampling. Of these, 33 (23%) had an outcome diagnosis of EIN (27 cases; 19%) or carcinoma (6 cases; 4%). Included were 18 cases (55%) diagnosed within the first year and presumed concurrent, and an additional 15 (45%) discovered after 1 year and interpreted as a later phase of disease or new events. The term ‘crowded glands’ is a highly significant finding that carries a substantial risk of an outcome of EIN and occasionally malignancy. It underscores the importance of follow-up when some but not all of the criteria for EIN are encountered in the appropriate clinical setting.

High-grade endometrioid and serous carcinomas of the ovary and fallopian tube are responsible for the majority of cancer deaths and comprise a spectrum that includes early or localized (tubal intraepithelial carcinoma) and advanced (invasive or metastatic) disease. We subdivided a series of these tumors into three groups, (1) classic serous, (2) mixed serous and endometrioid and (3) endometrioid carcinomas and determined: (1) the frequencies of coexisting tubal intraepithelial carcinoma, (2) frequency of a dominant ovarian mass suggesting an ovarian origin and (3) immuno-localization of WT-1, p53, PTEN, PAX2 and p16ink4. All tumors were analyzed for p53 mutations. Thirty six, 25 and 8% of groups 1–3 were associated with tubal intraepithelial carcinoma (P=0.09) and 34, 45 and 62% predominated in one ovary (P=0.028), respectively. Differences in frequencies of diffuse p53 immunostaining (85–93%), WT-1 (70–98%) and p16ink4positivity (69–75%) were not significant for all groups. Greater than 95% reduction in PAX2 and PTEN occurred in 67–75 and 5–12%, respectively; however, PAX2 and PTEN staining intensity, when present, was often heterogeneous, highlighting different tumor populations. PAX2 and PTEN expression were markedly reduced or absent in 12 of 12 and 4 of 12 tubal intraepithelial carcinomas. In summary, high-grade müllerian carcinomas share identical frequencies of altered or reduced expression of p53, PTEN and PAX2, all of which can be appreciated in tubal intraepithelial carcinomas. Because only a subset of these tumors appears to arise in the fallopian tube, attention to expression of these biomarkers in the ovary and other müllerian sites might facilitate the identification of other carcinogenic pathways. PAX2 and PTEN, in addition to p53 and p16ink4, comprise a potentially important gene combination in high-grade pelvic carcinogenesis.

High-grade endometrioid and serous carcinomas of the ovary and fallopian tube are responsible for the majority of cancer deaths and comprise a spectrum that includes early or localized (tubal intraepithelial carcinoma) and advanced (invasive or metastatic) disease. We subdivided a series of these tumors into three groups, (1) classic serous, (2) mixed serous and endometrioid and (3) endometrioid carcinomas and determined: (1) the frequencies of coexisting tubal intraepithelial carcinoma, (2) frequency of a dominant ovarian mass suggesting an ovarian origin and (3) immuno-localization of WT-1, p53, PTEN, PAX2 and p16ink4. All tumors were analyzed for p53 mutations. Thirty six, 25 and 8% of groups 1–3 were associated with tubal intraepithelial carcinoma (P=0.09) and 34, 45 and 62% predominated in one ovary (P=0.028), respectively. Differences in frequencies of diffuse p53 immunostaining (85–93%), WT-1 (70–98%) and p16ink4positivity (69–75%) were not significant for all groups. Greater than 95% reduction in PAX2 and PTEN occurred in 67–75 and 5–12%, respectively; however, PAX2 and PTEN staining intensity, when present, was often heterogeneous, highlighting different tumor populations. PAX2 and PTEN expression were markedly reduced or absent in 12 of 12 and 4 of 12 tubal intraepithelial carcinomas. In summary, high-grade müllerian carcinomas share identical frequencies of altered or reduced expression of p53, PTEN and PAX2, all of which can be appreciated in tubal intraepithelial carcinomas. Because only a subset of these tumors appears to arise in the fallopian tube, attention to expression of these biomarkers in the ovary and other müllerian sites might facilitate the identification of other carcinogenic pathways. PAX2 and PTEN, in addition to p53 and p16ink4, comprise a potentially important gene combination in high-grade pelvic carcinogenesis.

Recent evidence indicates that the distal fallopian tube is the principal site of early serous cancer in women with a hereditary risk for ovarian cancer. Moreover, the fimbria is involved by early cancer in a significant minority of pelvic serous carcinomas, irrespective of whether the patient has a hereditary BRCA1or BRCA2mutation. In addition, the distal tube has been identified occasionally as a site of concurrent endometrioid tumors in women with endometrial carcinoma. Although the risk of sporadic fimbrial tumors in otherwise healthy women without genetic risk is unknown, routine histological examination of the fimbria provides the opportunity to determine the risk of such an event. To illustrate this point, a case of a woman who underwent surgery for an ovarian fibroma is presented. The distal tube was submitted, and found to harbor a focus of serous tubal intraepithelial carcinoma (STIC) with a 2-mm invasive tumor. Incidentally discovered carcinomas underscore the potential risk, albeit low, of concurrent unsuspected malignancy in the distal fallopian tube and emphasize the importance of routine pathological examination of the fimbria in all salpingectomies. The rationale for this strategy, and its potential effect on early detection and in uncovering persons or families potentially at risk for ovarian cancer, is discussed.

Recent evidence indicates that the distal fallopian tube is the principal site of early serous cancer in women with a hereditary risk for ovarian cancer. Moreover, the fimbria is involved by early cancer in a significant minority of pelvic serous carcinomas, irrespective of whether the patient has a hereditary BRCA1 or BRCA2 mutation. In addition, the distal tube has been identified occasionally as a site of concurrent endometrioid tumors in women with endometrial carcinoma. Although the risk of sporadic fimbrial tumors in otherwise healthy women without genetic risk is unknown, routine histological examination of the fimbria provides the opportunity to determine the risk of such an event. To illustrate this point, a case of a woman who underwent surgery for an ovarian fibroma is presented. The distal tube was submitted, and found to harbor a focus of serous tubal intraepithelial carcinoma (STIC) with a 2-mm invasive tumor. Incidentally discovered carcinomas underscore the potential risk, albeit low, of concurrent unsuspected malignancy in the distal fallopian tube and emphasize the importance of routine pathological examination of the fimbria in all salpingectomies. The rationale for this strategy, and its potential effect on early detection and in uncovering persons or families potentially at risk for ovarian cancer, is discussed.Modern Pathology (2009) 22, 985–988; doi:10.1038/modpathol.2009.64; published online 1 May 2009

Diffuse p53 immunostaining distinguishes 85% of serous (Type II) from endometrioid (Type I) carcinomas and is an independent marker for poor prognosis. Interobserver reproducibility for the diagnosis of these entities, as well as selection and prediction of p53 immunostaining results, is unknown. Reproducibility of three pathologists regarding: (1) a two (I and II) and (2) three part classification (I, II or indeterminate); (3) recommendation for p53 staining and (4) expectations of p53 staining results were computed with the kappa (k) statistic. All cases were immunostained for p53 and independently scored. A two and three tiered classification scheme achieved high (k=0.71) and moderate (k=0.49) reproducibility. Non-unanimous cases were more likely to be reclassified into an ‘indeterminate' category (27 cases, 39% of passes) compared to those with unanimous (82 cases, 14% of passes) classification. Pathologists recommended p53 immunostaining with poor (k=0.28) reproducibility, but staining prediction was made with good concordance (69%, k=0.50). Moreover, p53 staining was more common in diagnostically discordant (46%) compared to concordant (16%) cases. A subset of endometrial cancers do not readily fit within a two-class system and can be culled from cases that (1) do not achieve interobserver concordance and (2) are more likely to be chosen for p53 immunostaining and (3) are more likely to stain positive for p53. Because p53 is an important marker for endometrial adenocarcinoma outcome, and cannot be predicted in advance in indeterminate cases, p53 immunostaining should be employed in cases with observer disagreement in a binary system.