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

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

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

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

Breast cancers in monoallelic MUTYH germline mutation carriers have clinicopathological features overlapping with those in BRCA1 germline mutation carriers.

PURPOSE: Breast cancer patients referred to genetic counseling often undergo genetic testing with broad panels that include both breast cancer susceptibility genes as well as genes more specific for extramammary sites. As a result, patients are often incidentally found to have germline mutations in genes that are not necessarily related to breast cancer risk. One such gene is MUTYH. To understand the role MUTYH may play in breast cancer, the clinicopathological features of patients with monoallelic MUTYH germline mutation and breast cancer were examined. METHODS: The clinicopathological characteristics of the breast cancers from patients with monoallelic MUTYH mutation were compared to breast cancer patients with other germline mutations in known breast cancer susceptibility genes, including ATM, BRCA1/2, CHEK2, and PALB2. The breast cancer patients who received genetic counseling but tested negative for the aforementioned gene mutations were used as a control group. RESULTS: Histologic characteristics of the breast cancers arising in monoallelic MUTYH mutation carriers had significantly larger tumor size, higher tumor grade, and more high-risk biomarker profiles (i.e., Her2-positive and triple-negative) than breast cancer patients with susceptibility genes, except for BRCA1. MUTYH mutation carriers also showed a trend of more frequent intratumoral divergency in terms of tumor grade and biomarker profiles. CONCLUSION: Although germline monoallelic MUTYH mutation is not thought to confer a meaningfully increased risk of breast cancer development, it may contribute to pathological aggressiveness and diversity of breast cancers when they sporadically arise in MUTYH carriers.

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

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

Spatial transcriptomics of a giant pilomatricoma.

Pilomatricomas (PMs) are common benign adnexal tumors that show a predilection for the head and neck region and are characterized at the molecular level by activating mutations in the beta-catenin (CTNNB1) gene. Giant PMs are a rare histopathological variant, according to the World Health Organization, which are defined by a size greater than 4 cm and are reported to show upregulation of yes-associated protein compared to PMs of typical 1-3 cm size. We describe the case of a 67-year-old man with an 8 cm giant PM involving his temporal scalp, whose PM we characterized by 10X spatial gene expression analysis. This revealed five total transcriptomic clusters, including four distinct clusters within the giant PM, each with a unique transcriptional pattern of hair follicle-related factors, keratin gene expression, and beta-catenin pathway activity.

A SOX10+/AR- immunoprofile may identify a subset of low positive ER carcinomas with a wider range of ER expression.

BACKGROUND: Invasive breast carcinomas (IBC) that strongly express SOX10 are almost always negative for androgen receptor (AR). Furthermore, this SOX10+/AR- subset of IBC is nearly always estrogen receptor and progesterone receptor negative (ER-/PR-), being most commonly seen in triple negative breast carcinomas (TNBC), but also in a small subset of HER2+/ER-/PR- IBC. Following our previous work demonstrating the expression of SOX10 in a subset of IBC with "low positive" ER expression (i.e. 1-10 % ER+ staining based on CAP guidelines, here referred to as "ER-low"), we sought to investigate the expression of both SOX10 and AR in a larger cohort of ER-low tumors. As our previous work also revealed occasional SOX10 expression in IBC with >10 % ER+ staining, we also included tumors with any percentage of ER staining, as long as the staining intensity was weak (this subset is referred to as "ER-weak"). METHODS: We screened cases of HER2-/ER+ IBC diagnosed at our institution over a 10 year period, identified both ER-low and ER-weak tumors and stained both groups with SOX10 and AR. RESULTS: Strong SOX10 expression was seen in 12/25 (48 %) ER-low tumors and 13/24 (54 %) ER-weak tumors. ER staining in the SOX10+ subset of ER-weak tumors ranged from 15 %-80 % (median 25 %). As expected, AR was negative in all but 1 of the SOX10+ tumors in both groups. While case numbers in these groups were too small for a meaningful statistical analysis, we did note that all SOX10+/AR- tumors within both the ER-low and ER-weak groups were histologic grade 3. CONCLUSION: The presence of a SOX10+/AR- profile in a significant subset of ER-low tumors confirms the findings of our previous work and provides further support for the proposed functionally ER negative status of this group. Furthermore, the fact that the same SOX10+/AR- profile is seen in a roughly equal subset of ER-weak tumors suggests that a wider range of ER staining may be acceptable as "low positive" in SOX10+/AR- tumors, as long as the ER staining is of weak intensity. However, given the small number of cases in this single institution study, we emphasize the need for larger studies to establish the biological and clinical significance of this tumor subset.

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

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

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

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

Fluorescein Mapping in Vulvar Paget Disease.

BACKGROUND: Vulvar Paget disease often requires extensive and, in some cases, multiple resections to treat. A fluorescein-mapping procedure followed by a staged vulvectomy may be an effective technique to tailor resection and identify clinically occult lesions. TECHNIQUE: We describe a two-step procedure; first, intravenous fluorescein sodium is injected, and the vulva is illuminated with a Wood's lamp. Representative biopsies are obtained and correlated on final pathology with the extent of disease to develop a final plan for excision. Second, using fluorescein to identify the confirmed areas of disease, the appropriate excisional procedure is performed once mapping biopsy pathology is known. EXPERIENCE: We describe our experience with eight patients with vulvar Paget disease undergoing fluorescein mapping biopsies and staged vulvectomy. Using intravenous fluorescein sodium, all patients were found to have Paget disease beyond the visible margins of their gross lesions. No patients experienced a recurrence of Paget disease within a median follow-up time of 32 months, comparable with other directed methods of surgical resection. CONCLUSION: We report a technique for the injection of fluorescein sodium for the visualization of vulvar Paget disease capable of providing accurate surgical margins and identification of occult satellite lesions with a high degree of safety and a favorable cost profile. This staged approach to vulvectomy could offer improved accuracy of resection for vulvar Paget disease with few drawbacks.

Clinicopathological characteristics of the SOX10+ subset of HER2+ breast cancer.

HER2-positive breast cancers (HER2+ BC) are a heterogeneous group of tumors with variable clinical behavior. SOX10, a biomarker that has been studied in the context of breast carcinomas, especially triple-negative breast carcinomas (TNBC), has yet to be systematically investigated in a cohort of HER2+ BC. Our aim was to investigate the clinicopathological features of the SOX10+ subset of HER2+ BC. 80 HER2+/ER- invasive breast carcinomas were stained for SOX10. All SOX10+ cases and a matched number of SOX10- cases were also stained for vimentin and androgen receptor (AR). 18 % (14/80) of our cases were SOX10+. SOX10 expression was seen in both IHC positive (3+) and equivocal (2+) but ISH-amplified cases. The SOX10+ tumors were significantly associated with both greater vimentin expression (36 % vs 0 %, p = 0.0407) and less AR expression (14 % vs 100 %, p = 0.0001) compared to SOX10- tumors. Interestingly, the vimentin+/AR- subset of our SOX10+ cases showed uniformly apocrine-like morphology, while all SOX10- cases, including those with apocrine-like morphology, were vimentin-/AR+. Our findings suggest that SOX10+/HER2+ BC are more likely to show a peculiar apocrine-like, vimentin+/AR- phenotype as compared to SOX10-/HER2+ BC.

HER2-positive/ER-low breast carcinoma shows a response to neoadjuvant chemotherapy similar to that of HER2-positive/ER-negative breast carcinoma.

Negative expression of estrogen receptor (ER) predicts response to chemotherapy in breast cancers (BCs). ER negative cancers are those with less than 1 % of nuclear staining. Tumors with 1-10 % staining are sub-classified as "low-positive" (ER-low). HER2 negative tumors with ER low staining are considered biologically and clinically equivalent to ER negative tumors. This study investigates whether ER low expression in HER2-positive (HER2+) BCs has different clinical behavior than ER negative HER2-positive tumors. We used a sample of 171 patients with HER2+ BCs to compare risk of residual cancer after neoadjuvant chemotherapy by different ER expression strength. Patients were classified into 3 groups: ER-negative (ER <1 %); ER-low (ER <10 %, any intensity or <33 % staining, weak intensity); and ER-high (ER = 10-33 %, moderate to strong intensity or >33 %, any intensity). The risk of residual cancer in patients with ER-low tumors was similar to the risk in patients with ER-negative tumors (RR = 0.76, 95 % CI: 0.30-1.93). Conversely, patients with ER-high tumors had twice the risk of residual cancer than patients with ER-negative tumors (RR = 2.20, 95 % CI: 1.46-3.31). These findings persisted after adjusting for tumor grade, clinical tumor and lymph node stage, chemotherapy regimen, and progesterone receptor status. In this cohort of patients with HER2+ BCs, ER-low tumors had a similar pathologic response to chemotherapy as ER-negative tumors suggesting similar clinical behavior. Future research should address biological explanations to these similarities between ER negative and ER low breast cancers such as HER2 enriched phenomenon.

Multi-modal Profiling of the Extracellular Matrix of Human Fallopian Tubes and Serous Tubal Intraepithelial Carcinomas.

Recent evidence supports the fimbriae of the fallopian tube as one origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). Therefore, we sought to determine the ECM composition of the benign fallopian tube and changes associated with serous tubal intraepithelial carcinomas (STICs), precursors of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets that identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies (COL2A1, COL4A5, COL16A1, elastin, LAMA5, annexin A2, and PAI1). To enable future in vitro studies, we investigated the levels and localization of ECM components included in tissue-engineered models (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan, and hyaluronic acid) using multispectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation. (J Histochem Cytochem XX: XXX-XXX, XXXX).

FIGO Grade 3 Endometrioid Adenocarcinomas With Diffusely Aberrant ß-Catenin Expression: An Aggressive Subset Resembling Cutaneous Pilomatrix Carcinomas.

Uterine endometrioid adenocarcinomas are known for their morphologic plasticity. In addition to a multiplicity of metaplasias, uterine endometrioid adenocarcinomas may also undergo high-grade divergent differentiation in the form of high-grade neuroendocrine carcinoma, neuroectodermal differentiation or carcinosarcoma; others may dedifferentiate completely. Here we describe 5 cases of uterine endometrioid adenocarcinomas with high-grade divergent differentiation showing a striking morphologic and immunophenotypic resemblance to cutaneous pilomatrix carcinoma. Specifically, the high-grade component in all cases exhibited solid, basaloid morphology with conspicuous tumor cell necrosis and the presence of shadow cells, accompanied by diffusely aberrant (nuclear and cytoplasmic) ß-catenin expression as well as variably diffuse CDX2 expression. In addition, the high-grade component in all cases showed loss of ER and PAX8 expression, retained MMR expression, wild-type p53 expression, patchy p16 expression, and diffusely positive cytokeratin expression (AE1/AE3 and CK7); at least focal neuroendocrine marker expression was present in all cases. CK20 was negative in all cases, with the exception of very focal staining in a single case (2% of tumor cells). All 5 of our tumors had at least a focal conventional FIGO grade 1 component. In all 4 cases tested, the low-grade component retained both PAX8 and ER expression and had, at best, focally aberrant ß-catenin expression. Two of our cases had molecular analysis performed and both harbored mutations in exon 3 of CTNNB1 as expected; molecular analysis also revealed that both cases lacked POLE or TP53 mutations and showed no microsatellite instability. The tumors in this series were uniformly aggressive. Four of the 5 patients in our cohort had available follow-up information; of these, 3/4 died of their disease within 14 mo of diagnosis and the fourth patient had distant metastatic disease at presentation and is alive with disease 1 mo following diagnosis. The 1 patient without follow-up information also had distant metastatic disease at presentation and was lost to follow-up 17 mo later. The cases described in this series (1) represent a highly aggressive CTNNB1-mutated subset of the "no specific molecular profile" category of endometrioid adenocarcinomas; (2) illustrate a form of high-grade divergent differentiation resembling cutaneous pilomatrix carcinoma already described in carcinomas at other anatomic sites; and (3) underscore the difficulty in recognizing this phenotype at distant metastatic sites, which are frequent even at the time of presentation, given the consistent loss of ER and PAX8 expression and concurrent CDX2 expression.

HER2 Expression in Endometrial Cancers Diagnosed as Clear Cell Carcinoma.

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

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

Evaluation of Collagen Alterations in Early Precursor Lesions of High Grade Serous Ovarian Cancer by Second Harmonic Generation Microscopy and Mass Spectrometry.

Background: The collagen architecture in high grade serous ovarian cancer (HGSOC) is highly remodeled compared to the normal ovary and the fallopian tubes (FT). We previously used Second Harmonic Generation (SHG) microscopy and machine learning to classify the changes in collagen fiber morphology occurring in serous tubal intraepithelial carcinoma (STIC) lesions that are concurrent with HGSOC. We now extend these studies to examine collagen remodeling in pure p53 signatures, STICs and normal regions in tissues that have no concurrent HGSOC. This is an important distinction as high-grade disease can result in distant collagen changes through a field effect mechanism. Methods: We trained a linear discriminant model based on SHG texture and image features as a classifier to discriminate the tissue groups. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with collagen fiber morphology alterations. Results: We quantified the differences in the collagen architecture between normal tissue and the precursors with good classification accuracy. Through proteomic analysis, we identified the downregulation of single α-chains including those for Col I and III, where these results are consistent with our previous SHG-based supramolecular analyses. Conclusion: This work provides new insights into ECM remodeling in early ovarian cancer and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach.

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

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

Genetic characterisation of adult primary pleomorphic uterine rhabdomyosarcoma and comparison with uterine carcinosarcoma.

AIMS: To characterise the genetic alterations in adult primary uterine rhabdomyosarcomas (uRMSs) and to investigate whether these tumours are genetically distinct from uterine carcinosarcomas (UCSs). METHODS AND RESULTS: Three tumours originally diagnosed as primary adult pleomorphic uRMS were subjected to massively parallel sequencing targeting 468 cancer-related genes and RNA-sequencing. Mutational profiles were compared with those of UCSs (n = 57) obtained from The Cancer Genome Atlas. Sequencing data analyses were performed using validated bioinformatic approaches. Pathogenic TP53 mutations and high levels of genomic instability were detected in the three cases. uRMS1 harboured a likely pathogenic YTHDF2-FOXR1 fusion. uRMS2 harboured a PPP2R1A hotspot mutation and amplification of multiple genes, including WHSC1L1, FGFR1, MDM2, and CCNE1, whereas uRMS3 harboured an FBXW7 hotspot mutation and an ANKRD11 homozygous deletion. Hierarchical clustering of somatic mutations and copy number alterations revealed that these tumours initially diagnosed as pleomorphic uRMSs and UCSs were similar. Subsequent comprehensive pathological re-review of the three uRMSs revealed previously unidentified minute pan-cytokeratin-positive atypical glands in one case (uRMS3), favouring its reclassification as UCS with extensive rhabdomyosarcomatous overgrowth. CONCLUSIONS: Adult pleomorphic uRMSs harbour TP53 mutations and high levels of copy number alterations. Our findings underscore the challenge in discriminating between uRMS and UCS with rhabdomyosarcomatous differentiation.