Mojgan Devouassoux‐Shisheboran

Challenges of a tailored immunohistochemistry algorithm for uterine leiomyosarcoma: an integrated analysis of leiomyomas with bizarre nuclei and fumarate hydratase (FH) deficiency

AimsLeiomyomas (LM) are the most common uterine mesenchymal neoplasms and encompass a variety of histological subtypes. Bizarre nuclei are described in both leiomyomas with bizarre nuclei (LM‐BN) and fumarate hydratase‐deficient leiomyomas (FH‐LM), which raise diagnostic concerns regarding leiomyosarcoma (LMS). Recently, an immunohistochemical algorithm to support the diagnosis of LMS based on the genomic landscape of these neoplasms was proposed. This study aimed to evaluate the algorithm's accuracy in distinguishing LM‐BN and FH‐LM from LMS.Methods and ResultsWe collected 68 LM (29 LM‐BN, 30 FH‐LM, and 9 LM) and 9 LMS, along with clinicopathological and molecular data. An immunohistochemical panel comprising p53, Rb, PTEN, ATRX, DAXX, and MDM2 was applied. Nine cases were non‐interpretable due to fixation issues. The algorithm demonstrated 100% accuracy for LM without bizarre nuclei (9/9) and for nonmyxoid LMS (5/5). Notably, 28.6% (14/49) of LM‐BN and FH‐LM exhibited at least two abnormalities, leading to potential misclassification as LMS. However, their clinical course, morphology, and genomic profile supported a benign diagnosis. Frequent alterations included Rb (20/49; 40.8%) and p53 (19/49; 38.8%), particularly in bizarre cells, while no abnormal staining was observed for ATRX, DAXX, or MDM2.ConclusionThe proposed algorithm has limitations in differentiating LMS from LM‐BN and FH‐LM, misclassifying 28.6% of the latter. Accurate interpretation requires proper internal controls, particularly for markers whose loss of expression favours malignancy. Morphology remains central for diagnosis, although integration of molecular data may provide additional insights for a definitive classification in challenging cases.

Role of gene sequencing in classifying struma ovarii: BRAF p.G469A mutation and TERT promoter alterations favour malignant struma ovarii

AimsStruma ovarii (SO) are rare, accounting for 0.3–1% of ovarian tumours, and include benign and malignant lesions. In most cases, histology is not predictive of clinical outcome and prognosis. The prognosis of histologically malignant thyroid‐type carcinomas can indeed be excellent, while SO, composed of normal thyroid tissue, can recur and are designated highly differentiated follicular carcinoma of the ovary. Clearer diagnostic criteria are therefore required.Methods and resultsWe retrospectively studied 31 SO using DNA and RNA sequencing with pan‐cancer gene panels, including eight biologically malignant SO (BMSO) defined based on ovarian serosal or extra‐ovarian dissemination at presentation or during follow‐up, 10 stage IA histologically malignant SO (HMSO) with thyroid‐type carcinoma morphology and 13 biologically and histologically benign SO (BSO), with none of the above‐mentioned characteristics. Molecular alterations were observed in 87.5% of BMSO, 70% of HMSO and 7.7% of BSO (P < 0.001). All patients with a peritoneal dissemination at presentation or during follow‐up had at least one gene alteration. BRAF mutations (44.5%) were only observed in malignant forms (HMSO and BMSO) and TERT promoter alterations (25%) only in cases of BMSO. The BRAF p.G469A mutation, which is extremely rare in thyroid carcinomas, was the molecular alteration most frequently associated with malignant SO (28.5%).ConclusionOur results highlight the clinical utility of molecular sequencing in SO, based on this limited number of cases. However, as malignant SO evolve slowly, more extensive molecular studies in SO with more than 10 years’ follow‐up are required to draw any conclusions on the prognostic value of the associated gene alterations.

Dissecting the Origin of Heterogeneity in Uterine and Ovarian Carcinosarcomas

Gynecologic carcinosarcomas (CS) are biphasic neoplasms composed of carcinomatous (C) and sarcomatous (S) malignant components. Because of their rarity and histologic complexity, genetic and functional studies on CS are scarce and the mechanisms of initiation and development remain largely unknown. Whole-genome analysis of the C and S components reveals shared genomic alterations, thus emphasizing the clonal evolution of CS. Reconstructions of the evolutionary history of each tumor further reveal that C and S samples are composed of both ancestral cell populations and component-specific subclones, supporting a common origin followed by distinct evolutionary trajectories. However, while we do not find any recurrent genomic features associated with phenotypic divergence, transcriptomic and methylome analyses identify a common mechanism across the cohort, the epithelial-to-mesenchymal transition (EMT), suggesting a role for nongenetic factors in inflicting changes to cellular fate. Altogether, these data accredit the hypothesis that CS tumors are driven by both clonal evolution and transcriptomic reprogramming, essential for susceptibility to transdifferentiation upon encountering environmental cues, thus linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences. Significance: We have provided a detailed characterization of the genomic landscape of CS and identified EMT as a common mechanism associated with phenotypic divergence, linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences.

Primary Vulvar and Vaginal Adenocarcinomas of Intestinal Type Are Closer To Colorectal Adenocarcinomas Than To Carcinomas of Müllerian Origin

Primary vulvar and vaginal adenocarcinomas of intestinal type (VVAIts) are very rare tumors, displaying morphologic and immunohistochemical overlap with colorectal adenocarcinomas. However, their immunoprofile and genomics are poorly studied, and their origin is still debated. Here, we studied a series of 8 VVAIts (4 vulvar and 4 vaginal) using a large panel of immunohistochemistry and DNA and RNA sequencing with clustering analyses. All tumors shared a similar morphology with colorectal adenocarcinomas and diffuse CK20 and CDX2 expression. SATB2 diffuse positivity was observed in 62.5% of tumors and CK7 in 82.5%, whereas PAX8, SOX17, p16, and estrogen and progesterone receptors were always negative. A p53 mutated-type expression was observed in 75% of tumors. All tumors were mismatch repair proficient. Neither human papillomavirus DNA nor pathogenic transcript fusions were detected. The most frequent molecular alterations were TP53 and KRAS variants in 71.4% and 42.9%, respectively. The transcriptomic analysis highlighted a robust VVAIts cluster distinct from endocervical, ovarian, lung, thyroid, salivary glands, breast, and renal carcinomas but failed to differentiate vulvar from vaginal intestinal-type tumors. On 2 different clustering analyses, VVAIts clustered altogether, very close to colorectal adenocarcinomas. Compared with endocervical adenocarcinomas of intestinal type, VVAIts had a significantly lower expression of SOX17 and epithelial-mesenchymal transition genes and a higher mitogen-activated protein kinase pathway gene expression. These results suggest that Müllerian structures leading to cervical adenocarcinomas may undergo intestinal-type transdifferentiation via an epithelial-mesenchymal transition phenomenon. Conversely, mitogen-activated protein kinase pathway activation in VVAIts, which plays a major role in colorectal adenocarcinomas, may indicate a close relationship in the carcinogenesis of these tumors. Our results indicate that adenocarcinomas of intestinal type, in the distal vagina or vestibular vulva, might be a unique and single entity, probably originating from cloacogenic embryonic remnants and/or ectopic colorectal mucosae inclusions. An open question would be to explore the efficacy of systemic drugs prescribed in colorectal cancers, in VVAIts.

The MEF2D::NCOA2 Fusion Defines a Distinct Emerging Vulvovaginal Myxoid Epithelioid Tumor With Smooth Muscle Differentiation

Myocyte-specific enhancer factor 2D gene and nuclear receptor coactivator 2 gene fusion (MEF2D::NCOA2) was recently reported in 2 vulvovaginal myxoid epithelioid smooth muscle tumors. We aimed to perform an integrated approach combining clinical, morphologic, immunohistochemical, and molecular profiling analyses, including targeted RNA sequencing, targeted gene expression analysis profiling with clustering, DNA mutational analysis, and array comparative genomic hybridization in a series of 3 MEF2D::NCOA2 fusion-associated vulvovaginal tumors, to better describe this entity. The median age at diagnosis was 45 years. Tumors were well circumscribed and located deeply within the vulva, vaginal wall, or between the bladder and the vagina (1/3, 33.3% each). The median size of tumors was 2.5 cm. All tumors had a similar morphology, reminiscent of smooth muscle tumor with prominent myxoid stromal changes (3/3, 100%). Tumor cells were haphazardly arranged in short fascicles and were mostly spindle cells. Microcystic spaces lined by epithelioid cells and/or sheets of epithelioid cells were observed in all tumors (3/3, 100%), associated with a myxoid background. Cytologic atypia was none to mild, and the mitotic counts were always low (≤1 mitosis/high-power fields). Immunohistochemistry found smooth muscle actin, desmin, h-caldesmon, estrogen receptors, and CD34 to be intensely and diffusely expressed in all tumors (3/3, 100%). A MEF2D::NCOA2 transcript was observed in all tumors (3/3, 100%), which was the driver of molecular alteration. No pathogenic variants were found, and array comparative genomic hybridization found simple genomic profiles for all tumors (3/3, 100%). On targeted gene expression analysis, MEF2D::NCOA2 fusion-associated tumors clustered distinctly from other gynecologic mimickers and neoplasms with myxoid stromal changes (vulvovaginal leiomyomas, myxoid vulvovaginal leiomyomas, deep angiomyxomas, myxoid leiomyosarcomas, myxoid endometrial stromal sarcomas, and inflammatory myofibroblastic tumors). The signaling pathways involved in this entity included the expression of genes encoding smooth muscle phenotype proteins, favoring a smooth muscle (myoid) differentiation. All patients were alive and free of disease at the last follow-up. To conclude, vulvovaginal MEF2D::NCOA2 fusion-associated tumors are distinct and emerging entities, with a rather indolent behavior.

Assessing the Status of Cyclin E1 (CCNE1) From Gene to Protein Level in Ovarian and Endometrial Carcinomas: A Systematic Review

Twenty percent and 45.4% of high-grade ovarian carcinomas (OC) and endometrial carcinomas (EC) exhibit CCNE1 amplification (CCNE1-amp), respectively, which is related to poor prognosis, but could serve as predictive biomarker for response to innovative targeted therapies. However, there is no consensus regarding how to evaluate the CCNE1 status (at the DNA, RNA, and/or protein level). Therefore, we conducted a systematic review of CCNE1 status testing in tubo-ovarian neoplasms and EC, comparing their performance for clinical purposes and highlighting the test's interpretation criteria (CRD420250651291). Among the 734 records initially found on PubMed and Google Scholar, 48 reports were finally included. Molecular analyses and immunohistochemistry (IHC) were reported on 9774 tubo-ovarian neoplasms and 750 EC, and 6966 tubo-ovarian neoplasms and 856 EC, respectively. The most frequently morphological used method to detect CCNE1-amp was fluorescent in situ hybridization (13/16 studies, 81.3%), with quite consensual criteria to defined amplification (ie, CCNE1/chromosome 19 ratio ≥2, and/or >8/≥8 copies of CCNE1 per nucleus, and/or ≥4 CCNE1 copies in ≥40% of cells). The proportion of tubo-ovarian neoplasms with CCNE1 immunohistochemical overexpression varied from 13.5% to 96%, and 14.6% to 86.1% in EC. The sensitivity and specificity of CCNE1 IHC to detect/exclude CCNE1-amp varied from 54.5% to 100% and 59.3% to 90.1%, respectively. Given the reported data, CCNE1 overexpression should be considered either when an H-score is ≥100 or when the staining is >60% with >5% of cells strongly stained. Both CCNE1-amp and CCNE1 overexpressions were associated with poor prognosis and with response to Wee1 and CDK2 inhibitors in high-grade serous OC (overall response rate up to 53%, objective response rate of 32%-40%). In contrast, CCNE1 messenger RNA overexpression had no prognostic value. Thus, both CCNE1-amp detection by fluorescent in situ hybridization and CCNE1 protein levels quantification using IHC represent today the most validated tools to determine the CCNE1 status in OC/EC.

Pan‐TRK immunohistochemistry in gynaecological mesenchymal tumours: diagnostic implications and pitfalls

AimsNTRK‐rearranged sarcomas of the female genital tract mainly occur in the uterus (more commonly cervix than corpus) and are characterized by a “fibrosarcoma‐like” morphology and NTRK gene rearrangements. These neoplasms may exhibit histological overlap with other entities and can present diagnostic difficulties without molecular confirmation. Pan‐TRK immunohistochemistry was developed to identify tumours harbouring NTRK rearrangements. The aim of this study was to characterize pan‐TRK immunohistochemical expression in a large cohort of gynaecological mesenchymal neoplasms and investigate the utility of pan‐TRK immunohistochemistry to distinguish NTRK‐rearranged sarcoma from its mimics.Methods and resultsA total of 473 gynaecological mesenchymal tumours (461 without known NTRK fusions and 12 NTRK‐rearranged sarcomas) were selected. Pan‐TRK immunohistochemistry (EPR17341, Abcam) was performed on whole tissue sections and tissue microarrays. Molecular interrogation of pan‐TRK positive tumours was performed by RNA sequencing or fluorescence in situ hybridization (FISH). Of the 12 NTRK‐rearranged sarcomas, 11 (92%) exhibited diffuse (≥70%) cytoplasmic pan‐TRK staining with moderate/marked intensity, while the other was negative. Eleven (2.4%) additional tumours also exhibited pan‐TRK immunohistochemical expression: three low‐grade endometrial stromal sarcomas, seven high‐grade endometrial stromal sarcomas, and an undifferentiated uterine sarcoma. Molecular confirmation of the absence of NTRK rearrangements was possible in nine of these tumours. Of these nine neoplasms, seven exhibited focal/multifocal (<70%) pan‐TRK cytoplasmic staining with weak/moderate intensity.ConclusionEven though pan‐TRK immunohistochemical expression is not entirely sensitive or specific for NTRK‐rearranged sarcomas, these neoplasms tend to exhibit diffuse staining of moderate/strong intensity, unlike its mimics. Pan‐TRK should be performed in monomorphic uterine (corpus and cervix) spindle cell neoplasms that are negative for smooth muscle markers and hormone receptors and positive for CD34 and/ or S100. Ultimately, the diagnosis requires molecular confirmation.