Sarcomas With RAD51B Fusions Are Associated With a Heterogeneous Phenotype.

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 women with a median age of 57 years 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, whereas 2 tumors exhibited 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 and high mitotic activity (median: 30/10 high-power fields), whereas coagulative necrosis was noted in 6 cases and lymphovascular invasion in 2. By immunohistochemistry, 2 showed myoid and melanocytic markers in keeping with PEComa, whereas 4 cases were only positive for smooth muscle markers consistent with LMS (including 3 myxoid). The remaining 2 cases had a nonspecific immunoprofile. Five cases tested by targeted RNA sequencing (Archer FusionPlex, Illumina TruSight) showed different fusion partners (HMGA2, PDDC1, and CEP170). RAD51B rearrangements were identified by FISH in the remaining 3 cases. Targeted DNA sequencing in 2 cases was negative for TSC gene alterations. Clinical outcome, available in 5 patients (median follow-up, 19 months), revealed 3 local recurrences, 2 lung metastases, and 4 deaths due to 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.

Pattern A endocervical adenocarcinomas with ovarian metastasis are indolent and molecularly distinct from destructively invasive adenocarcinomas.

AIMS: The invasive pattern in HPV-associated endocervical adenocarcinoma (HPVA) has prognostic value. Non-destructive (pattern A) HPVA has excellent prognosis mirroring adenocarcinoma in-situ (AIS). However, the rare occurrence of ovarian spread in these tumours suggests aggressiveness in a subset of patients with these otherwise indolent lesions. We hypothesise that AIS/pattern A HPVA with ovarian metastases are biologically different than metastatic destructively invasive HPVA. METHODS AND RESULTS: Samples from patients with HPVA and synchronous or metachronous metastases were retrieved and reviewed to confirm diagnosis and determine the Silva pattern in the primary lesion. For each case, normal tissue, cervical tumour and at least one metastasis underwent comprehensive sequencing using a 447-gene panel. Pathogenic single-nucleotide variants and segmental copy-number alterations (CNA), tumour mutational burden and molecular signatures were evaluated and compared between primary and metastases and among invasive pattern categories. We identified 13 patients: four had AIS/pattern A primaries, while nine had pattern B/C tumours. All AIS/pattern A lesions had metastasis only to ovary; 50% of patients with ovarian involvement, regardless of invasive pattern, also had involvement of the endometrium and/or fallopian tube mucosa by HPVA. In the ovary, AIS/pattern A HPVA showed deceptive well-differentiated glands, often with adenofibroma-like appearance. Conversely, pattern C HPVAs consistently showed overt infiltrative features in the ovary. Sequencing confirmed the genetic relationship between primary and metastatic tumours in each case. PIK3CA alterations were identified in three of four AIS/pattern A HPVAs and three of eight pattern B/C tumours with sequenced metastases. Pattern C tumours showed a notably higher number of CNA in primary tumours compared to pattern A/B tumours. Only one metastatic AIS/pattern A HPVA had a novel pathogenic variant compared to the primary. Conversely, five of eight pattern B/C tumours with sequenced metastases developed novel pathogenic variants in the metastasis not seen in the primary. All four AIS/pattern A patients were alive and free of disease at 31, 47, 58 and 212 months after initial diagnosis. Conversely, cancer-related death was documented in five of nine pattern B/C patients with follow-up at 7, 20, 20, 43 and 87 months. CONCLUSION: Morphologically and genomically, AIS/pattern A HPVA with secondary ovarian involvement appears distinct from destructively invasive tumours. In at least a subset of these cases, ovarian spread appears to occur via trans-Mullerian superficial extension, different from the stromal and lymphatic vascular spread typical of more aggressive tumours (pattern C). These differences may explain the indolent outcome observed in the rare subset of patients with AIS/pattern A HPVA and ovarian metastasis. Our data underscore the potential for conservative surgical management approaches to pattern A HPVA.

Lesions sub-diagnostic of endometrioid intra-epithelial neoplasia/atypical hyperplasia: value of morphology and immunohistochemistry in predicting neoplastic outcome.

AIMS: Areas of gland crowding that do not fulfil diagnostic criteria of endometrioid intra-epithelial neoplasia (EIN) are often encountered in endometrial biopsies. In this study, we document the prevalence of neoplastic outcome in patients with these subdiagnostic lesions (SL) and assess the utility of morphological features and a three-marker immunohistochemistry panel (PAX2, PTEN, beta-catenin) to predict outcome. METHODS AND RESULTS: Of 430 women with SL on endometrial sampling at Brigham and Women's Hospital between 2001 and 2021 with available follow-up biopsy, 72 (17%) had a neoplastic outcome (EIN or endometrioid carcinoma). Multilayered epithelium and mitoses in SL were statistically associated with a neoplastic outcome. Abnormal three-marker staining was observed in 93% (53 of 57) of SL with neoplastic outcome and 60% (37 of 62) of a control group with benign outcome. Among the 72 patients with neoplastic outcome, EIN/carcinoma tissue was available in 33; of these, 30 (91%) showed abnormal staining for one or more markers. Remarkably, in 84% of these cases the EIN/carcinoma had the aberrant expression seen in the preceding SL. Based on a prevalence of 17%, the positive and negative predictive values of abnormal staining in one or more markers were 24 and 97%, respectively. CONCLUSIONS: The presence of SL warrants clinical surveillance and repeat sampling because it is followed by endometrioid neoplasia in a significant subset of patients. Normal three-marker staining identifies women with a very low risk of neoplastic outcome. Conversely, abnormal staining is frequent in SL with benign outcome leading to poor specificity and positive predictive value.

Practical guidance for assessing and reporting lymphovascular space invasion (LVSI) in endometrial carcinoma.

Lymphovascular space invasion (LVSI) is an important prognostic parameter in endometrial carcinoma (EC) and has gained increasing interest in recent years due to an expanding body of evidence of its independent prognostic value, especially when the presence of LVSI is quantified. A key strength of LVSI as a prognostic factor is that it can be detected on routine microscopic examination, without ancillary tests, and thus can be used in low-resource settings. A weakness, however, is the lack of uniformly applied criteria for assessment and quantification of LVSI, resulting in interobserver variation in diagnosis. This is confounded by artefacts and other morphological features that may mimic LVSI (commonly referred to as pseudo-LVSI). Despite these issues, multiple studies have shown that LVSI is strongly associated with lymph node (LN) metastasis and is an independent risk factor for LN recurrence and distant metastasis. Consequently, the presence of substantial/extensive LVSI has become an important consideration in formulating adjuvant treatment recommendations in patients with EC, and this has been incorporated in the recent International Federation of Gynecology and Obstetrics (FIGO) 2023 staging system. Herein, we review the current literature on LVSI in EC and discuss its role as a prognostic marker, the reproducibility of LVSI assessment and distinction between LVSI and its mimics. We provide illustrations of key diagnostic features and discuss the two-tiered (none/focal versus substantial) system of LVSI classification. This work is intended to provide guidance to practising pathologists and unify the approach towards LVSI assessment in EC.

STK11 (LKB1) immunohistochemistry is a sensitive and specific marker for STK11 adnexal tumours.

AIMS: ST11 adnexal tumour is a rare, recently described malignant neoplasm that is associated with Peutz-Jeghers syndrome. It predominantly originates from the para-adnexal soft tissues and often shows secondary involvement of the fallopian tube and ovary. STK11 adnexal tumours have a broad differential diagnosis due to their variable morphology and non-specific immunoprofile, and diagnostic confirmation currently requires sequencing to identify an STK11 mutation. We investigate the diagnostic utility of STK11 (LKB1) immunohistochemistry (IHC) in a cohort of STK11 adnexal tumours and morphological mimics. METHODS AND RESULTS: IHC for STK11 was performed on 122 tumours, including 17 STK11 adnexal tumours and 105 morphological mimics (10 female adnexal tumours of Wolffian origin, 22 adult granulosa cell tumours, 10 juvenile granulosa cell tumours, four Sertoli-Leydig cell tumours, two Leydig cell tumours, one Sertoli cell tumour, one steroid cell tumour, four extra-ovarian sex cord-stromal tumours, 16 ovarian endometrioid carcinomas, eight tubo-ovarian high-grade serous carcinomas, five ovarian mesonephric-like adenocarcinomas, 14 ovarian carcinosarcomas, five peritoneal malignant mesotheliomas, two pelvic plexiform leiomyomata and one ovarian solid pseudopapillary tumour). All STK11 adnexal tumours showed complete loss of cytoplasmic staining for STK11. All other tumour types showed retained cytoplasmic staining, except for one endometrioid carcinoma with mucinous differentiation which showed complete loss of STK11 expression and a high-grade serous carcinoma with subclonal loss. CONCLUSIONS: STK11 is a highly sensitive and specific immunohistochemical marker for distinguishing STK11 adnexal tumour from its histological mimics, and can obviate the need for confirmatory molecular studies in the appropriate morphological context.

Atypical Endometriosis: Comprehensive Characterization of Clinicopathologic, Immunohistochemical, and Molecular Features.

Atypical endometriosis (A-EMS), defined by cytologic atypia and/or crowded glands resembling endometrial intraepithelial neoplasia, remains poorly understood. We aimed to refine the morphologic, immunohistochemical, and molecular features of A-EMS in an institutional series. Cases were identified through a structured search and reviewed by 2 pathologists. Immunohistochemistry and comprehensive sequencing using a panel 447-gene coverage were performed in suitable cases. A-EMS with synchronous and/or subsequent EMS-related neoplasia were compared with those without. Of 4598 EMS cases over an 11-yr period, 36 A-EMS were identified. The mean age at presentation was 46 (range 26-68) yr. Locations included the ovary (24, 66%), tubo-ovary (6, 17%), fallopian tube (3, 8%), and peritoneum (3, 8%). The mean size was 6.5 (range 0.5-40) mm. Cytologic atypia was mild in 4 (11%), moderate in 21 (58%), and severe in 11 (31%). Most lesions were partially or completely flat (28, 78%); of these, 66% showed hobnail nuclei. Crowded/cribriform and micropapillary/papillary patterns were seen in 11 (31%) and 16 (44%) A-EMS, respectively. Immunohistochemistry, performed in 33 A-EMS, showed wildtype p53 (100%) retained PMS2/MSH6 (100%), and positive estrogen receptor (97%, mean 65% cells), progesterone receptor (76%, mean 30% cells), and Napsin A (39%). Ki67 labelling was <1% to 10% (median 5%). Nine (25%) patients presented with concurrent or subsequent ipsilateral endometrioid, seromucinous, or clear cell neoplasia (4 borderline tumors and 4 carcinomas). The only A-EMS feature statistically more frequent in this subset was crowded/glands (6/9 vs. 2/27 A-EMS without, P =0.001 Fisher exact test). Sequencing showed pathogenic variants in 5 of 6 cases analyzed, involving ATM , BRCA2 , KRAS , AKT , CTNNB1 , PTEN , and ARID1A among other genes. In 2 cases, synchronous neoplasia showed an accumulation of additional variants. A-EMS is characterized by cytologic atypia and crowded architecture but low proliferation index, positive estrogen receptor, and normal p53 and MMR, which can be helpful in the distinction from malignancy. The prevalence of synchronous/subsequent tubo-ovarian neoplasia in our series was 25%, significantly higher than the reported 1% in conventional EMS. Moreover, A-EMS harbors genomic alterations seen in EMS-related tumors and shares pathogenic variants with synchronous ipsilateral neoplasia. Therefore, it is important to report A-EMS as currently defined and describe its architectural features, especially gland crowding as this appears to increase the risk of EMS-related epithelial neoplasia. Napsin-A is often positive in A-EMS and should be interpreted with caution.

Localized Endometrial Proliferations of Pregnancy are Clonal Glandular Outgrowths Characterized by PTEN Loss and PIK3CA Pathogenic Variants.

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 biological and clinical importance, we explore the pathologic, immunophenotypic, and molecular features of this entity. Nine cases of LEPP identified in 15 years were retrieved from departmental archives and reviewed. Immunohistochemistry and next-generation sequencing using a comprehensive 446-gene panel were performed when the material was available. Eight cases were identified in curettage specimens performed after first-trimester pregnancy loss, and 1 in the basal plate of a mature placenta. The mean patient age was 35 (range 27-41) years. The 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 2. 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 estrogen receptor (mean 71%) and progesterone receptor (mean 74%). All were negative for p40 except 1 case (focal weak positivity). PTEN was markedly reduced in background secretory glands in all cases; in 5/7, LEPP foci showed a complete absence of PTEN expression. PIK3CA pathogenic variants were identified in 4/4 cases sequenced; 3/4 had inactivating PTEN mutations. 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 estrogen receptor/progesterone receptor, PTEN loss, and PIK3CA and PTEN mutations. Although our findings indicate that LEPP is neoplastic, for now, we advise against diagnosing LEPP as endometrial carcinoma or hyperplasia because LEPP has a particular clinicopathologic 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.

Molecular Landscape of Mullerian Clear Cell Carcinomas Identifies The Cancer Genome Atlas-like Prognostic Subgroups.

Mullerian clear cell carcinoma (CCC) is often aggressive and chemoresistant. 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 peritoneum/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 was 6.8 mutations/megabase (range, 1.3-185, 21% were ≥10 mutations/Mb). The most frequently mutated genes were ARID1A (48%), PIK3CA (45%), TP53 (23%), and PTEN (10%). ERBB2 amplification occurred in 4%. When classified according to the Cancer Genome Atlas/the Proactive Molecular Risk Classifier for Endometrial Cancer endometrial carcinoma molecular subgroups, 3 (3%) were POLE ultramutated, 5 (5%) were microsatellite instability-high (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 were not associated with the 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 ARID1A mutations, and/or harbored a TERT-promoter mutation. The Cancer Genome Atlas and NSMP-specific stratifications were prognostic for both the entire cohort and the subset of stage I ovarian tumors. On multivariable analysis, stage, lymphovascular invasion, and tumor mutational burden were prognostic for disease-free survival, whereas advanced stage and TP53-mutant subgroup - but not a TP53 mutation 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 treatments, such as immunotherapy and anti-HER2 agents.

Expanding the Clinicopathologic and Molecular Spectrum of Lipoblastoma-Like Tumor in a Series of 28 Cases.

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 RB1 were identified. The metastatic LLT showed concurrent pathogenic PIK3CA and MTOR activating mutations, both in the primary and in the lung/pleural metastasis; the latter also harbored TERT promoter mutation. One tumor had a pathogenic TSC1 mutation, and one tumor showed 2-copy deletion of CDKN2A, CDKN2B, and MTAP. 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.

Molecular correlates of invasion pattern in HPV-associated endocervical adenocarcinoma: emergence of two distinct risk-stratified tiers.

BACKGROUND: The pattern-based (Silva) classification of invasive human papilloma virus (HPV)-associated endocervical adenocarcinomas (HPVA) is an established and reproducible method to predict outcomes for this otherwise stage-dependent group of tumours. Previous studies utilising targeted sequencing have shown a correlation between mutational profiles and an invasive pattern. However, such correlation has not been explored using comprehensive molecular testing. DESIGN: Clinicopathologic data including invasive pattern (Silva groups A, B, and C) was collected for a cohort of invasive HPVA, which previously underwent massive parallel sequencing using a panel covering 447 genes. Pathogenic alterations, molecular signatures, tumour mutational burden (TMB), and copy number alterations (CNA) were correlated with pattern of invasion. RESULTS: Forty five HPVA (11 pattern A, 17 pattern B, and 17 pattern C tumours) were included. Patients with pattern A presented at stage I with no involved lymph nodes or evidence of recurrence (in those with >2 months of follow-up). Patterns B and C patients also mostly presented at stage I with negative lymph nodes, but had a greater frequency of recurrence; 3/17 pattern B and 1/17 pattern C HPVAs harboured lymphovascular space invasion (LVI). An APOBEC mutational signature was detected only in Silva pattern C tumours (5/17), and pathogenic PIK3CA changes were detected only in destructively invasive HPVA (patterns B and C). When cases were grouped as low-risk (pattern A and pattern B without LVI) and high-risk (pattern B with LVI and pattern C), high-risk tumours were enriched in mutations in PIK3CA, ATRX, and ERBB2. There was a statistically significant difference in TMB between low-risk and high-risk pattern tumours (P = 0.006), as well as between Pattern C tumours with and without an APOBEC signature (P = 0.002). CNA burden increased from pattern A to C. CONCLUSION: Our findings further indicate that key molecular events in HPVA correlate with the morphologic invasive properties of the tumour and their aggressiveness. Pattern B tumours with LVI clustered with pattern C tumours, whereas pattern B tumours without LVI approached pattern A genotypically. Our study provides a biologic foundation for consolidating the Silva system into low-risk (pattern A + B without LVI) and high-risk (pattern B with LVI and pattern C) categories.