Virchows Archiv

Evaluation of the prognostic potential of histopathological subtyping in high-grade serous ovarian carcinoma

Abstract High-grade serous ovarian carcinoma (HGSOC) can be categorized into four gene expression-based subtypes, with supposedly distinct prognoses and treatment responses. Murakami et al. translated these gene expression-based subtypes into the histopathological mesenchymal, immunoreactive, solid and proliferative, and papilloglandular subtypes, showing differences in survival outcomes. Miyagawa et al. refined these criteria to improve the interobserver concordance. The current retrospective study evaluated the interobserver variability and the prognostic differences between the histopathologic subtypes using the criteria of both Murakami et al. and Miyagawa et al. in 208 HGSOC cases. The mesenchymal subtype was considered first, followed by the immunoreactive subtype. Non-conforming cases were categorized as solid and proliferative or papilloglandular. The mesenchymal subtype was identified in 122 patients (58.7%) for both criteria. Using the criteria of Murakami et al., 10 cases (4.8%) were immunoreactive, 26 (12.5%) solid and proliferative, and 50 (24%) papilloglandular, with a concordance rate of 62.5% (κ = 0.34, p < .001). Using the Miyagawa et al. criteria, 23 cases (11%) were immunoreactive, 20 (9.6%) solid and proliferative, and 43 (20.7%) papilloglandular. No survival differences were observed between the subtypes. The fair reproducibility of the histopathological subtype classification of HGSOC and the lack of survival differences among these subtypes indicate the need for further refinement of the criteria and exploration of their correlation with overall survival outcomes before clinical application.

Endometrial endometrioid adenocarcinoma with a malignant squamous component: is the unusual metastatic pattern unique of these tumors?

The FIGO scheme is currently applied for tumor grading of endometrioid adenocarcinoma. The current report presents a series of ten cases of endometrioid carcinomas that when applying the FIGO grading does not fully convey the true biological nature of the disease. The squamous component of these tumors is malignant; it constitutes the predominant invasive component, and it often metastasizes to unconventional sites. Half of the cohort developed distant disease recurrence within 2 years, even those with early-stage disease. Somatic mutations were analyzed, targeting 101 genes in all ten cases, and mutations in PTEN, MMR, PIK3CA, ATM, RB1, and TP53 genes were detected, often multiple mutations in the same case. None of the cases revealed unique molecular signatures or previously unreported gene mutations. Immunohistochemical staining for beta-catenin showed aberrant nuclear staining in eight of ten cases and remaining two showed cytoplasmic and membranous staining. Aggressive behavior and unusual sites of metastases are observed in this series even in low-grade tumor. The FIGO grading on smaller samples may be deceptive for these cases. Even if FIGO is applied, the pathology report should emphasize the malignant squamous component and its potential significance so that the gynecologic oncology team can formulate appropriate adjuvant treatment upfront. This case series argues that this histology should be regarded as a high-grade endometrioid carcinoma and can show unusual metastatic patterns. Further research is needed with more cases within this histologic subtype to guide recommendations on adjuvant therapies for this aggressive tumor type.

CTNNB1 mutation represents a recurrent driver molecular alteration in a subset of endometrial stromal tumors

Abstract Low-grade endometrial stromal sarcomas (LGESS) and endometrial stromal nodules represent a distinct entity with characteristic morphology, immunohistochemical profile, and molecular features. It has been suggested that the activation of the Wnt signaling pathway is a potential driver in some of these tumors. Approximately 70–75% of LGESS are characterized by non-random recurrent fusions mostly involving JAZF1 and PHF1 genes. Although the exact mechanism remains unclear, activation of the Wnt signaling pathway appears to be related to the functional deregulation of chromatin remodeling complexes caused by these fusion proteins. However, knowledge about other possible mechanisms and recurrent molecular alterations occurring at the DNA level in LGESS remains limited. We report three cases of LGESS in patients aged 39, 49, and 50, lacking recurrent fusions but harboring CTNNB1 mutations in exon 3 as the sole detectable molecular alteration. One case had morphological features of typical LGESS (in some areas with perivascular whorling, which was not a dominant feature), the second case showed features of the fibroblastic variant of LGESS, and the third case comprised a LGESS with a peculiar morphology with diffuse whorling morphologically identical to the recently described entity endometrial stromal tumor with GREB1::CTNNB1 fusion. RNA-Seq-based clustering analysis of the three cases and a set of 193 uterine tumors showed that the CTNNB1 -mutated cases clustered near LGESS cases. This study adds to the growing body of evidence that CTNNB1 mutations represent a driver molecular event in a small subset of endometrial stromal tumors. Moreover, the results of our study suggest that tumors with CTNNB1 mutation and GREB1::CTNNB1 fusion may exhibit identical morphology and potentially represent the same category of tumor. Characterization and reporting of additional cases are needed to determine whether these are part of the spectrum of low-grade endometrial stromal tumors (either endometrial stromal nodule or LGESS) or represent a separate category of tumors characterized by CTNNB1 alteration as a driver event.

ERBB2/ERBB3-mutated S100/SOX10-positive uterine sarcoma: something new

A distinctive subset of uterine mesenchymal tumors display recurrent genetic fusions involving receptor tyrosine kinases, including NTRK, PDGFB, FGFR1, and RET, presumably leading to aberrant pathway activation. A pair of recent studies have highlighted the existence of a genetic fusion-negative uterine sarcoma that is characterized by activating mutations in ERBB2/ERBB3, CDKN2A deletion, inactivating ATRX mutation, and a S100 + /SOX10 + immunohistochemical profile. This report describes another case of this emerging entity that was diagnosed in a 57-year-old woman. The 8-cm tumor was centered in the uterine cervix and was comprised mostly of spindle cells configured in fascicles. The tumor was diffusely immunoreactive for SOX10 and S100, with more localized staining for CD68, CD56, MITF, and PRAME. HMB-45, ER, PR, HER2, Melan-A/MART1, STAT6, pan-TRK, ALK, CD34, desmin, CD10, myogenin, and pancytokeratins were all negative, and there was retained expression of H3K27me3. The following molecular alterations were found: ERBB2 p.Val777Leu, ATRX p.F2113Sfs*, CDKN2A deep deletion, NF1 p.W2317*, SMARCA4 p691Sfs*. The authors review the sparse literature on molecular-genetic aberrations involving the epidermal growth factor receptor family of receptor tyrosine kinases (ERBB1/EGFR, ERBB2, ERBB3, and ERBB4) in uterine mesenchymal tumors, a review that suggests that such tumors may be pathologically heterogeneous. The potential clinical significance of demonstrating a targetable ERBB2/ERBB3 tyrosine kinase mutation or other EGFR family aberrations, as well as its distinctive pathologic profile, supports the segregation of the tumor reported herein as a distinct and emerging entity.

PAX8 expression in cancerous and non-neoplastic tissue: a tissue microarray study on more than 17,000 tumors from 149 different tumor entities

AbstractPAX8 plays a role in development of the thyroid, kidney, and the Wolffian and Mullerian tract. In surgical pathology, PAX8 immunohistochemistry is used to determine tumors of renal and ovarian origin, but data on its expression in other tumors are conflicting. To evaluate PAX8 expression in normal and tumor tissues, a tissue microarray containing 17,386 samples from 149 different tumor types and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. PAX8 results were compared with previously collected data on cadherin 16 (CDH16). PAX8 positivity was found in 40 different tumor types. The highest rate of PAX8 positivity was found in thyroidal neoplasms of follicular origin (98.6–100%), gynecological carcinomas (up to 100%), renal tumors (82.6–97.8%), and urothelial neoplasms (2.3–23.7%). Important tumors with near complete absence of PAX8 staining (< 1%) included all subtypes of breast cancers, hepatocellular carcinomas, gastric, prostatic, pancreatic, and pulmonary adenocarcinomas, neuroendocrine neoplasms, small cell carcinomas of various sites, and lymphomas. High PAX8 expression was associated with low tumor grade in 365 non-invasive papillary urothelial carcinomas (p < 0.0001) but unrelated to patient outcome and/or tumor phenotype in clear cell renal cell carcinoma, high-grade serous ovarian cancer, and endometrioid endometrial carcinoma. For determining a renal tumor origin, sensitivity was 88.1% and specificity 87.2% for PAX8, while sensitivity was 85.3% and specificity 95.7% for CDH16. The combination of PAX8 and CDH16 increased specificity to 96.8%. In conclusion, PAX8 immunohistochemistry is a suitable diagnostic tool. The combination of PAX8 and CDH16 positivity has high specificity for renal cell carcinoma.

Molecular characteristics of tubo-ovarian carcinosarcoma at different anatomic locations

AbstractCarcinosarcoma (CS) is an uncommon and clinically aggressive malignancy. The objective of the present study was to characterize the molecular features of CS at various anatomic locations, including serous effusions. Specimens (n = 32) consisted of 25 biopsies/surgical resection specimens and 7 serous effusions (6 peritoneal, 1 pleural) from 25 patients. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. A total of 31 mutations were found in 25 of the 32 tumors studied, of which 1 had 3 mutations, 4 had 2 different mutations, and 20 had a single mutation. The most common mutations were in TP53 (n = 25 in 24 tumors; 1 tumor with 2 different mutations), with less common mutations found in RB1 (n = 2), MET (n = 1), KRAS (n = 1), PTEN (n = 1), and KIT (n = 1). Patient-matched specimens harbored the same TP53 mutation. Tumors with no detected mutations were more common in serous effusion specimens (3/7; 43%) compared with surgical specimens (4/25; 16%). In conclusion, the molecular landscape of CS is dominated by TP53 mutations, reinforcing the observation that the majority of these tumors develop from high-grade serous carcinoma. Whether CS cells in serous effusions differ from their counterparts in solid lesions remains uncertain.

Ovarian endometrioid carcinoma with a sex cord-like pattern: a morphological, immunohistochemical, and molecular analysis

Abstract Sex cord-like endometrioid carcinoma (SCLEC) is an uncommon entity which may constitute a diagnostic challenge. This study aimed to perform a clinicopathological, immunohistochemical, and molecular reappraisal of ovarian SCLEC. Consecutive ovarian SCLECs cases from a single institution were reviewed during a 13-year period. Twenty-three immunohistochemical markers were tested; 10 genes were analyzed by next-generation sequencing. Nine cases of ovarian SCLEC were identified. Mean patient age was 65.7 years; three cases showed extraovarian extension. Architectural pattern included sertoliform (n = 2), granulosa-like (n = 2), and mixed granulosa-like/sertoliform (n = 5). Eosinophilic changes accompanied by increased nuclear atypia were observed in four tumors. Endometrioid features (glands, squamous/morular differentiation) were observed in six cases. Most tumors were positive for cytokeratin-7 (8/9), EMA (9/9), estrogen and progesterone receptor (9/9), CD10 (7/9, including a luminal pattern reminiscent of mesonephric neoplasms), nuclear β-catenin (8/9), and CDX2 (8/9). A minority of cases showed block-type p16 pattern (2/9), PAX8-positivity (3/9), and non-diffuse positivity for WT1 (1/9), inhibin (1/9), chromogranin (1/9), and synaptophysin (2/9). All cases were negative for GATA3, TTF1, calretinin, and SF1. Ki67 range was 15–90%. Six cases showed CTNNB1 exon 3 mutation. Eight cases were of “no specific molecular profile” (NSMP) and one was p53-abnormal. In conclusion, SCLECs frequently exhibit a mixed sertoliform/granulosa-like architecture and express epithelial markers, hormone receptors, nuclear β-catenin, and CDX2, with luminal CD10 positivity and CTNNB1 mutations. PAX8 expression is often lost, while other mesonephric, sex cord, and neuroendocrine markers are negative.

Morphological and immunohistochemical evaluation in distinguishing post-radiotherapy serous-like endometrial change (PoRSEC) and serous endometrial intraepithelial carcinoma (SEIC)

AbstractUteri from women undergoing chemoradiotherapy (CRT) may show reactive atypia which may mimic serous endometrial intraepithelial carcinoma (SEIC). We aimed to assess the prevalence and morphological/immunohistochemical features of post-radiotherapy serous-like endometrial changes (PoRSEC) in women undergone CRT for locally advanced cervical cancer, with a focus on the differential diagnosis with SEIC. Consecutive patients with locally advanced cervical cancer undergone CRT between 2011 and 2018 were reviewed. Endometrial histological specimens were assessed for the presence of PoRSEC. Twenty-two cases of SEIC were included for comparison. Immunohistochemistry for p53, p16, and Ki67 was performed. Out of 244 reviewed patients, 36 (14.7%) showed PoRSEC. The degree of nuclear atypia was similar between PoRSECs and SEIC. However, a papillary architecture with areas of confluent papillae was only observed in SEIC. SEIC cases showed a high mitotic activity as opposed to PoRSEC cases. The expression of p53 was aberrant in all SEICs but in none of the PoRSECs; however, 13/36 PoRSECs showed p53 positivity in most tumor cells, potentially mimicking a mutation pattern. A block-type p16 expression was observed in all SEICs and in 16/36 PoRSECs. Mean Ki67 expression was 26.9% in SEIC (range 5–70%) and 8.16% in PoRSEC (range 5–35%). While SEIC showed sharp morphological and immunohistochemical demarcation, PoRSEC were more heterogenous and merged imperceptibly with normal endometrium. In conclusion, PoRSEC may mimic SEIC both morphologically and immunohistochemically. However, a papillary architecture with cytological demarcation is typically observed in SEIC but not in PoRSEC.

The effect of the peritoneal tumor microenvironment on invasion of peritoneal metastases of high-grade serous ovarian cancer and the impact of NEOADJUVANT chemotherapy

Peritoneal metastases of high-grade serous ovarian cancer (HGSOC) are small-sized deposits with superficial growth toward the peritoneal cavity. It is unknown whether integrity of the peritoneal elastic lamina (PEL) correlates with the peritoneal tumor microenvironment (pTME) and whether neoadjuvant chemotherapy (NACT) affects the pTME. We explored integrity of PEL, composition of pTME, effects of NACT, and the prognostic implications in patients with extensive peritoneal metastases of HGSOC. Peritoneal samples (n = 69) were collected during cytoreductive surgery between 2003 and 2016. Clinical data were collected from medical charts. Integrity of PEL was evaluated with elastic stains. T cell (CD3, CD8) and M2-macrophage markers (CD163) were scored using algorithms created in definiens tissue studio. Patients with a disrupted PEL (n = 39; 57%), more often had residual disease after surgery (p = 0.050), compared to intact PEL. An intact PEL was associated with increased intraepithelial (ie) CD8+ cells (p = 0.032), but was not correlated with improved survival. After NACT, increased ieCD3+ cells were shown, compared to no-NACT (p = 0.044). Abundance of total CD3+ and CD8+ cells were associated with PFS (multivariate HR 0.40; 95%CI 0.23-0.69 and HR 0.49; 95%CI 0.29-0.83) and OS (HR 0.33; 95%CI 0.18-0.62 and HR 0.36; 95%CI 0.20-0.64). M2-macrophage infiltration was not correlated with survival. NACT increases abundance of ieCD3+ cells in peritoneal metastases of HGSOC. Increase of CD3+ and CD8+ cells is associated with improved PFS and OS. This suggests that CD3+ and CD8+ cells may function as prognostic biomarkers. Their role as predictive biomarker for chemotherapy or immunotherapy response in HGSOC warrants further research.

EPM2 AIP1 immunohistochemistry as a surrogate of promoter methylation analysis in endometrial carcinoma

Abstract Mismatch repair (MMR) status in endometrial carcinoma (EC) is crucial for diagnosis, prognosis, treatment, and Lynch syndrome pre-screening. MLH1 loss is the most frequent cause of MMR deficiency and usually by promoter hypermethylation. We tried to confirm the role of EPM2 AIP1 immunohistochemistry as a surrogate of MLH1 promoter methylation in EC. Case series from two different institutions were analyzed by comparable methods using immunohistochemistry for MMR proteins and EPM2 AIP1, and pyrosequencing for MLH1 methylation. In the first series of 70 cases, concordance was 100%, after reassessing three cases with methylation scores close to cut-off, by tumor cell enrichment. In the second series of 29 MLH1-deficient ECs, concordance was 96.5%, while in the control group of 30 MMR-proficient EC, one MLH1-positive case was EPM2 AIP1-negative. EPM2 AIP1 immunoreactivity was qualitatively superior in curettages and biopsies compared to hysterectomy. We conclude that EPM2 AIP1 immunohistochemistry is a good surrogate for MLH1 promoter methylation analysis, cost-effective with short turnaround time, but needs attention regarding preanalytical handling, normal tissue contamination, or low tumor percentage.

Uterine sarcoma with KAT6B/A::KANSL1 fusion: a molecular and clinicopathological study on 9 cases

Abstract Uterine sarcomas with KAT6B/A::KANSL1 fusion represent a new entity characterized by bland morphology, commonly with hybrid features of low-grade endometrial stromal sarcoma (LG-ESS) and tumors with smooth muscle differentiation. In our study, we performed a detailed morphological, immunohistochemical, and molecular analysis of 9 cases of these tumors. Six of those had been originally diagnosed as LG-ESS, one as leiomyoma, one as leiomyosarcoma, and the remaining case as sarcoma with the KAT6B/A::KANSL1 fusion. Seven cases showed overlapping features between endometrial stromal and smooth muscle tumors, one case resembled cellular leiomyoma, and one case resembled high-grade endometrial stromal sarcoma. Immunohistochemically, the tumors showed a common expression of smooth muscle markers and endometrial stromal markers. Molecular findings showed the KAT6B/A::KANSL1 fusion in all cases (by NGS and FISH). In addition, mutations affecting genes such as TP53, PDGFRB, NF1, RB1, PTEN, ATM, RB1, FANCD2, and TSC1 were present in all 5 cases with aggressive behavior. One patient with no evidence of disease showed no additional mutations, while another harbored a mutation of a single gene (ERCC3). Of the 8 patients with available follow-up, two died of disease, 3 are currently alive with disease, and 3 have no evidence of disease. The correct recognition of tumors with the KAT6B/A::KANSL1 fusion is essential because despite the bland morphological features of most cases, these tumors have a propensity for aggressive behavior.

Immunohistochemical analysis of 147 cases of low-grade endometrial stromal sarcoma: refining the immunohistochemical profile of LG-ESS on a large, molecularly confirmed series

Abstract Low-grade endometrial stromal sarcoma (LG-ESS) can present diagnostic challenges, due to its overlapping morphological features with other uterine mesenchymal tumors. Misdiagnosis rates remain significant, and immunohistochemical data for LG-ESS are limited to small series and inconsistent antibody panels. This study aimed to refine the IHC profile of LG-ESS by analyzing a large, molecularly confirmed series of 147 cases using a panel of 24 antibodies, including newer markers like transgelin and smoothelin. CD10 and IFITM1, key endometrial stromal markers, were expressed in 86% (92% of those extensively) and 69% (60% of those extensively) of cases, with fusion-positive tumors showing significantly higher expression. Smooth muscle markers (α-SMA, desmin, h-caldesmon, calponin, transgelin) were variably expressed, predominantly in focal or low-intensity patterns, with α-SMA reaching the highest frequency of expression (44%). However, the intensity of smooth muscle marker expression was usually very low. Smoothelin was rarely expressed. Hormone receptors were frequently positive, with PR showing a higher frequency (92% vs. 83%) and intensity than ER. Markers like S-100, HMB45, and CD117 were largely negative; all tumors were p53 wild-type, with preserved SMARCB1/SMARCA4 expression and ALK and ROS1 negativity. This work represents the largest molecularly validated IHC study on LG-ESS, providing a robust diagnostic profile for routine pathology. By addressing key diagnostic limitations and examining newer markers, our study supports a more standardized approach to diagnosing LG-ESS and underscores the value of immunohistochemical panels, particularly in fusion-negative tumors where diagnosis relies on morphological and immunohistochemical interpretation. These findings contribute critical data for improving diagnostic accuracy.

Endometrial carcinomas with ambiguous histology often harbor TP53 mutations

Abstract The objective of the present study was to characterize the molecular features of endometrial carcinomas with ambiguous histology. Eighteen carcinomas that could not be conclusively typed based on morphology and immunohistochemistry underwent analysis of mismatch repair (MMR) status, microsatellite status, and whole-exome sequencing. None of the tumors had pathogenic POLE mutation. Twelve tumors (67%) were microsatellite stable, and 6 (33%) had microsatellite instability. Fourteen tumors (78%) harbored TP53 mutations, and 2 (11%) had mutations in MMR genes. Eleven carcinomas (61%) were classified as copy number high and 7 (39%) as MSI-hypermutated, the latter including 3 tumors with TP53 mutation who concomitantly had MSI or mutation in a MMR gene. Other mutations that were found in > 1 tumor affected MUC16 (7 tumors), PIK3CA (6 tumors), PPP2R1A (6 tumors), ARID1A (5 tumors), PTEN (5 tumors), FAT1 (4 tumors), FAT4 (3 tumors), BRCA2 (2 tumors), ERBB2 (2 tumors), FBXW7 (2 tumors), MET (2 tumors), MTOR (2 tumors), JAK1 (2 tumors), and CSMD3 (2 tumors). At the last follow-up (median = 68.6 months), 8 patients had no evidence of disease, 1 patient was alive with disease, 8 patients were dead of disease, and 1 patient died of other cause. In conclusion, based on this series, the molecular landscape of endometrial carcinomas with ambiguous histology is dominated by TP53 mutations and the absence of POLE mutations, with heterogeneous molecular profile with respect to other genes. A high proportion of these tumors is clinically aggressive.

Molecular substratification of endometrial carcinomas with no special molecular profile (NSMP) by using a limited NGS custom panel may facilitate effective patient selection for the PIK3CA-targeted therapy

Abstract Endometrial carcinomas (EC) of no special molecular profile (NSMP) represent the largest molecular category of EC, comprising a mixture of tumors with different histology and molecular profiles. These facts likely point to different tumor biology, clinical outcomes, and targeted therapy responses within this molecular category. The PIK3CA is currently the only targetable kinase oncoprotein directly implicated in EC carcinogenesis. Investigating a unique single-institution cohort, we attempted to stratify NSMP ECs based on the presence of the PIK3CA pathogenic mutation. Those cases were further analyzed for other well-established-associated oncogenic driver gene mutations. Histological and clinical variables were also correlated in each case. Altogether, 175 ECs were prospectively tested by a limited custom NGS panel containing ARID1A, BCOR, BRCA1, BRCA2, CTNNB1, KRAS, MLH1, MSH2, MSH6, NRAS, PIK3CA, PMS2, POLD1, POLE, PTEN,and TP53 genes. We identified 24 PIK3CA mutated cases in the group of 80 NSMP ECs, with another co-occurring mutation in at least one oncogenic driver gene (CTNNB1, PTEN, ARID1A, KRAS, BCOR, PMS2) in 19 cases. In conclusion, a limited NGS panel can effectively test EC tissue for specific pathogenetically relevant oncogene mutations. The NSMP EC category contains 30% of the PIK3CA mutated cases. Of those, 21% contain the PIK3CA mutation as a sole EC-associated oncogene mutation, while 79% harbor at least one more mutated gene. These findings may inform future healthcare planning and improve the effectiveness of EC patient selection for the PIK3CA-targeted therapy.

Validation of Modaplex POLE mutation assay in endometrial carcinoma

AbstractThe TCGA-based molecular classification of endometrial cancer has emerged as an important tool to stratify patients according to prognosis. A simplified scheme has been proposed, by using immunohistochemistry for p53, MSH6, and PMS2 and a molecular test for POLE mutations (NGS or Sanger sequencing, techniques that are not available in many centers worldwide). In this study, we validate a novel method that allows simultaneous analysis of multiple pathogenic POLE mutations. The Modaplex technology integrates polymerase chain reaction and capillary electrophoresis. The design of this study encompassed 4 different steps: (1) a retrospective-pilot phase, with 80 tumors, balancing the four molecular subgroups. (2) A retrospective phase of 25 tumors obtained between 2016 and 2020, and 30 tumors obtained between 2000 and 2015. (3) An inter-laboratory corssavalidation step with 19 cases (belonging to phases 1 and 2). (4) A prospective cohort of 123 tumors, of unknown POLE status, with simultaneous validation by Sanger sequencing. A total of 258 samples were analyzed. In the first and second phases, the test showed positive/negative predictive values of 100%, by correctly identifying POLE mutation status in 79/79 and 55/55 cases. Phase 3 showed 100% of inter-laboratory consistency. Phase 4 showed 16 positive samples out of the 123 prospective cases. Overall, the test has revealed sensitivity and specificity of 100%, identifying a total of 47 POLE-mutated tumors. We have shown that this technique allows faster and easier identification of multiple pathogenic POLE mutations with high robustness and confidence when comparing to other tests such as Sanger sequencing.

Histological classification of mucinous ovarian tumors: inter-observer reproducibility, clinical relevance, and role of genetic biomarkers

The morphological distinction between the various types of mucinous ovarian tumors has major prognostic implications but may be challenging. The aims of our study were to describe inter-observer reproducibility in the morphological diagnosis of mucinous ovarian tumors, to evaluate the clinical relevance of possible diagnostic discrepancies, and to identify molecular abnormalities correlated with the histological type. Seventy-nine ovarian mucinous borderline tumors (MOB) and either expansile or infiltrative carcinomas (MOC) were independently reviewed by two gynecological pathologists. Molecular analysis was performed in 32 cases. Concordance between the two pathologists was reached in 67 cases (k: 0.78). The main discrepancies (8/12) were the evaluation of nuclear grade 3 or that of microfoci ( 5 mm) infiltrative-type pattern of invasion. No specific or sensitive molecular profile might help in the differential diagnosis of mucinous ovarian tumors.

Pathological processing of sentinel lymph nodes in endometrial carcinoma — routine aspects of grossing, ultra-staging, and surgico-pathological parameters in a series of 833 lymph nodes

AbstractSentinel lymph nodes are widely accepted in the treatment of endometrial carcinoma. Whereas surgical aspects are well studied, the pathological work-up in terms of grossing, frozen section, and the so-called ultra-staging is still a matter of debate. This results in conflicting national or center-based recommendations. In a series of consecutive 833 sentinel lymph nodes from 206 patients in endometrial carcinomas, we compared three different grossing techniques and the use of frozen section in terms of anatomy, detection rates, and survival. In total, 42 macro-metastases, 6 micro-metastases, and 25 nodes with isolated tumor cells were found. Lymph nodes affected at least with micro-metastasis were about 0.5cm enlarged. Detection rates in lamellation technique increased with a step of 5.9% to 8.3% in comparison to bi-valved or complete embedding. The lamellation technique presented with a slight beneficial prognosis in pN0 subgroup (OS, p=0.05), which besides size effects might be attributed to trimming loss. In frozen section, this effect was less pronounced than expected (OS, p=0.56). Ultra-staging only revealed additional micro-metastases and isolated tumor cells. Exclusively, macro-metastases showed poor survival (p<0.001). In multivariate analysis, T-stage, subtype, and lympho-vascular invasion status outperformed this staging parameter significantly. Grossing of sentinel lymph nodes is the most essential step with evidence to prefer lamellation in 2 mm steps. Step sectioning should consider widely spaced protocols to exclude macro-metastases. Frozen sections might add value to the intra-operative assessment of endometrial carcinoma in selected cases. The excellent biological behavior of cases with isolated tumor cells might question the routine application of pan-cytokeratin as ultra-staging method.

Recommendations for diagnosing STIC: a systematic review and meta-analysis

AbstractOur understanding of the oncogenesis of high-grade serous cancer of the ovary and its precursor lesions, such as serous tubal intraepithelial carcinoma (STIC), has significantly increased over the last decades. Adequate and reproducible diagnosis of these precursor lesions is important. Diagnosing STIC can have prognostic consequences and is an absolute requirement for safely offering alternative risk reducing strategies, such as risk reducing salpingectomy with delayed oophorectomy. However, diagnosing STIC is a challenging task, possessing only moderate reproducibility. In this review and meta-analysis, we look at how pathologists come to a diagnosis of STIC. We performed a literature search identifying 39 studies on risk reducing salpingo-oophorectomy in women with a known BRCA1/2 PV, collectively reporting on 6833 patients. We found a pooled estimated proportion of STIC of 2.8% (95% CI, 2.0–3.7). We focused on reported grossing protocols, morphological criteria, level of pathologist training, and the use of immunohistochemistry. The most commonly mentioned morphological characteristics of STIC are (1) loss of cell polarity, (2) nuclear pleomorphism, (3) high nuclear to cytoplasmic ratio, (4) mitotic activity, (5) pseudostratification, and (6) prominent nucleoli. The difference in reported incidence of STIC between studies who totally embedded all specimens and those who did not was 3.2% (95% CI, 2.3–4.2) versus 1.7% (95% CI, 0.0–6.2) (p 0.24). We provide an overview of diagnostic features and present a framework for arriving at an adequate diagnosis, consisting of the use of the SEE-FIM grossing protocol, evaluation by a subspecialized gynecopathologist, rational use of immunohistochemical staining, and obtaining a second opinion from a colleague.

Uterine mesenchymal tumour with a novel EWSR1::CTBP1 gene fusion

Abstract A growing number of mesenchymal uterine tumours are defined by a specific molecular aberration. We present the case of a 65-year-old woman who presented with postmenopausal bleeding and a large intramural uterine tumour. Histopathological analysis of the resected uterus revealed an intramural, mesenchymal neoplasm comprising of spindle and epithelioid cells, with no immunohistochemical expression of lineage-specific markers and low proliferative activity. Molecular testing using next-generation sequencing (NGS) revealed an EWSR1::CTBP1 gene fusion, which was confirmed by the presence of EWSR1 gene rearrangement detected using fluorescence in situ hybridisation (FISH). Staging revealed no further tumour manifestations, and the 3-year follow-up was uneventful. The EWSR1::CTBP1 gene fusion has never previously been reported in uterine tumours, having been reported in the literature only once, in the context of a gastroblastoma. The presented case expands the range of the gene-fusion-associated mesenchymal tumours of the uterus.

HPV-independent squamous sell carcinoma of cervix: a clinicopathological, immunohistochemical, and molecular analysis of six cases

Abstract HPV-independent squamous cell carcinoma (SCC) of the cervix is a rare subtype associated with aggressive clinical behavior and poor prognosis, as reported in limited studies with small sample sizes. This study aims to investigate the clinicopathological, immunohistochemical, and molecular characteristics of six cases of HPV-independent SCC. Formalin-fixed, paraffin-embedded tissues from six patients were analyzed using immunohistochemistry and Next-Generation Sequencing (NGS). Human papillomavirus (HPV) testing was performed using the Aptima HPV assay, and immunohistochemical markers, including p16, p53, PD-L1, HER2 and CK5/6 were evaluated. The median age at diagnosis was 62 years. Key histopathological features included an infiltrative-destructive growth pattern, pronounced keratinization, and extensive necrosis in the majority of cases. All tumors were p16-negative and high-risk HPV-negative by the Aptima HPV assay, confirming their HPV-independent status. One case exhibited p53 overexpression and HER2 positivity (score 3 +). Five out of six patients succumbed to the disease. FIGO stages ranged from IIB to IVB. Molecular analysis identified mutations in TP53 and PIK3CA, as well as novel CTNNB1 and TERT promoter mutations, and HER2 amplification . In five of six patients PD-L1 expression with at least CPS score of 2,5 was observed. HPV-independent cervical SCC typically affects older patients and is associated with poor clinical outcomes. This study highlights TP53 and PIK3CA mutations and novel findings, including CTNNB1, TERT promoter mutations, and HER2 amplification. The observed PD-L1 expression patterns may suggest potential benefits from immunotherapy. Further research is warranted to better understand the clinicopathologic characteristics and molecular drivers of HPV-independent SCC and to explore novel therapeutic strategies.

SWI/SNF protein expression in anaplastic carcinomatous mural nodules of ovarian mucinous tumors

Primary mucinous cystadenocarcinoma of the breast commonly harbours TP53 mutations and PI3K/AKT pathway alterations

Uterine mesenchymal tumours harboring the KAT6B/A::KANSL1 gene fusion represent a distinct type of uterine sarcoma based on DNA methylation profiles

AbstractUterine mesenchymal tumours harboring KAT6B/A::KANSL1 gene fusions typically exhibit histological and immunophenotypic overlap with endometrial stromal and smooth muscle tumours. To date it remains uncertain whether such neoplasms should be regarded as variants of smooth muscle or endometrial stromal neoplasm, or if they constitute a distinct tumour type. In this study we investigated DNA methylation patterns and copy number variations (CNVs) in a series of uterine tumours harboring KAT6B/A::KANSL1 gene fusions in comparison to other mesenchymal neoplasms of the gynecological tract. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a distinct cluster for 8/13 KAT6B/A::KANSL1 tumours (herein referred to as core cluster). The other 5 tumours (herein referred to as outliers) did not assign to the core cluster but clustered near various other tumour types. CNV analysis did not identify significant alterations in the core cluster. In contrast, various alterations, including deletions at the CDKN2A/B and NF1 loci were identified in the outlier group. Analysis of the DNA methylation clusters in relation to histological features revealed that while tumours in the core KAT6B/A::KANSL1 cluster were histologically bland, outlier tumours frequently exhibited “high-grade” histologic features in the form of significant nuclear atypia, increased mitotic activity and necrosis. Three of the five patients with outlier tumours died from their disease while clinical follow-up in the remaining two patients was limited (less than 12 months). In comparison, none of the 7 out of 8 patients with tumors in the core KAT6B/A::KANSL1 sarcoma cluster, where follow-up was available, died from disease. Furthermore, only 1 out of 7 patients recurred (mean follow-up of 30 months). In conclusion, KAT6B/A::KANSL1 uterine sarcoma is a molecularly unique type of uterine tumour that should be recognized as a distinct entity. These tumors typically exhibit low-grade histologic features but are occasionally morphologically high-grade; the latter have a DNA methylation profile different from the typical low-grade neoplasms and may be associated with aggressive behaviour.

S100 and CD34 positive spindle cell tumors of the uterine cervix with EGFR mutation: a hitherto unrecognized neoplasm phenotypically and epigenetically overlapping with “NTRK-rearranged spindle cell neoplasms” of the uterus

Abstract NTRK-rearranged spindle cell neoplasm represents an emerging entity included in the latest 5th edition of WHO classification of both soft tissue and female genital tumors. By immunohistochemistry, they are commonly positive for CD34, S100 protein, and CD30 and typically harbor fusions of kinase genes such as NTRK1/2/3, RET, and BRAF. In the gynecological tract, they typically affect the uterine cervix or uterine body. Most of the reported cases had fibrosarcoma-like morphology, occasionally showing perivascular and stromal hyalinization with only a few cases showing a less cellular spindle cell proliferation. Except for one case with RET fusion, all other gynecological cases harbored exclusively NTRK1/2/3 fusions. Besides kinase gene fusions, the analogous tumors in soft tissues may also harbor activating EGFR or BRAF point mutations, but no such case has been described in the uterus. Herein we are reporting two cases from the uterine cervix showing morphology and molecular features previously unreported at this anatomic site. The patients were 46 and 34 years old and clinically presented with unremarkable cervical polyps each measuring 8 mm in diameter. Histologically, both cases had a rounded polypoid outline and were composed of hypocellular proliferation of bland spindle cells lacking mitotic activity and growing in a fibrotic stroma which was punctuated by prominent small vessels with thick hyalinized walls. Immunohistochemically, both showed a diffuse expression of CD34, CD30, and S100 protein, whereas SOX10 was negative. Both cases harbored exon 20 EGFR mutation and did not reveal any fusions or significant copy number changes. The patient in case 1 was treated by hysterectomy with salpingectomy with no other residual tumor detected, and she was alive and well 27 months after the diagnosis. The patient in case 2 had no other known tumors at the time of diagnosis, but no follow-up is available. We believe the reported cases represent a hitherto unrecognized variant of “NTRK-rearranged spindle cell neoplasms” of the uterine cervix with novel EGFR mutations.

ERBB2/ ERBB3-mutated S100/ SOX10-positive unclassified high-grade uterine sarcoma: first detailed description of a novel entity

AbstractWith the increasing use of innovative next generation sequencing (NGS) platforms in routine diagnostic and research settings, the genetic landscape of uterine sarcomas has been dynamically evolving during the last two decades. Notably, the majority of recently recognized genotypes in uterine sarcomas represent gene fusions, while recurrent oncogene mutations of diagnostic and/ or therapeutic value have been rare. Recently, a distinctive aggressive uterine sarcoma expressing S100 and SOX10, but otherwise lacking diagnostic morphological, immunophenotypic and molecular features of other uterine malignancies has been presented in a scientific abstract form (USCAP, 2023), but detailed description and delineation of the entity is still missing. We herein describe two high-grade unclassified uterine sarcomas characterized by spindle to round cell morphology and diffuse expression of S100 and SOX10, originating in the uterine body and cervix of 53- and 45-year-old women and carrying an ERBB3 (p.Glu928Gly) and an ERBB2 (p.Val777Leu) mutation, respectively. Both tumors harbored in addition genomic HER2 amplification, ATRX mutation and CDKN2A deletion. Methylation studies revealed a methylome most similar to MPNST-like tumors, but distinct from melanoma, MPNST, clear cell sarcoma, and endometrial stromal sarcoma. Case 1 died of progressive peritoneal metastases after multiple trials of chemotherapy 47 months after diagnosis. Case 2 is a recent case who presented with a cervical mass, which was biopsied. This study defines a novel heretofore unrecognized aggressive uterine sarcoma with unique phenotypic and genotypic features. Given the potential value of targeting HER2, recognizing this tumor type is mandatory for appropriate therapeutic strategies and for better future delineation of the entity.

CD34-positive pleomorphic uterine sarcoma with NUDT3::RAD51B fusion

Sarcomas with RAD51B fusions are rare, recently recognized neoplasms that predominantly arise in the uterus. They showed heterogeneous phenotypic features and are typically associated with aggressive biological behaviors. To date, only 14 cases of RAD51B-rearranged sarcomas have been reported in English literature, including 6 perivascular epithelioid cell tumors, 6 leiomyosarcomas, and 2 undifferentiated sarcomas (both uterine in origin). We present an additional case of uterine sarcoma with RAD51B rearrangement. The patient was a 71-year-old woman with a 5.0-cm polypoid mass in the uterine cavity. Histologically, the tumor was composed of spindle to pleomorphic cells arranged in storiform and fascicular patterns within variably myxoid and collagenous stroma. The neoplastic cells had vesicular to hyperchromatic nuclei with occasional multinucleation, with a mitotic rate of 3/10 high-power fields. Notable stromal features included staghorn shaped blood vessels, thick band-like collagen deposition, and prominent chronic inflammatory infiltrates. Immunohistochemically, the tumor cells showed diffuse and strong positivity for CD34, P53, and P16, with focal expression of pan-TRK and smooth muscle actin. Targeted RNA-sequencing revealed NUDT3::RAD51B fusion, which was confirmed by fluorescence in situ hybridization of RAD51B rearrangement. The patient had been free of disease at 22 months' follow-up. This case further illustrates the phenotypic heterogeneity of RAD51B-rearranged sarcomas and expands their clinicopathological and molecular genetic spectrum.

Analysis of HNF1β expression suggests that its downregulation is involved in the sarcomatoid transformation of yolk sac tumor

Sarcomatoid yolk sac tumor postpubertal-type (YSTpt) is a rare phenotype of germ cell tumor that occurs mostly after chemotherapy. Its diagnosis is clinically relevant but challenging, due to its somewhat inconspicuous histologic features and negative/low expression of classical YSTpt makers (α-fetoprotein (AFP), glypican-3 (GPC3), and GATA3)). HNF1β is likely a key inducer of the YSTpt phenotype, acting in part by regulating the binding of FOXA2 to its target genomic sequences. Recently, immunohistochemical studies have shown that HNF1β has a sensitivity comparable to FOXA2 (higher than GPC3 and AFP) for non-sarcomatoid YSTpt. We found that sarcomatoid YSTpt did not express HNF1β (0: 8/8 (100%)), suggesting that combined downregulation of FOXA2 and HNF1β may underlie the sarcomatoid transformation in this rare phenotype of germ cell tumor.

Practical guidelines of the EOTTD for pathological and genetic diagnosis of hydatidiform moles

Hydatidiform moles are rare and thus most pathologists and geneticists have little experience with their diagnosis. It is important to promptly and correctly identify hydatidiform moles given that they are premalignant disorders associated with a risk of persistent gestational trophoblastic disease and gestational trophoblastic neoplasia. Improvement in diagnosis can be achieved with uniformization of diagnostic criteria and establishment of algorithms. To this aim, the Pathology and Genetics Working Party of the European Organisation for Treatment of Trophoblastic Diseases has developed guidelines that describe the pathological criteria and ancillary techniques that can be used in the differential diagnosis of hydatidiform moles. These guidelines are based on the best available evidence in the literature, professional experience and consensus of the experts' group involved in its development.

Renal cell tumor with sex-cord/gonadoblastoma-like features: analysis of 6 cases

Renal tumors are one of the most diverse groups of tumors in pathology. Many emerging and important entities have been described recently. Here, we describe a series of renal tumors occurring in adult patients, with distinct histologic features, and with a striking resemblance to gonadal sex cord-stromal tumors. Patients were three males and three females aged 39-82 years; tumor size ranged from 0.9 to 3.6 cm. Five tumors were organ-confined, while one case had a focal perinephric invasion. No aggressive behavior was noted. Microscopically, all the tumors were composed of loose or compact tubular structures with elongated or angulated shapes. The tumor cells were cylindrical or cuboidal, with pale eosinophilic cytoplasm, irregular nuclear membranes, and ISUP/WHO grade 2-3 nuclei. The stroma showed focal or prominent collagen deposition with prominent basement membrane-like material. In all cases, the tumor cells were positive for PAX8, CD10, and vimentin and retained positivity for FH and SDHB. Cathepsin K and AMACR were variably positive. Tumors were negative for HMB45, Melan A, TFE3, SF1, inhibin, calretinin, ER, PR, CD117, OCT3/4, SALL4, ALK, and WT1. Molecular studies showed no abnormalities in TFEB, TFE3, or FH genes. In 3/4 tested cases, mutation of the NF2 gene was present. In all the tested male cases, loss of the Y chromosome was found. In the relatively short follow-up, these tumors appear to have indolent behavior. This study expands the clinicopathologic diversity of renal cell tumors by describing a series of potentially novel tumors morphologically resembling gonadal sex-stromal tumors, with negativity for sex cord-stromal markers. Potential relationship to recently described biphasic hyalinizing psammomatous renal cell carcinoma is discussed.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

The role of FOXL2, SOX9, and β-catenin expression and DICER1 mutation in differentiating sex cord tumor with annular tubules from other sex cord tumors of the ovary

Sex cord tumor with annular tubules (SCTAT) is a highly rare type of ovarian sex cord-stromal tumor (SCST), the diagnosis of which remains to be challenging. The aim of this study was to scrutinize the utility of three immunohistochemical markers including Forkhead box protein 2 (FOXL2), SOX9, and β-catenin and DICER1 mutation status in distinguishing SCTATs from other ovarian SCSTs. Nine cases of SCTAT, 10 Sertoli-Leydig cell tumor (SCLT), 10 adult-type granulosa cell tumor (AGCT), and 8 juvenile-type granulosa cell tumor (JGCT) were included in the study. SCTATs were characterized by diffuse and strong expression of SOX9, focal and weak expression of FOXL2, and the absence of DICER1 mutation. However, AGCTs and JGCTs displayed strong and diffuse expression of FOXL2, focal/no immunoreaction for SOX9. SLCTs generally showed moderate intensity of FOXL2 and SOX9 expression. Nuclear β-catenin expression was observed in none of SLCT, 1/9 of SCTAT, 6/8 JGCT, and 4/10 AGCT cases, respectively. DICER1 hotspot mutation was detected in only 3 cases of SLCT and 2 cases of JGCT. We conclude that in addition to strong and diffuse SOX9 expression, weak/absent expression of FOXL2 is suggestive for the diagnosis of SCTAT. Hence, we suggest that inclusion of these two markers, SOX-9 and FOXL2, to the immunohistochemical panel helps in differentiation of SCTAT from other SCSTs in addition to morphologic findings. We also conclude that SCTATs of the ovary do not harbor DICER1 hotspot mutation.

Neuroendocrine tumor (NET) of the vagina in the light of WHO 2020 2-tiered grading system: clinicopathological report of the first described case

Up to now, a vaginal well-differentiated neuroendocrine tumor (NET) has never been well described in the literature. A 2-cm vaginal nodule morphologically revealed a proliferation of mild to moderately atypical eosinophilic epithelioid cells, without tumor cell necrosis. Immunohistochemistry (IHC) showed positivity for CK (AE1/AE3), chromogranin A, and synaptophysin, focal positivity for CDX2, and negativity for PAX8 and TTF1. Metastatic origin was excluded by imaging and the morphological context with benign mucinous glands as present in the surgical resection specimen. Considering mitotic index and Mib1/Ki67 (8 mitoses/2 mm

The cytokeratin 17 expression in primary ovarian tumors has diagnostic but not prognostic significance

We assessed the value of cytokeratin 17 (CK17) expression for the differential diagnosis between primary ovarian mucinous tumors and metastases from the gastrointestinal tract (GIT) and the significance of CK17 expression in a broad spectrum of primary ovarian tumors with respect to their prognosis. The sample set consisted of 554 primary ovarian tumors and 255 GIT tumors. In the primary ovarian tumors, a higher CK17 expression (in > 10% of tumors cells) was present only in 0-11.4% of all tumors (including mucinous tumors, micropapillary serous borderline tumors, clear cell, endometrioid, and high-grade serous carcinomas). The only exception was low-grade serous carcinoma, where higher CK17 expression was present in 24% of cases. Concerning GIT tumors, the higher levels of CK 17 expression (in > 10% of tumor cells) were observed in the upper GIT tumors (68.5% of pancreatic ductal adenocarcinoma, 61.6% of gallbladder adenocarcinoma, and 46% of gastric adenocarcinoma), which differs substantially not only from most of the primary ovarian tumors, but also from colorectal carcinoma (3.7%; p < 0.001). The results of our study suggest that expression of CK17 can potentially be used as an adjunct marker in differential diagnosis between primary ovarian mucinous tumors and metastases from the upper GIT, but not from colorectal carcinoma. However, in GIT tumors, CK17 can be used in the differential diagnosis between adenocarcinomas of the upper and lower GIT. Statistical analysis did not reveal strong association of CK17 expression with clinicopathological variables or patient outcomes in any primary ovarian tumors.

Microcystic stromal tumor of the ovary: a recurrent case with somatic CTNNB1 missense mutation

Microcystic stromal tumors (MCSTs) of the ovary are rare sex cord-stromal tumors that are considered benign neoplasms because almost all cases display unilateral, localized lesions and have benign outcomes, except for one recurrent case with familial adenomatous polyposis and another initial metastatic case with a CTNNB1 mutation. We report herein a sporadic case that relapsed as intra-abdominal spread 9 years and 1 month after primary left salpingo-oophorectomy for torsion of the ovarian tumor pedicle. The tumor relapsed as multiple disseminations at the subabdominal wall, Douglas pouch, and omentum. Histologically, the tumor cells formed microcysts and infiltrated the surrounding adipose tissue, similar to the invasive implants of ovarian epithelial borderline tumors. Mutation analysis of the recurrent tumor revealed a somatic CTNNB1 p.S33Y activated missense mutation and a germline KDR p.Q472H variant. In conclusion, long-term clinical follow-up may be needed to detect any recurrence of MCST, irrespective of familial adenomatous polyposis.

The evolving spectrum of endocervical adenocarcinoma in situ (AIS)

Immunophenotype analysis using CLDN18, CDH17, and PAX8 for the subcategorization of endocervical adenocarcinomas in situ: gastric-type, intestinal-type, gastrointestinal-type, and Müllerian-type

A classification system for invasive endocervical adenocarcinoma (ECA) focusing on high-risk human papillomavirus (HPV) detection has been recently developed. However, precursor lesions of each ECA subtype and immunohistochemical markers that effectively subcategorize ECAs with gastric and intestinal differentiation have not been fully described. Here, we aimed to subcategorize endocervical adenocarcinoma in situ (AIS) by immunophenotype and to characterize the histopathology of each AIS subtype. We immunohistochemically analyzed 36 AIS and 25 lobular endocervical glandular hyperplasia (LEGH) samples using three cell lineage-specific markers (CLDN18, gastric epithelial cells; CDH17, intestinal epithelial cells; and PAX8, Müllerian epithelial cells). The AISs were immunophenotypically classified as gastric-type (G-AIS; n = 2), intestinal-type (I-AIS; n = 10), gastrointestinal-type (GI-AIS; n = 3), Müllerian-type (M-AIS; n = 18), and AIS, not otherwise specified (AIS-NOS; n = 3). All 25 LEGHs were categorized as gastric-type. G-AIS had pale eosinophilic or clear cytoplasm with a small amount of apical mucin and fewer mitotic bodies. I-AIS comprised various numbers of goblet cell-type tumor cells. GI-AIS showed intermediate or mixed features of G-AIS and I-AIS. M-AIS, as with the usual-type ECA, was typically characterized by mucin depletion; however, several lesions had abundant cytoplasmic mucin. High-risk HPV was detected in most AISs but was negative in 100% (2/2) of G-AIS, 10% (1/10) of I-AIS, and 6% (1/18) of M-AIS lesions. In summary, the AIS subtypes defined by immunophenotype had distinct histopathological and etiological characteristics. Thus, immunophenotyping with CLDN18, CDH17, and PAX8 might improve the diagnostic accuracy of histopathological classifications of ECAs.

Relevance of routine pathology review in cervical carcinoma

To determine the impact of pathology review on the management of patients with cervical carcinoma, 264 reports of pathology review from 230 patients referred to Erasmus MC (2010-2012) were studied retrospectively. Discrepancies between pathologic diagnoses were classified as 'major' if they led to changes in treatment, and as 'minor' where there was no change. Patient and tumor characteristics were analyzed to identify the factors influencing these discrepancies. Fifty-eight (25.2%) discrepancies were identified; 28 (12.2%) were major, these resulted frequently from missing essential information, or discordant assessment of tumor invasion. Pathology review prevented under-treatment of 3.5%, over-treatment of 1.3%, treatment for incorrect malignancy of 1.3%, and enabled definitive treatment of 6.1% of patients. This highlights the importance of pathology review for appropriate management. Major discrepancies were rare (1%) for patients with macroscopic tumor and histologic diagnosis of squamous cell carcinoma (n = 100). For these patients, yield of pathology review may be limited.

Immunohistochemical expression of PRAME in 485 cases of epithelial tubo-ovarian tumors

Preferentially expressed antigen of melanoma (PRAME) is a cancer/testis antigen selectively expressed in somatic tissues and various solid malignant tumors and is associated with poor prognostic outcome. Our research aimed to comprehensively compare its expression in a large cohort of tubo-ovarian epithelial tumors and examine its correlation with our clinico-pathologic data, as well as to assess its potential use in diagnostics and therapy.We examined 485 cases of epithelial tubo-ovarian tumors including 107 clear cell carcinomas (CCC), 52 endometroid carcinomas (EC), 103 high grade serous carcinomas (HGSC), 119 low grade serous carcinomas (LGSC)/micropapillary variant of serous borderline tumors (mSBT), and 104 cases of mucinous carcinomas (MC)/mucinous borderline tumors (MBT). The immunohistochemical analysis was performed using TMAs.The highest levels of expression were seen in EC (60%), HGSC (62%), and CCC (56%), while expression in LGSC/mSBT (4%) and MC/MBT (2%) was rare. The clinico-pathologic correlations and survival analysis showed no prognostic significance.The results of our study showed that PRAME is neither prognostic nor a suitable ancillary marker in the differential diagnosis of tubo-ovarian epithelial tumors. Nevertheless, knowledge about the PRAME expression may be important concerning its potential predictive significance, because targeting PRAME as a potential therapeutic option is currently under investigation.

Histomorphologic assessment and distribution of high-risk human papillomavirus (HPV) types in cervical high-grade squamous intraepithelial lesions with unusual histomorphologic features

In rare cases, equivocal histomorphology ('deceiving dysplasia') does not allow immediate diagnosis of cervical high-grade squamous intraepithelial lesion (HSIL). We studied whether these cases are correlated with specific high-risk human papillomavirus (hr HPV) types. During 2011-2017, 39 cases of p16-positive cervical tissue biopsies with unusual ('deceiving') dysplastic histomorphology were identified and matched with the same number of controls (typical HSIL samples). Histomorphological characteristics were reviewed blindly and HPV testing was performed using the clinically validated RealTime test (Abbott) and Anyplex HPV 28 (Seegene). HPV 16 and HPV 31 were the two most frequent HPV types in both groups, although minimum, proportional, hierarchical and any etiological attribution estimates for HPV 16 were significantly lower in the deceiving group (13.2%, 21.3%, 23.7% and 23.7%) than in the control group (32.4%, 48.1%, 48.6% and 48.6%). In addition, the distribution of other hr HPV types differed between the two study groups, with five HPV types (HPV 56, 58, 59, 73 and 82) detected only in the deceiving group. Histomorphologic review of both groups (regardless of HPV type) confirmed significant differences in nuclear atypia, maximum lesion thickness and cellularity, although these were diminished when cross-comparisons between HPV16/18 and non-HPV16/18 cases pooled from both study groups were evaluated. Different attribution estimates for HPV 16, HPV 16/18 and non-16/18 hr HPV types in deceiving and control groups were observed, in particular for HPV 16. However, an unusual (deceiving) histomorphology may also depend on unknown HPV-related molecular changes.

A novel tumor budding and cell nest size-based grading system outperforms conventional methods in vulvar squamous cell carcinoma

The tumor budding and tumor cell nest size-based (TBNS) grading scheme is an emerging prognostic indicator for squamous cell carcinoma (SCC) across various organs; however, its significance in vulvar SCC (VSCC) remains poorly investigated. In this study, we applied the TBNS grading system to an institutional cohort of 62 consecutive surgically resected VSCC cases (39 HPV-independent and 23 HPV-associated), excluding patients with neoadjuvant chemotherapy or FIGO stage IA disease. High tumor budding activity, small cell nest size, and high TBNS grade were significantly associated with reduced overall survival (OS) and disease-free survival (DFS) in VSCC, as well as with adverse clinicopathologic features such as lymphovascular space invasion, perineural involvement, lymph node metastasis, and advanced FIGO stage (p  0.05). We conclude that the TBNS grading system is a promising prognostic indicator for VSCC, outperforming conventional grading systems. Further validation in larger cohorts is needed to expand the clinical applicability of this grading system in VSCC.

Utility of special AT-rich sequence-binding protein 2 (SATB2) immunohistochemistry as a marker for secondary perianal paget disease

A panel-based approach using immunohistochemistry (IHC) is currently used for subtyping perianal Paget disease (PPD) in the absence of a synchronous neoplasm. Special AT-rich Sequence Binding Protein 2 (SATB2) has been established as a sensitive and specific marker for lower gastrointestinal tract carcinomas. We evaluated its performance as a marker of secondary PPD. A panel of IHCs including CK7, CK20, GCDFP-15, CDX2, and SATB2 were performed on fifteen cases of PPD (identified between 1991-2001) and seven cases of primary vulvar Paget disease with perianal involvement. Eight cases (53%) were classified as secondary PPD based on the presence of a synchronous (n = 7) or a metachronous neoplasm (n = 1). There was no differential staining for CK7 (positive in 7/7 primary vs. 7/8 secondary PPD; P = 1.00) and CK20 (positive in 4/7 primary vs. 8/8 secondary PPD; P = .08). GCDFP-15 was positive in 5/7 cases of primary PPD while negative in all cases of secondary PPD (P = .01). CDX2 was positive in all cases of secondary PPD (P = .001) while SATB2 was positive in 7/8 cases of secondary PPD (P = .01). Both CDX2 and SATB2 were positive in 1/7 cases of primary PPD. The addition of an IHC panel in conjunction with clinical/imaging findings can help definitively classify PPD as either primary or secondary in most cases. We show that SATB2 has comparable performance to CDX2 and can be a helpful additional tool.

Clinicopathological and prognostic significance of immunophenotypic characterization of endocervical adenocarcinoma using CLDN18, CDH17, and PAX8 in association with HPV status

In 2020, the WHO published a new system for classifying invasive endocervical adenocarcinoma based on histological features and high-risk human papillomavirus (HPV) infection. However, immunophenotypes of each histological subtype require further investigation. We immunohistochemically analyzed 66 invasive endocervical adenocarcinomas using three cell-lineage-specific markers: claudin 18 (CLDN18) for gastric, cadherin 17 (CDH17) for intestinal, and PAX8 for Müllerian epithelial cells. We identified five immunophenotypes of endocervical adenocarcinoma: gastric (21%); intestinal (14%); gastrointestinal (11%); Müllerian (35%); and not otherwise specified (NOS) (20%). Adenocarcinomas with gastric immunophenotype, characterized by aging (p = 0.0050), infrequent HPV infection (p < 0.0001), concurrent lobular endocervical glandular hyperplasia (p = 0.0060), lymphovascular invasion (p = 0.0073), advanced clinical stage (p = 0.0001), and the poorest progression-free (p < 0.0001) and overall (p = 0.0023) survivals, were morphologically compatible with gastric-type adenocarcinoma of the WHO 2020 classification. Conversely, most adenocarcinomas with Müllerian (91%) and intestinal (89%) immunophenotypes were HPV associated and morphologically compatible with usual- or intestinal-type adenocarcinomas of the WHO 2020 classification. The morphology of adenocarcinomas with gastrointestinal immunophenotype was intermediate or mixed between those of gastric and intestinal immunophenotypes; 57% were HPV associated. Adenocarcinomas with NOS immunophenotype were mainly HPV associated (85%) and histologically poorly differentiated. Multivariate analysis revealed that gastric (p = 0.008), intestinal + gastrointestinal (p = 0.0103), and NOS (p = 0.009) immunophenotypes were independent predictors of progression-free survival. Immunophenotypes characterized by CLDN18, CDH17, and PAX8 exhibited clinicopathological relevance and may improve the diagnostic accuracy and prognostic value of conventional histological classification.

Gastric-type cervical adenocarcinoma with squamous differentiation: buried in adenosquamous carcinomas?

Gastric-type adenocarcinoma (GAS) of the cervix is a human papilloma virus (HPV)-independent, aggressive, and chemo-resistant adenocarcinoma. However, although the histopathological features of GAS have been extensively investigated, squamous differentiation has not been mentioned. This study aimed to elucidate the frequency of GAS with squamous differentiation and describe their clinicopathological characteristics. We retrospectively evaluated 78 patients with GAS (n = 13) and adenosquamous carcinoma (n = 65) diagnosed between 2000 and 2020. Two patients with GAS with squamous differentiation were identified. Both tumors showed advanced stage (pT2bN1) and had predominant GAS and merged squamous cell carcinoma components without p16-block positivity and HPV DNA. Gastric-type adenocarcinoma in situ was confirmed in both cases. Some cases of GAS could show squamous differentiation mimicking the usual, HPV-associated, adenosquamous carcinoma. GAS with squamous differentiation is recognized as an HPV-independent cancer.

Primary lymphoma of the uterine cervix: a clinicopathologic study of 13 cases with review of additional 54 cases in the literature

Primary lymphoma of the uterine cervix (LUCX) is extremely rare, and its diagnosis is challenging. However, its clinicopathological features have not been well characterized. Thirteen primary LUCX patients were retrospectively studied, and 54 patients from the literature were reviewed. Primary LUCX was identified in 0.22% (13/6000) of patients with uterine cervical malignancies in our institution. The patients' median age was 51 years (range: 22-85 years). All patients had a bulk of neoplasms in the uterine cervix. The median tumour diameter was 6 cm (range: 1.5-10 cm). Approximately 78.0% (39/50) of the patients initially presented with irregular vaginal bleeding or discharge. Moreover, 86.7% (39/45) had Ann Arbor stage I or II. Diffuse large B-cell lymphoma, not otherwise specified, was the most common type, accounting for 85.0% (57/67) of primary LUCX cases. Follicular lymphoma (7.5%, 5/67), extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (4.5%, 3/67), mantle cell lymphoma (MCL), blastoid variant (1.5%, 1/67), and peripheral T-cell lymphoma (1.5%, 1/67) were occasionally observed. Three patients (7.1%, 3/42) with DLBCL, NOS died from the disease during the follow-up period. Their 5-year overall survival (OS) rate was 93.5%. The patient with MCL, blastoid variant in our present cohort died of the disease 33 months after diagnosis. Primary LUCX is an extremely rare condition. The clinical symptoms are non-specific. DLBCL, NOS is the most common histologic type, showing a favourable outcome with accurate diagnosis and timely and optimal treatment.

SATB2 is expressed in neuroendocrine carcinoma of the uterine cervix

Neuroendocrine carcinoma (NEC) of the uterine cervix is less characterized than neuroendocrine neoplasms of other sites such as of the digestive system and the lung. Special AT-rich sequence-binding protein 2 (SATB2) recently emerged as a marker of well-differentiated neuroendocrine tumors of the lower gastrointestinal (GI) tract. Among NECs, SATB2 is more frequently expressed in cutaneous Merkel cell carcinoma than in NEC of other anatomical sites. In our study, we performed an immunohistochemical study of SATB2 in 16 NECs of the uterine cervix, where the expression of these markers is still undefined. SATB2 was expressed in 12/16 cervical NECs (75%), with 7/16 cases (44%) showing SATB2 positivity in ≥ 50% of cells. In 7 cervical NECs associated with a non-neuroendocrine component, the expression of SATB2 was restricted to the neuroendocrine component. SATB2 was positive in all cases that expressed CDX2 (n = 7) and TTF1 (n = 5), with no evident association with p16 and p53. Our study demonstrated that SATB2 is often expressed in NECs of the uterine cervix. This information should be taken into account when assessing the origin of a NEC.

Epithelioid leiomyosarcoma harboring HMGA2::RAD51B fusion: expanding the spectrum of RAD51B-rearranged uterine sarcoma

Endometrial carcinomas with solid basaloid morphology and geographic necrosis: clinicopathological and molecular features of 18 cases

Pilomatrix-like high-grade endometrioid carcinoma (PiMHEC) is characterized by solid basaloid morphology, geographic necrosis, and shadow cells, often associated with CTNNB1 mutations and aberrant β-catenin expression, exhibiting aggressive behavior. However, high-grade endometrial carcinomas with similar solid basaloid/geographic necrosis (SB-GN) features may overlap diagnostically with PiMHEC. This study aimed to investigate the clinicopathological and molecular characteristics of endometrial carcinomas with SB-GN and compare them with PiMHEC. Eighteen endometrial carcinomas with SB-GN (including 6 PiMHECs) were diagnosed in Fudan University Shanghai Cancer Center. Histopathological, immunohistochemical, and molecular features based on next-generation sequencing were reviewed. In our cohort, recurrence/metastasis occurred early (median: 5 months; range: 0-21). Most cases had classic basaloid tumor cells with solid growth pattern. Spindle-shaped cells or tumor cells with more cytoplasm were occasionally observed. Twenty-eight percent of patients had squamous differentiation and 33% had shadow cells. In solid components, aberrant β-catenin expression was observed in 77.8% of cases. Eighty-three percent harbored CTNNB1 exon 3 mutations (frequent co-mutations: PTEN, ARID1A, PIK3CA). Molecular classification revealed 28% MMR-deficient, 22% POLE-mutated, 6% p53-abnormal, and 44% no specific molecular profile (NSMP). Endometrial carcinomas with SB-GN showed no significant differences compared to PiMHECs, except for higher PR positivity in solid components (58.3% vs. 0%, p = 0.046). Endometrial carcinomas with SB-GN represent a distinct and aggressive tumor group characterized by solid growth pattern, prominent necrosis, frequent basaloid morphology, high rates of CTNNB1 mutations, and aberrant β-catenin staining. Our results demonstrate that endometrial carcinomas with SB-GN and PiMHECs share similar clinicopathologic and molecular profiles, supporting a unified biological spectrum.

FOXA1 expression and its association with mucin expression and KRAS mutation in ovarian mucinous tumors: implications for tumor progression and differentiation

This study aimed to investigate forkhead box A1 (FOXA1) expression in ovarian mucinous tumors and its association with mucin expression and KRAS mutation status to clarify its role in tumor progression and differentiation. We analyzed 57 normal tissues or benign ovarian lesions, 110 mucinous ovarian tumors, including mucinous carcinomas, and 214 other ovarian epithelial carcinomas using immunohistochemistry for FOXA1, MUC2, MUC5AC, and MUC6. We also performed KRAS mutation analysis. Strong nuclear staining of FOXA1 was observed in Walthard nests, Brenner tumors, and fallopian tube ciliated epithelium. FOXA1 expression was significantly associated with mucinous histology in ovarian epithelial carcinomas (P < 0.001). In mucinous tumors, FOXA1 was expressed in 73.6% of cystadenomas, 91.4% of borderline tumors, 100% of borderline tumors with intraepithelial carcinomas, and 87.5% of carcinomas. MUC2 expression progressively increased from mucinous cystadenomas to borderline tumors (P < 0.050) and significantly correlated with FOXA1 expression (P = 0.024). The prevalence of KRAS mutations also tended to increase with the malignancy of mucinous tumors (P < 0.050); however, KRAS mutations were significantly enriched in FOXA1-negative cystadenomas compared with FOXA1-positive cystadenomas (P < 0.050). A stepwise increase was noted in the percentage of both KRAS mutations and FOXA1 expression from cystadenoma to carcinoma. Mucinous ovarian tumors commonly express FOXA1. The co-occurrence of KRAS mutations and FOXA1 expression may be important for driving the progression and intestinal differentiation of mucinous ovarian tumors.

Endometrial assessment for fertility-sparing treatment of young women with early-stage endometrial carcinoma: adaptation of a standardized synoptic reporting

Recent guidelines emphasize regular endometrial assessment for fertility-sparing treatment (FST) in endometrial carcinoma (EC), but no consensus exists on histological reporting. We reviewed 14 patients under 40 years old with early-stage, low-grade endometrial endometrioid carcinoma (EEC) treated with FST. Our patients received oral therapy (28.6%) or combined oral therapy with intrauterine device (71.4%), and were followed for 11-76 months (median, 31.5 months). Using a classification proposed by Wheeler et al., treatment outcomes included resolution (50%), persistence (14.3%), progression (14.3%), and recurrence (21.4%). Resolution occurred within 4-15 months. Two patients achieved successful pregnancies. Three underwent hysterectomy, including one with progression and distant metastasis. We find that young women with EEC often extend FST beyond recommended durations due to strong fertility desires, increasing progression risk. Standardized synoptic reporting with routine reviews of prior biopsies, obtaining deeper tissue sections, and seeking second opinions can improve endometrial assessment and guide treatment.

An extensive immunohistochemical analysis of 290 ovarian adult granulosa cell tumors with 29 markers

The current knowledge about the immunohistochemical features of adult granulosa cell tumor (AGCT) is mostly limited to the "traditional" immunohistochemical markers of sex cord differentiation, such as inhibin, calretinin, FOXL2, SF1, and CD99. Knowledge about the immunohistochemical markers possibly used for predictive purpose is limited. In our study, we focused on the immunohistochemical examination of 290 cases of AGCT classified based on strict diagnostic criteria, including molecular testing. The antibodies used included 12 of the "diagnostic" antibodies already examined in previous studies, 10 antibodies whose expression has not yet been examined in AGCT, and 7 antibodies with possible predictive significance, including the expression of HER2, PD-L1, CTLA4, and 4 mismatch repair (MMR) proteins. The results of our study showed expression of FOXL2, SF1, CD99, inhibin A, calretinin, ER, PR, AR, CKAE1/3, and CAIX in 98%, 100%, 90%, 78%, 45%, 41%, 94%, 82%, 26%, and 9% of AGCT, respectively. GATA3, SATB2, napsin A, MUC4, TTF1, and CD44 were all negative. PTEN showed a loss of expression in 71% of cases and DPC4 in 4% of cases. The aberrant staining pattern (overexpression) of p53 was found in 1% (3/268) of cases, 2 primary tumors, and 1 recurrent case. Concerning the predictive markers, the results of our study showed that AGCT is microsatellite stable, do not express PD-L1, and are HER2 negative. The CTLA4 expression was found in almost 70% of AGCT tumor cells.

Immunophenotypic analysis of angiomyolipoma with epithelial cysts, comparison to mixed epithelial and stromal tumors and epithelial and stromal elements of normal kidney and ovaries

Angiomyolipoma with epithelial cysts (AMLEC) is a rare variant of renal angiomyolipoma (AML). It is characterized by a conventional AML component admixed with epithelial cysts within an "ovarian-like" stroma. Mixed epithelial and stromal tumor (MEST) is another renal neoplasm featuring epithelial cysts and "ovarian-like" stroma. While there is consensus that in MEST the epithelial and stromal components are neoplastic, in AMLEC it has been hypothesized that the epithelial component may represent renal tubular entrapment or ovarian-like transdifferentiation of tumor cells. The aim of this study was to compare the immunophenotypes of the epithelial-stromal components of AMLEC and MEST, with normal kidney and ovary to provide additional insights into the pathogenesis and relationships of these entities. In this study, we analyzed eight cases of AMLEC and 14 cases of MEST from 2003 to 2023. We used tissue microarrays, full sections, or unstained slides with an immunohistochemical panel including renal and ovarian markers: SF1, ER, PR, AR, PAX8, WT1, GATA3, CA-IX, p16, inhibin A, and BCL2. We compared these cases with ten non-neoplastic ovary and kidney samples. Our findings indicate that the epithelial component of AMLEC and MEST resembles hormone receptor positive renal tubular epithelium (AR + /ER - /PR -). AMLEC's stromal component resembled hormone receptor positive renal stroma, while MEST's resembled ovarian stroma, supporting mullerian transdifferentiation. Our study showed that the epithelial and stromal components of AMLEC and MEST are immunophenotypically different and also differ from normal tissues. Our findings suggest that in AMLEC, the epithelial-stromal component represents a hormonally driven proliferation of non-neoplastic renal elements within a dysregulated tumor microenvironment.

Spatial expression of claudin 18.2 in matched primaries and metastases of tubo-ovarian carcinoma of all subtypes

AbstractPhysiologically, claudin 18 splice variant 2 (CLDN18.2) expression is restricted to the gastric epithelium, but its expression has been detected in solid cancers. Zolbetuximab, a chimeric IgG1 antibody targeting CLDN18.2, has demonstrated promising effects in patients suffering from CLDN18.2-positive, HER2-negative locally advanced gastric cancer and is currently being studied further. To date, little is known about CLDN18.2 expression in other histological subtypes of tubo-ovarian carcinoma (TOC) and their matching metastases.Using a cohort of all histological TOC subtypes, we investigated the immunohistochemical (IHC) CLDN18.2 expression in both TOCs (n = 536), their matching metastatic tissue (n = 385) and in 93 metastases without primary. Tissue microarrays comprised both the tumor center and periphery. IHC positivity was defined as biomarker expression of ≥ 75% in tumor cells with moderate-to-strong membranous staining.Overall CLDN18.2 positivity was 4.1% (21/515) in the TOC centers and 3.6% (18/498) in their peripheries. In primaries of mucinous tubo-ovarian carcinoma (MTOC), CLDN18.2 positivity rates were 45% (18/40) and 36.6% (15/41), respectively. Positivity rates for the corresponding metastases were 33% (4/12, center) and 27% (3/11, periphery). The expression was relatively homogenous throughout all tumor sites. With no expression in 99.5% of nonmucinous tumors, CLDN18.2 positivity was almost exclusively seen in the mucinous subtype.In tubo-ovarian carcinoma, CLDN18.2 expression was, with rare exceptions, restricted to the mucinous subtype. Among them, 33% of metastasized MTOCs presented with CLDN18.2 positivity. Hence, CLDN18.2 might display a promising target for personalized therapy in patients with advanced MTOC.

Molecular features for timely cancer diagnosis and treatment – tumors of the ovary, fallopian tube and endometrium

Gynecologic pathology has moved, within only a few years, from being a diagnostic area devoid of molecular testing into a diagnostic discipline in which such analyses are becoming routine. The direct relevance of molecular characterization to the choice of treatment of patients with carcinomas originating in both the uterus and adnexae makes it likely that such testing will only expand along with our understanding of the molecular make-up of these tumors. As a consequence, gynecologic pathologists have become an integral part of patient management, rather than lab personnel providing external services.In parallel, molecular testing is expanding as a tool for diagnosing rare tumors affecting these organs, including soft tissue tumors, sex cord-stromal tumors and germ cell tumors, as well as other rare entities. Increased knowledge in this area bears directly on the ability to diagnose these tumors in a reproducible manner, as well as recognize and consult on genetic diseases. Hopefully, despite the inherent difficulty in studying rare cancers, it will also translate into new therapeutic options for the malignant ones among these rare cancers.

An update on the role of SMARCA4 deficiency in gynecological neoplasms: how and where

SMARCA4, encoding the BRG1 protein, is a crucial component of the SWI/SNF chromatin remodeling complex, essential for regulating gene expression and maintaining genomic integrity; our knowledge regarding the role of these genes continues to evolve. Deficiency or loss of SMARCA4 expression has been implicated in the development and progression of various gynecological neoplasms, notably small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), SMARCA4-deficient uterine sarcoma, and some subtypes of endometrial carcinoma such as undifferentiated or dedifferentiated carcinoma. Given its central role in oncogenesis, targeting SMARCA4-deficient tumors has become an area of active research, with potential therapeutic strategies above all in these aggressive gynecological tumor subtypes. This review summarizes current knowledge of the role of SMARCA4 deficiency in gynecological cancers, discussing the morphological and immunophenotypic characteristics of the different entities, emphasizing clinical implications and potential targeted therapies. Further understanding of its structure, function, and therapeutic vulnerabilities is crucial for improving patient outcomes.

Endometrial stromal tumor with whorling and GREB1::CTNNB1 fusion: expanding the knowledge on a recently described entity

Endometrial stromal tumors with whorling and CTNNB1 rearrangement (ESTW-CTNNB1) are recently described gynecological mesenchymal neoplasms with unique clinicopathological and molecular features. To date, only four of these tumors have been reported. Herein, we describe a case of ESTW-CTNNB1 in a 67-year-old woman. The tumor measured 10 cm and had well-defined margins. On histology, the neoplasm was mostly composed of whorls of epithelioid cells immersed in a loose fibroblastic background, with a delicate vasculature and focal sex cord-like pattern. Tumor cells were mildly atypical, and mitotic index was < 1/10HPF. The tumor showed areas of ischemic necrosis and hydropic changes; no coagulative tumor cell necrosis was observed. On immunohistochemistry, tumor cells were diffusely positive for β-catenin (nuclear), CD10, WT1, calretinin, SATB2, estrogen, and progesterone receptor and negative for CDX2, CK8/18, and p63, with Ki67 = 10%. Molecular analysis revealed a GREB1::CTNNB1 fusion. In conclusion, we reported we 5th case of ESTW-CTNNB1, confirming the previously described features and highlighting positivity for SATB2.

Residual disease volume and prognosis in endometrioid precancer after progestin therapy

Progestin therapy is a conservative treatment option for atypical hyperplasia/endometrioid intraepithelial neoplasia (AEH/EIN), particularly for patients seeking fertility preservation or for whom surgery is not feasible. However, approximately 30% of patients exhibit resistance to therapy, underscoring the need for early identification of responders and non-responders. We conducted a retrospective study of 252 AEH/EIN patients who underwent progestin therapy, with serial follow-up endometrial biopsies yielding 892 samples with quantifiable residual disease (RD). The amount of RD was evaluated as a predictor of therapeutic response, with a focus on its prognostic significance. Among the 252 patients, 194 (77%) were classified as responders, while 58 (23%) were non-responders. Responders exhibited a progressive reduction in RD across follow-up biopsies, with all achieving complete decidualization by the final biopsy. In contrast, non-responders consistently demonstrated persistent RD, with more RD in initial biopsies post progestin therapy significantly correlating with non-response. An amount of RD exceeding 20% in the initial biopsy or a less than 50% reduction in subsequent biopsies strongly predicted therapeutic failure (p < 0.001). The amount of RD is a valuable predictive marker for progestin therapy outcomes in AEH/EIN patients. Incorporating RD volume assessment in pathology reports can enhance clinical decision-making, facilitating more personalized and effective treatment strategies. Early identification of non-responders may prevent prolonged ineffective therapy and enable timely alternative interventions.

Mesonephric-like adenocarcinoma of the endometrium: detailed molecular characterization of the first case showing a morphological continuum from mesonephric-like metaplasia and atypical mesonephric-like hyperplasia to adenocarcinoma

Mesonephric-like adenocarcinoma is a rare subtype of endometrial cancer, characterized by its histological and molecular overlap with mesonephric carcinoma of the uterine cervix. This study presents a unique case of mesonephric-like adenocarcinoma in the endometrium, accompanied by adjacent mesonephric-like hyperplasia and atypical mesonephric-like hyperplasia. Histologically, the transition from mesonephric metaplasia to adenocarcinoma was evident, and molecular analysis revealed shared mutations, including KRAS (p.G12D) in both atypical hyperplasia and adenocarcinoma, and a novel BCOR mutation (p.A1314Nfs*49) in the adenocarcinoma. These findings suggest a Müllerian origin of mesonephric-like adenocarcinoma via mesonephric transdifferentiation, challenging the traditional understanding of mesonephric histogenesis. The identification of these mutations underscores the potential for targeted therapies, particularly in cases with BCOR and KRAS mutations. This study is the first to document preneoplastic lesions in mesonephric-like adenocarcinoma, offering new insights into its pathogenesis and therapeutic implications.

Next-generation sequencing in the molecular classification of endometrial carcinomas: Experience with 270 cases suggesting a potentially more aggressive clinical behavior of multiple classifier endometrial carcinomas

Molecular classification of endometrial carcinomas (EC) divides these neoplasms into four distinct subgroups based on their molecular background. Given its clinical significance, genetic examination is becoming integral to the diagnostic process. This study aims to share our experience with the molecular classification of EC using immunohistochemistry (IHC) and next-generation sequencing (NGS). We included all ECs diagnosed at two institutions from 2020 to the present. All cases were prospectively examined by IHC for MMR proteins and p53, followed by NGS using a customized panel covering 18 genes, based on which ECs were classified into four molecular subgroups: POLE mutated, hypermutated (MMR deficient), no specific molecular profile (NSMP), and TP53 mutated. The cohort comprised 270 molecularly classified ECs: 18 (6.6%) POLE mutated, 85 (31.5%) hypermutated, 137 (50.7%) NSMP, and 30 (11.1%) TP53 mutated. Twelve cases (4.4%) were classified as 'multiple classifier' EC. Notably, most of these cases with available follow-up (6/9) behaved aggressively. Within the POLEmut EC group, 3/4 cases had advanced tumors, including one patient who died of the disease. Similarly, in the MMRd/TP53mut group, 3/5 patients with available follow-up had metastatic disease, leading to death of the patient in 1 case. ECs of NSMP showed multiple genetic alterations, with the most common mutations being PTEN (44% within the group of NSMP), followed by PIK3CA (30%), ARID1A (21%), and KRAS (9%). Our findings suggest that combining immunohistochemistry with NGS offers a more reliable classification of ECs, including 'multiple classifier' cases, which, based on our observations, tend to exhibit aggressive behavior. Additionally, our data highlight the complex genetic background of NSMP ECs, which can facilitate further stratification of tumors within this group and potentially help select patients for dedicated clinical trials.

Endometrial stromal tumor with whorling and GREB1::CTNNB1 fusion—a case report on a rare entity

Endometrial stromal tumors are rare lesions with a diverse morphology, which may make achieving the correct diagnosis challenging in some cases. We report a case of a uterine mesenchymal tumor diagnosed as endometrial stromal nodule with a peculiar whorled morphology and GREB1::CTNNB1 fusion confirmed by transcriptome RNA sequencing. The tumor was sharply demarcated, lacked invasive growth, and had benign behavior, as the patient remained without disease recurrence 15 years later. Immunohistochemically, the tumor cells showed diffuse nuclear expression of beta-catenin, confirming the activation of the beta-catenin pathway. Our case represents only the 4th reported case of CTNNB1-rearranged endometrial stromal tumor with extensive whorling. The biological nature of uterine tumors characterized by whorled morphology and rearrangement of CTNNB1 is not yet clear, which underscores the importance of genetic profiling for accurate diagnosis and potential targeted therapies in malignant cases.

Corded and hyalinized endometrioid endometrial carcinoma with high-grade features: a clinicopathological and TCGA-based molecular analysis

Corded and hyalinized endometrioid carcinoma (CHEC) typically shows low-grade features and "no specific molecular profile" (NSMP). This study aimed to perform a clinicopathological and molecular characterization of endometrial CHEC with high-grade features. Immunohistochemistry for cytokeratin AE1/AE3, e-cadherin, β-catenin, estrogen receptor, progesterone receptor, p53, p16, and mismatch repair proteins was performed. A next-generation sequencing kit was used to assess POLE, POLD1, APC, MLH1, MSH2, MSH6, PMS2, MUTYH, EPCAM, and CTNNB1. Molecular groups, i.e., POLE-mutant, mismatch repair deficient (MMRd), p53-abnormal, and NSMP, were assigned according to the TCGA classifier. Six high-grade endometrial CHECs were identified. The mean age was 57.5 years; 5/6 cases were uterine-confined. Five cases showed a diffusely and markedly atypical corded component and a MMRd or p53-abnormal signature; additional features included single-cell keratinization, necrosis, osteoid or myxoid/chondro-myxoid matrix, foci of anaplasia, and nuclear β-catenin expression. The remaining case showed a low mitotic count and a NSMP phenotype, with focal bizarre cells in an otherwise classical CH endometrioid carcinoma. All cases showed variably reduced expression of epithelial markers and hormone receptors in the corded component. No mutations were found in any of the analyzed genes. In conclusion, high-grade CHECs are a heterogeneous subset of biphasic endometrial carcinoma which show similarities and differences with classical CHEC and carcinosarcoma. These cases often show MMRd or p53-abnormal signatures.

H3K27me3 deficiency in dedifferentiated carcinoma and carcinosarcoma of the endometrium

The present study immunohistochemically investigated trimethylation of lysine 27 of histone 3 (H3K27me3) expression in 769 endometrial carcinomas and 196 uterine mesenchymal tumors. One dedifferentiated endometrial carcinoma (DEC) and one carcinosarcoma showed H3K27me3 deficiency that was limited to undifferentiated and sarcomatous components, respectively. Switch/sucrose nonfermenting (SWI/SNF) complex subunits (SMARCA4, SMARCB1, and ARID1A/1B) and mismatch repair proteins were proficient in both tumors. The dimethylation of H3K27 (H3K27me2) was deficient in the undifferentiated component, whereas the sarcomatous component had scattered H3K27me2-positive cells. CXorf67, which inhibits PRC2 function, was diffusely expressed in the sarcomatous component. CXorf67 was negative in the undifferentiated component, which was submitted to a genetic analysis and showed no alterations in PRC2 core subunits or H3K27. The present results suggest H3K27 methylation dysregulation as a cause of SWI/SNF-proficient DEC and carcinosarcoma and imply differences in their level of and the mechanisms underlying H3K27 methylation dysregulation.

Endometrial carcinomas with dyshesive, eosinophilic, and vacuolated (histiocyte-like) tumor cells: a reactive-like phenotype associated with aggressive behavior

Herein, we present a clinicopathological and molecular analysis of four cases of endometrial carcinoma (EC) diffusely exhibiting dyshesive cells with wide eosinophilic and vacuolated cytoplasm (histiocyte-like tumor cells, HLTCs). We compared these HLTCs to similar cells found in microcystic, elongated and fragmented (MELF) pattern (n = 20) or after neoadjuvant chemotherapy (NAC) (n = 5). The four cases were endometrioid, serous, clear cell, and gastric-type; all were at FIGO stage ≥ III. HLTCs showed an epithelial Müllerian phenotype and at least focal CK20, HNF1β, and CK5/6 expression, with aberrant e-cadherin and β-catenin expression; two cases were MMR-deficient, and one was p53-abnormal; all were POLE wild type. MELF-associated and NAC-associated HLTCs showed similar morphological/immunophenotypical features. However, MELF-associated HLTCs were mainly intraglandular and inflammation-associated, did not form a distinct tumor component, and showed no relationship with lymph node metastases. In conclusion, different histotypes of EC may show a prominent HLTC component, which shows peculiar morphological/immunophenotypical features and appears associated with aggressive behavior.

Genetic features of endometrioid-type endometrial carcinoma arising in uterine adenomyosis

This study aimed to clarify the genetic features of endometrioid-type endometrial cancer arising in adenomyosis (EC-AIA) using targeted sequencing and immunohistochemistry (IHC) for both carcinoma and adjacent adenomyosis tissues. We identified three endometrioid-type EC-AIAs in 689 patients with endometrial cancer; two exhibited grade 3 endometrioid carcinoma. IHC revealed retained expression of PMS2, MSH6, ARID1A, and PAX2. Two of them showed diffuse strong p53 expression only in the carcinoma. PTEN expression was lost in carcinoma of only one of these cases. Carcinoma had many gene mutations than adjacent adenomyosis in all cases. KRAS and TP53 mutations were found in two of them. The other patient had mutations in KRAS, PIK3CA, and PPP2R1A. They were classified as two "p53-mutated" and one "non-specific molecular profile." These molecular alterations in endometrioid-type EC-AIA imply similar carcinogenesis to a subset of endometrial endometrioid carcinoma and might be used as targets of liquid biopsy after further validation.

Uterine cellular leiomyomas are characterized by common HMGA2 aberrations, followed by chromosome 1p deletion and MED12 mutation: morphological, molecular, and immunohistochemical study of 52 cases

Cellular leiomyoma (CL) represents an uncommon variant of uterine leiomyoma with limited data concerning its immunohistochemical and molecular profile. We performed a comprehensive analysis of 52 CL cases all of which were analyzed immunohistochemically. Molecular analysis was possible in 32 cases with sufficient DNA, and 38 cases with sufficient RNA. The immunohistochemical results showed a high expression of smooth muscle markers (calponin (100%), desmin (100%), smooth muscle actin (98.1%), caldesmon (96.1%), transgelin (96.1%), smooth muscle myosin heavy chain (86.5%), and smoothelin (61.5%)). Concerning markers of endometrial stromal differentiation, the expression of CD10 was observed in 65.4% cases (42.2% with H-score > 50), and IFITM1 in 36.5% cases (1.9% with H-score > 50). 36.5% showed HMGA2 overexpression at the IHC level, associated with increased mRNA expression in 14/14 cases. The rearrangement of the HMGA2 gene was detected in 13.2%. Chromosome 1p deletion was found in 19.3%, while 9.4% of tumors showed a pathogenic mutation in the MED12 gene. In conclusion, CL is immunohistochemically characterized by a high expression of "smooth muscle" markers commonly associated with a co-expression of "endometrial stromal" markers, where IFITM1 shows superior performance compared to CD10 regarding its specificity for differentiation from endometrial stromal tumors. The sensitivity of smoothelin in CL seems rather low, but no data is available to assess its specificity. On a molecular level, the most common mutually exclusive aberration in CL affects HMGA2, followed by chromosome 1p deletions and MED12 mutations.

MicroRNA-21 expression in cancer cells is an independent biomarker of progression-free survival of endometrioid endometrial carcinoma

Endometrial carcinoma is one of the most common gynecological cancers. MicroRNA-21 (miR-21) is the most consistently overexpressed miRNA in almost all human cancer types, and it might be a useful clinical biomarker and therapeutic target. However, its precise localization and significance in endometrial carcinoma have not been clarified. This study aimed to examine miR-21 expression in endometrial carcinoma and reveal its clinicopathological importance. We investigated miR-21 expression by in situ hybridization (ISH) using locked nucleic acid (LNA)-modified probes in 230 endometrial carcinoma patients. We evaluated miR-21 expression in cancer cells and stroma separately. High miR-21 expression in cancer cells was significantly associated with higher histological grade and lymph node metastasis. In Kaplan-Meier analysis, high miR-21 expression in cancer cells was significantly associated with poor progression-free survival. In particular, in endometrioid carcinoma, high miR-21 expression in cancer cells was an independent prognostic factor associated with poor progression-free survival, as well as older age and higher International Federation of Gynecology and Obstetrics (FIGO) stage.

Prognostic impact of histological review of high-grade endometrial carcinomas in a large Danish cohort

The aim of this study was to investigate the outcome of histological subtype review of high-grade endometrial carcinoma (EC) and its prognostic impact in a large well-documented Danish nationwide cohort. From the Danish Gynecological Cancer Database (DGCD) 2005-2012 cohort, we included 425 patients with an original diagnosis of high-grade EC, independent of histologic subtype. Of these, at least one hematoxylin and eosin (H&E)-stained slide from 396 cases (93.2%) was available for review. The histologic subtype was reviewed by specialized gynecopathologists blinded to the original diagnosis and clinical outcome. Interobserver variability between original and revised histologic subtypes was analyzed using simple Kappa statistics. Hazard ratios (HR), recurrence-free survival (RFS), and overall survival were calculated for original and revised subtypes, respectively. Overall histologic subtype agreement was moderate (kappa = 0.42) with the highest agreement for endometrioid-type EC (EEC; 75.5%) and serous-type EC (SEC; 63.8%). For clear cell carcinoma and un-/dedifferentiated EC, agreement was significantly lower: 30.1% and 33.3% respectively. Of the 396 reviewed cases, only two (0.5%) were re-classified as low-grade EEC upon revision. Interestingly, GR3 EEC had better RFS than SEC with stronger significance after revision (HR 2.36 (95% CI 1.43-3.89), p = 0.001), compared to original diagnosis (HR 1.74 (95% CI 1.07-2.81), p = 0.024). In conclusion, this study confirmed that pathology review results in substantial shift in histological subtype in high-grade EC. After review, a stronger prognostic benefit for GR3 EEC as compared to other histological subtypes was observed. This work supports maintaining a low threshold for pathology revision of high-grade EC in clinical practice.

Detection of microsatellite instability with Idylla MSI assay in colorectal and endometrial cancer

AbstractUniversal testing of microsatellite instability (MSI) is recommended for colorectal cancer (CRC) and endometrial cancer (EC) to screen for Lynch syndrome and to aid in assessing prognosis and optimal treatment. We compared the performance of Idylla MSI test to immunohistochemistry (IHC) of mismatch repair (MMR) proteins in consecutive series of 100 CRC and 108 EC samples, as well as in retrospective series of 28 CRC and 33 EC specimens with known deficient MMR protein expression. The concordance between the Idylla test and IHC was 100% in all CRC samples (n=128) but lower in EC samples (87.2%; n=141). In the EC samples, sensitivity of Idylla test was 72.7% and specificity 100%. EC MSI/dMMR agreement was 85.4% for MLH1, 87.5% for MSH2, and only 35.3% for MSH6. When we analyzed 14 EC samples that were discrepant, i.e., dMMR using IHC and microsatellite stable using Idylla, with microsatellite markers BAT25 and BAT26, we found four cases to be replication error (RER) positive. All RER positive cases were deficient for MSH6 protein expression. We also re-analyzed EC samples with variable tumor cellularity to determine the limit of detection of the Idylla test and found that a 30% or higher tumor cellularity is required. We conclude that Idylla MSI test offers a sensitive and specific method for CRC diagnostics but is less sensitive in EC samples especially in the case of MSH6 deficiency.

Correlation of PD-L1 expression with immunohistochemically determined molecular profile in endometrial carcinomas

Endometrial carcinoma programmed death-ligand 1 (PD-L1) expression in tumor cells (TCs) and tumor-associated inflammatory cells (ICs) have recently been reported in several studies which vary in terms of their cohort size, design, and methodology. We aimed to assess PD-L1 staining in endometrial carcinomas and correlate this with clinical and pathological factors and PTEN, ARID1A, p53, and MMR protein expression. PD-L1 immunohistochemistry was performed on whole tissue sections of all tumor blocks of 59 consecutive unselected endometrial carcinomas between November 2018 and September 2019. TC and IC PD-L1 positivity with a 1% cut-off value was observed in 10.2% and 67.8% of cases, respectively, and with a 5% cut-off value in 3.4% and 42.4% of cases, respectively. TC PD-L1 positivity with both 1% and 5% cut-off values was significantly related to ARID1A loss (p = 0.001 and p = 0.046, respectively). IC PD-L1 positivity with 1% and 5% cut-off values and combined score were significantly associated with MMR protein deficiency (p = 0.041, p = 0.031, and p = 0.028, respectively). Advanced stage tumors exhibited more frequent PD-L1 expression in ICs (p = 0.039). MELF-type myometrial invasion pattern was more common in tumors with ARID1A loss (p = 0.047). We observed higher rates of IC PD-L1 positivity in endometrial carcinomas than documented in prior studies; this may be related to our usage of "recent" paraffin blocks and whole tissue sections of all tumor blocks. There was a much higher PD-L1 expression in the ICs compared to TCs in our cases. We confirm a previously documented association between MMR deficiency and PD-L1 expression and show a novel association between ARID1A loss and PD-L1 expression in endometrial carcinomas. ARID1A loss represents a potential biomarker of immune checkpoint inhibitor response in endometrial carcinoma.

Correlation of high LAT1 expression with the prognosis of endometrioid carcinoma of the uterine corpus

The expression of L-type amino acid transporter 1 (LAT1) has been described to play essential roles in cancer cell growth and survival. To determine the significance of LAT1 in the prognosis of endometrial endometrioid carcinoma, we investigated LAT1 expression in 353 endometrioid carcinoma patients by immunohistochemical analysis using tissue microarray. The tumors in which stained tumor cells made up more than 25% of the tumor were graded as high expression. High expression of LAT1 was detected in 29 (8.2%) of patients. The ratio of high LAT1 expression did not significantly differ by age (< 60 vs. ≥ 60), FIGO stage (stage I/II vs. III/IV), histological grade (grade 1 vs. grade 2/3), or lymph node metastasis (positive vs. negative). However, high LAT1 expression in endometrioid carcinoma was associated with a poorer progression-free survival and overall survival, as per the results of the log-rank test (P = 0.0263 and 0.0404, respectively). Cox univariate and multivariate analyses revealed that high LAT1 expression is an independent marker of poor progression-free survival (hazard ratio = 2.598, P = 0.0137), in addition to a higher age (≥ 60 years vs. < 60 years), FIGO stage (stage III/IV vs. I/II), and histological grade (grade 2/3 vs. grade 1). In conclusion, we demonstrate that LAT1 is associated with a poor prognosis of endometrioid carcinoma of the uterine corpus.

The clinical, morphological, and genetic heterogeneity of endometrial stromal sarcoma

ZC3H7B–BCOR high-grade endometrial stromal sarcoma may present as myoma nascens with cytoplasmic signet ring cell change

We report on 51-year-old woman who presented with brown discharge and postcoital bleeding due to myoma nascens-like polypoid mass distending cervical canal. Histologically, the tumor consisted of high-grade spindle cell component with up to 15 mitotic figures per 10 HPF and also low-grade leiomyoma-like areas with focal myxoid change and so far undescribed cytoplasmic signet ring cell change. Immunohistochemically Desmin, actin, and h-caldesmon were negative. Conversely, BCOR positive expression was coupled with Cyclin D1 positivity and was antibody clone dependent. The molecular NGS and FISH study identified reciprocal fusion gene ZC3H7B-BCOR. In conclusion, these findings further support the idea of routine reflex molecular testing of uterine mesenchymal tumors with unusual clinical presentation or in case malignancy is suspected. Lastly, we suggest ZC3H7B-BCOR rearranged high-grade endometrial stromal sarcoma might be considered as a tumor suitable for BCL6-targeted treatment.

Histotype-specific analysis of acid ceramidase expression in ovarian cancer

Acid ceramidase (ASAH1) is a key player in sphingolipid metabolism and signaling. It has prognostic value for several cancers, but histotype-specific analyses of ovarian cancer are not yet available. We used three retrospective TMA cohorts encompassing a total of 1106 ovarian cancers with follow-up data for immunohistochemical analysis of acid ceramidase (ASAH1) expression. Patients with sub-optimal debulking and persistent residual tumor after surgery introduced bias in the prognostic analysis and were excluded from further studies. Overall, we detected an association of ASAH1 expression with better prognosis in ovarian cancer patients. ASAH1 expression differed between histological ovarian cancer histotypes with most frequent expression in endometrioid and clear cell ovarian cancer, which are both associated with good prognosis. Stratified subgroup analyses within these histotypes did not reveal significant survival differences, but the power of the analysis may be limited by smaller sample sizes. In contrast to breast cancer, we found only a modest concordance between estrogen receptor status and ASAH1 expression within the endometrioid ovarian cancer histotype. In an exploratory analysis of estrogen receptor negative endometrioid ovarian cancer, ASAH1 expression was associated with significantly better overall survival (P = 0.007). Acid ceramidase is most frequently expressed in endometrioid and clear cell histotypes and could add independent prognostic value to estrogen receptor in endometrioid ovarian cancer. Modulating sphingolipid metabolism may lead to novel therapeutic intervention strategies for this disease.

Chemotherapy-associated foam cell aggregates as a prognostic factor in patients with pelvic high-grade serous carcinoma receiving neo-adjuvant chemotherapy

High-grade serous carcinoma (HGSC) tends to recur after treatment; therefore, the Chemotherapy Response Score (CRS) has been proposed as a histopathological prognostic scoring system for measuring the response to neo-adjuvant chemotherapy and the risk of recurrence. This study aimed to evaluate the CRS in only those with an R0 debulking status and to investigate new prognostic factors for progression-free survival (PFS). We reviewed the CRS of HGSC patients with R0 using surgical specimens of the omental sections. Patients were categorized according to foam cell change (FCC), defined as foam cells occupying more than half of the area of the chemotherapy-associated scar. In total, 100 HGSC patients were evaluated. PFS was significantly different according to the CRS. For CRSs of 1/2 and 3, the median PFS were 18 and 27 months, respectively (HR, 1.84; 95% CI 1.01-3.33, p = 0.045). Moreover, the FCC group showed significantly longer PFS than did the non-FCC group (20 vs 59 months; HR 2.43; 95% CI 1.15-5.14; p = 0.020). The present study validated the CRS of those in the R0 cohort. Furthermore, an increase in foam cells in the regression scar reflects the chemotherapy response and the FCC may be a useful novel prognostic factor for patients undergoing R0 resection. This finding must be further validated independently.

STR genotyping diagnosis of hydatidiform moles: an assessment of 2871 consecutive products of conceptions

This study evaluated the incidence of hydatidiform moles in a tertiary care setting and examined the efficacy of short tandem repeat genotyping (STR) as a primary ancillary diagnostic tool for molar gestations. Consecutive products of conceptions undergoing genotyping were analyzed. A total of 2871 cases were analyzed, comprising 1693 in-house and 1178 consultation specimens. Patient ages ranged from 10 to 57 years (mean 32, median 33). Laser microdissection was performed in 138 specimens. STR genotyping yielded a definitive diagnosis in 99.5% of cases (2857/2871), classifying them as 282 complete moles (241 monospermic, 41 dispermic), 564 partial moles (546 dispermic, 12 monospermic, 6 triandric tetraploid), and 2006 non-molar gestations (including 125 trisomies, 24 digynic triploids, 4 egg donor conceptions, 1 trigynic tetraploid, 3 androgenetic/biparental mosaics, 2 chimeric twin gestations with a molar component of either monospermic complete mole or heterozygous partial mole, and 1 trigynic tetraploid). Among molar gestations, complete and partial moles accounted for 38.6% and 61.4% of in-house cases and 28.2% and 72.0% of consultation cases, respectively. Notably, complete moles occurred exclusively in patients younger than 15 or older than 50. The mean turnaround time for STR genotyping was 5.3 days overall (5.7 days for in-house and 4.8 days for consultation cases). Hydatidiform moles accounted for 29.4% of the study cohort in our tertiary care setting, with partial hydatidiform moles comprising two-thirds of these cases. With an average turnaround time of 5.3 days and a 99.5% success rate, STR genotyping demonstrated high efficiency in the accurate diagnosis and classification of hydatidiform moles.

HER2 status as a potential predictive biomarker for ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a subtype of ovarian carcinoma characterized by unique biological features and highly malignant characteristics including low chemosensitivity. Therefore, new therapeutic targets are needed. These could include the downstream pathways of receptor tyrosine kinases, especially the human epidermal growth factor receptor 2 (HER2). Our main objective was to characterize the HER2 status using immunohistochemistry (IHC) and FISH on 118 OCCCs, also considering the novel paradigm of HER2-zero and HER2-low status. Other aims included determination of the association between HER2 status and survival, HER2 gene DNA and RNA NGS analysis, HER2 gene expression analysis, and correlation between IHC and gene expression in HER2-zero and HER2-low cases. Cases with HER2 overexpression/amplification accounted for 5.1% (6/118), with additional 3% harbouring HER2 gene mutation. The remaining 112 (94.9%) cases were HER2-negative. Of these, 75% were classified as HER2-zero and 25% as HER2-low. This percentage of HER2 aberrations is significant concerning their possible therapeutic influence. Cases from the HER2-zero group showed significantly better survival. Although this relationship lost statistical significance in multivariate analysis, the results have potential therapeutic significance. HER2 gene expression analysis showed a significant correlation with HER2 IHC status in the entire cohort (HER2-positive vs. HER2-negative), while in the cohort of only HER2-negative cases, the results did not reach statistical significance, suggesting that gene expression analysis would not be suitable to confirm the subdivision into HER2-low and HER2-zero. Our results also emphasize the need for standardized HER2 testing in OCCC to determine the best predictor of clinical response.

Endocervical adenocarcinoma with a micropapillary component: a clinicopathologic analysis in the setting of current WHO classification

Our study aimed to investigate the clinicopathologic and molecular features of endocervical adenocarcinoma with a micropapillary component (EAC-MP) in the setting of current classification schema. We investigated 26 EAC-MP from consecutive 511 adenocarcinomas. HER2 status was analyzed by immunohistochemistry and fluorescence in situ hybridization. Four cases were performed with targeted next-generation sequencing (NGS). We found that HPV-associated adenocarcinomas (HPVA) with a micropapillary component (HPVA-MP) (n = 12) had a higher frequency of large tumor size (> 2 cm), Silva pattern C (12/12, 100%), invasion of the deep cervical wall (> 2/3) (8/12, 66.7%), lymphovascular space invasion (LVSI) (11/12, 91.7%), lymph node metastasis (4/11, 36.4%), FIGO stage III/IV (4/12, 33.3%), and HER2 amplification (3/12, 25%, P = 0.015), compared to those without (HPVA-NMP (all P  0.05). NGS identified significant mutations in STK11, TERT, ERBB2, TP53, PIK3CA, ARID1A, and NTRK2. We conclude that the micropapillary structure is an indicator for unfavorable clinical outcomes in HPVA, and can aid in the prognostic stratification of Silva pattern C EAC. The presence of HER2 amplification and specific gene mutations raise the possibility for targeted therapy in the future.

Human epidermal growth factor 2 (HER2) amplification in uterine serous carcinoma: an analysis of prognosis and immune microenvironment

Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. Anti-human epidermal growth factor receptor 2 (HER2) therapy has demonstrated its promising effects on HER2-positive USC. However, data on prognostic relevance and immune microenvironment are limited in HER2-positive USC. This study aimed to determine the clinicopathologic features, prognosis, and the immune microenvironment trait in HER2 status in USC. We applied immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and multi-color immunofluorescence to investigate HER2 expression and amplification, PD-L1 expression, and tumor infiltration lymphocytes (TIL) in 77 USC (61 pure and 16 mixed-type USC). HER2 IHC 1 + , 2 + , and 3 + were found in 26, 18, and 10 USC, respectively. HER2 staining frequently had an incomplete membrane (basolateral or "U"-shaped) pattern. Twenty-three cases (23/54, 42.6%) showed an intra-tumor heterogeneous staining. HER2 amplification was present in 16/77 (20.8%) USC. HER2 amplification was significantly associated with deep myometrial invasion (> 1/2), and increased intra-epithelial and stromal density of CD20 + or CD8 + TIL (all P  0.05). HER2 amplification was associated with poor overall and progression-free survival in USC, but lost the prognostic significance on multivariate analysis. We concluded that HER2 amplified USC had adverse clinical outcomes, but showed the potential active immune microenvironment. Our findings raised the possibility of the combined anti-HER2 and immunotherapy for HER2-positive USC in the future.

Association of CD133, ALDH1, CD117 and OCT4 expression with prognosis of patients with cervical cancer

Cancer stem cells (CSC), a small population of neoplastic cells, are associated with worse prognosis. The aim of this study was to evaluate the expression of ALDH1, CD117, CD133 and OCT4; potential markers of CSC; and their associations with the prognosis of women diagnosed with cervical cancer. This retrospective cohort study included 126 women diagnosed with cervical cancer whose biopsies were analyzed by immunohistochemistry. Median values of marked cells were used to define cutoff points for low and high expression. For specific survival, multivariate analyses showed statistical significance for lymph node metastases (HR 8.15; 95% CI 3.00-22.18) and borderline significance for high CD133 expression (p = 0.058). For overall survival, multivariate analyses showed statistical significance for IIA-IVB staging (HR 4.60; 95% CI 1.46-14.56), lymph node metastases (HR 5.13; 95% CI 12.02-13.03) and high CD133 expression (2.67; 95% CI 1.11-6.43). Considering only women with SCC, the same clinicopathological variables were associated with worse specific and overall survival in univariate analyses. However, higher expression of CD 133 (HR 11.10; 95% CI 2.42-50.94 and 6.00; 95% CI 2.02-17.87) and staging IIA-IVB (HR 5.96; 95% CI 1.30-27.34 and HR 12.47; 95% CI 2.45-63.54) respectively impacted negatively specific and overall survival, as multivariate analyses showed. Secondarily, it was observed that ALDH1 expression was associated with adenocarcinoma and CD117 expression with squamous cells carcinoma. Higher expression of CD133 was associated with worse specific and overall survival, indicating that it could have relevance as a clinical marker and therapeutic target.

Mature cystic teratoma of the ovary with malignant transformation of tall cell subtype of papillary thyroid carcinoma

Malignancies rarely occur in somatic parts of mature cystic teratoma of the ovary. Squamous cell carcinoma is the most common form of cancer that can develop in mature cystic teratoma. Other less frequent malignancies include melanoma, sarcoma, carcinoid, and germ cell neoplasms. Only three cases have been reported as papillary thyroid carcinoma arising in struma ovarii. We present a unique case of a 31-year-old female patient who presented with a left ovarian cyst and underwent conservative surgical management in the form of cystectomy. Histopathological examination confirmed the diagnosis of a tall cell subtype of papillary thyroid carcinoma arising from a small focus of thyroid tissue in a mature cystic teratoma of the ovary. The patient was followed up for 60 months with an uneventful clinical course. For a better understanding of such rare cancers, collaborative retrospective studies on large databases with other medical centers are required.

Claudin-10 is a new candidate prognostic marker in metastatic high-grade serous carcinoma

AbstractThe objective of this study was to analyze the expression and prognostic role of the tight junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p &lt; 0.001), and was higher in HGSC surgical specimens compared to effusions (p &lt; 0.001). qRT-PCR confirmed the presence of CLDN10 mRNA in HGSC effusions. High (&gt; 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

NTRK-rearranged spindle cell sarcoma of the uterine cervix with a novel NUMA1::NTRK1 fusion

AbstractNTRK-rearranged uterine sarcoma is a recently described entity that represents a subset of uterine sarcomas with distinct clinicopathological features. From a molecular point of view, this tumour is defined by NTRK gene rearrangement, resulting in overexpression or constitutive activation of Trk receptors. The presence of NTRK fusion is indicative of treatment response with a selective small-molecule inhibitor of the Trk kinases. Here, we report a case of an NTRK-rearranged sarcoma of the uterine cervix in a 43-year-old patient, measuring 80 mm in its largest dimension, with a novel NUMA1-NTRK1 fusion, not previously reported in NTRK-rearranged uterine sarcomas or other NTRK-rearranged tumours. The fusion, involving NUMA1 exon 14 (NM_006185.4) and NTRK1 exon 11 (NM_002529.4), was identified by next-generation sequencing (NGS) studies (FusionPlex Pan Solid Tumor v2 panel). Although the presence of NTRK fusion has been reported in a variety of neoplasms, a fusion involving NUMA1 (nuclear mitotic apparatus protein 1) and a tyrosine kinase partner has previously been reported in human neoplasms only in a handful of cases. The resulting fusion protein comprises the oligomerization domain of NUMA1, which is predicted to cause constant activation of the tyrosine kinase domain of NTRK1. The recognition and accurate diagnosis of these tumours are important due to the availability of potential targeted therapeutic options.

Expression profiles of metallothionein-I/II and megalin/LRP-2 in uterine cervical squamous lesions

AbstractMetallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which are implicated in a variety of physiological and pathological processes. Their growth-regulating, anti-apoptotic and anti-inflammatory functions have been attributed not only to intracellular free radical scavenging and to zinc and copper regulation but also to the ability of secreted MT to bind on surface lipoprotein receptor-megalin/LRP2, which enables the endocytosis of MT-I/II and a wide range of other functionally distinct ligands. In the present study, we analysed the expression pattern of both proteins in 55 cases of premalignant transformation of cervical squamous cells, i.e. in low- and high-grade squamous intraepithelial lesion (LSIL and HSIL). The data showed that in LSIL (cervical intraepithelial neoplasia CIN1; N = 25) MTs were present only in basal and parabasal cells and that megalin was only weakly expressed. In HSIL (CIN2; N = 15 and CIN 3/carcinoma in situ; N = 15), however, overexpression and co-localization of MT with megalin were found in the entire hyperplastic epithelium. Moreover, megalin immunoreactivity appeared on the glandular epithelium and vascular endothelium, as well as on lymphatic cells in stroma. Besides, multiple megalin-positive cells expressed phosphorylated Akt1, implying that MT- and/or megalin-dependent prosurvival signal transduction pathways might contribute to the development of severe cervical dysplasia. The data emphasize the diagnostic power of combined MT/megalin analysis in pre-cancer screening.

Does endometrial morular metaplasia represent odontogenic differentiation?

AbstractThe nature of endometrial morular metaplasia (MorM) is still unknown. The nuclear β-catenin accumulation and the not rare ghost cell keratinization suggest a similarity with hard keratin-producing odontogenic and hair matrix tumors rather than with squamous differentiation. We aimed to compare MorM to hard keratin-producing tumors. Forty-one hard keratin-producing tumors, including 26 hair matrix tumors (20 pilomatrixomas and 6 pilomatrix carcinomas) and 15 odontogenic tumors (adamantinomatous craniopharyngiomas), were compared to 15 endometrioid carcinomas with MorM with or without squamous/keratinizing features. Immunohistochemistry for β-catenin, CD10, CDX2, ki67, p63, CK5/6, CK7, CK8/18, CK19, and pan-hard keratin was performed; 10 cases of endometrioid carcinomas with conventional squamous differentiation were used as controls. In adamantinomatous craniopharyngiomas, the β-catenin-accumulating cell clusters (whorl-like structures) were morphologically similar to MorM (round syncytial aggregates of bland cells with round-to-spindled nuclei and profuse cytoplasm), with overlapping squamous/keratinizing features (clear cells with prominent membrane, rounded squamous formations, ghost cells). Both MorM and whorl-like structures consistently showed positivity for CD10 and CDX2, with low ki67; cytokeratins pattern was also overlapping, although more variable. Hard keratin was focally/multifocally positive in 8 MorM cases and focally in one conventional squamous differentiation case. Hair matrix tumors showed no morphological or immunophenotypical overlap with MorM. MorM shows wide morphological and immunophenotypical overlap with the whorl-like structures of adamantinomatous craniopharyngiomas, which are analogous to enamel knots of tooth development. This suggests that MorM might be an aberrant mimic of odontogenic differentiation.

Programmed death-ligand 1 (PD-L1) expression in cervical intraepithelial neoplasia and cervical squamous cell carcinoma of HIV-infected and non-infected patients

AbstractProgrammed death-ligand 1 (PD-L1) is overexpressed in cervical carcinoma, hindering tumor destruction. The aim of this study was to assess PD-L1 expression by immunohistochemistry in cervical squamous cell carcinoma (SCC) and squamous intraepithelial lesions (SILs) from human immunodeficiency virus–positive (HIV+) and human immunodeficiency virus-negative (HIV-) patients. A total of 166 SCC and SIL samples of HIV+ and HIV- patients were included and analyzed for PD-L1 expression through tumor proportion score (TPS), and results were stratified in five TPS groups using SP263 antibody and, combined positive score (CPS) using 22C3 antibody. In cohort 1 (SP263 clone), all HIV+ patients were negative for intraepithelial lesion or malignancy (NILM), and low-grade squamous intraepithelial lesions (LSILs) scored &lt; 1; and 87.5% of high-grade squamous intraepithelial lesions (HSILs) adjacent to SCC, 19% of HSILs non-adjacent to SCC, and 69% of SCCs scored ≥ 1 (15.4% scored 5). In HIV- patients, all NILM, LSILs, HSILs adjacent to SCC, and two HSILs non-adjacent to SCC scored &lt; 1. SCC: 88.2% scored ≥ 1 and 5.9% scored 5. In cohort 2 (SP263 and 22C3 clones), 16.7% of HIV+ patients with SCC were positive with both clones, CPS ≥ 1 (22C3) or score 5 (≥ 50%) (SP263), showing no significant differences in positivity between both clones. These results indicate that a relatively low percentage of SCCs (16.7%; both in HIV+ and in HIV- patients) express PD-L1 (TPS ≥ 50% and CPS &gt; 1), which may be due to some samples being archival material, sample characteristics, or use of different methodologies, highlighting the need for standardization of PD-L1 assessment in SCC of the cervix. The fact that PD-L1 is overexpressed in SILs of HIV+ patients suggests potential additional applications for immunotherapy in this disease.

Death domain-associated protein (DAXX) expression is associated with poor survival in metastatic high-grade serous carcinoma

AbstractThe objective of this study was to analyze the expression and clinical role of mitosis regulators α-thalassemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX) in metastatic high-grade serous carcinoma (HGSC). ATRX and DAXX protein expression by immunohistochemistry was analyzed in 400 HGSC effusions. DAXX expression was additionally studied in 15 cancer cell lines, including 4 ovarian carcinoma lines, and in 81 of the 400 HGSC effusions using Western blotting. ATRX and DAXX were expressed in HGSC cells in 386/400 (96%) and 348/400 (87%) effusions, respectively. Western blotting showed DAXX expression in all 15 cell lines and in 70/81 (86%) HGSC effusions. DAXX expression by immunohistochemistry was higher in pleural compared to peritoneal effusions (p = 0.006) and in post-chemotherapy compared to pre-chemotherapy effusions (p = 0.004), and its expression was significantly associated with poor overall survival in univariate of the entire cohort (p = 0.014), as well as analysis limited to chemo-naïve effusions tapped at diagnosis (p = 0.038). The former association retained its prognostic role in Cox multivariate survival analysis (p = 0.011). ATRX expression was unrelated to clinicopathologic parameters or survival. In conclusion, DAXX is associated with disease progression and could be a prognostic marker in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

Expression of the cancer stem cell marker SSEA1 is associated with poor survival in metastatic high-grade serous carcinoma

AbstractThe objective of the present study was to perform a quantitative analysis of cancer stem cell (CSC) marker expression in ovarian carcinoma effusions. The clinical role of SSEA1 in metastatic high-grade serous carcinoma (HGSC) was additionally analyzed. CD133, Nanog, SOX2, Oct3/4, SSEA1, and SSEA4 protein expressions were quantitatively analyzed using flow cytometry (FCM) in 24 effusions. SSEA1 expression by immunohistochemistry was analyzed in 384 HGSC effusions. Highly variable expression of CSC markers by FCM was observed, ranging from 0 to 78% of Ber-EP4-positive cells in the case of CD133, with the largest number of negative specimens seen for SSEA4. SSEA1 expression by immunohistochemistry was found in HGSC cells in 336/384 (89%) effusions, most commonly focally (&lt; 5% of cells). SSEA1 was overexpressed in post-chemotherapy disease recurrence specimens compared with chemo-naïve HGSC effusions tapped at diagnosis (p = 0.029). In univariate survival analysis, higher SSEA1 expression was significantly associated with poor overall survival (p = 0.047) and progression-free survival (p = 0.018), though it failed to retain its prognostic role in Cox multivariate survival analysis in which it was analyzed with clinical parameters (p = 0.059 andp = 0.111 for overall and progression-free survival, respectively). In conclusion, CSC markers are variably expressed in ovarian carcinoma effusions. SSEA1 expression is associated with disease progression and poor survival in metastatic HGSC. Silencing this molecule may have therapeutic relevance in this cancer.

PD-1/PD-L1 expression and tumor-infiltrating lymphocytes are prognostically favorable in advanced high-grade serous ovarian carcinoma

AbstractThe response rate to checkpoint inhibitors for women with high-grade serous carcinoma of the ovary, fallopian tube, and peritoneum (HGSC) is modest, and development of predictive biomarkers is needed. The main focus has been on tumor cell PD-L1 expression, but its assessment alone is insufficient for patient selection in most malignancies. We mapped the presence of macrophages (CD68 and CD163) and lymphocytes (CD3) located within the tumor epithelium, the cell type–specific expression of PD-L1 and PD-1, and their impact on 5-year overall survival (OS) in a consecutive cohort of 130 women diagnosed with advanced HGSC between 2011 and 2015. PD-L1 was expressed mainly by macrophages (not by tumor cells) and PD-1 by lymphocytes. Women with higher CD3, PD-L1, and PD-1 expression had improved OS (P = 0.03,P = 0.007, andP = 0.02, respectively). In the external data set (203 women), high expression ofCD274(encoding PD-L1) was associated with improved OS (P = 0.03), in accordance with our results. Furthermore, higher CD163 expression was associated with better outcome in women with no residual tumor after primary surgery (P = 0.02). Thus, women with greater lymphocyte tumor infiltration had better outcome and PD-L1/PD-1 expression, regardless of PD-1/PD-L1 being markers for immune suppressive pathways, conferred a survival benefit in our cohort. Our results highlight that tumor immunity may be harnessed in subsets of HGSC.

Mutational Screening of BRCA1/2 Genes as a Predictive Factor for Therapeutic Response in Epithelial Ovarian Cancer: A Consensus Guide from the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH)

AbstractGermline/somatic BRCA-mutated ovarian carcinomas (OC) are associated to have better response with platinum-based chemotherapy and long-term prognosis than non-BRCA-associated OCs. In addition, these mutations are predictive factors to response to Poly(ADP-ribose) polymerase (PARP) inhibitors. Different positioning papers have addressed the clinical recommendations for BRCA testing in OC. This consensus guide represents a collection of technical recommendations to address the detection of BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations and their experience in the molecular characterization of these genes. Since the implementation of BRCA testing as a predictive factor can initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue, distinctive features of both strategies are discussed. Additionally, the recommendations included in this paper provide some references, quality parameters, and genomic tools aimed to standardize and facilitate the clinical genomic diagnosis of OC.

The presence of clear cell glands around the ovarian endometrioid cyst has an association with clear cell carcinoma

We found some clear cell glands appeared in the endometrioid cysts (ECs) of the ovary (EC-CCG). To explore the clinicopathological features, molecular biological changes, and prognosis in EC-CCG and analyze the association with ovarian clear cell borderline tumors (CCBT) and clear cell carcinoma (CCC). We retrospectively examined 35 cases of EC-CCG, compared them to 13 cases of clear cell cystadenomas, 14 cases of CCBT, and 49 cases of CCC. We analyzed the differences in clinicopathological features and prognosis between the four groups. Data on clinicopathology and survival were gathered. Immunohistochemistry (IHC) was performed in all cases, and we analyzed the molecular changes of 2 cases of EC-CCG and 1 case of CCC by whole-exome sequencing (WES). EC-CCG shared some common clinicopathological features with CCBT: they occurred before menopause, had an elevated serum CA125 level in some cases, had an ovarian cystic mass on B-ultrasound, and had a risk of recurrence. Microscopically, both diseases were based on typical EC, and clear cell glands in the EC cyst wall were seen in varying numbers. Some cases of EC-CCG had IHC results similar to those of CCBT and CCC, with positive expression of HNF1β and NapsinA; decreased expression of ER, PR, and ARID1A; and increased expression of Ki67 (> 5%). WES results revealed that EC-CCG had mutations in TP53BP1, ZNF462, FN1, and FTL (which was also mutated in CCC). In summary, we found that clear cell glands appearing around EC in the ovary have an association with CCC.

A 2-tier subdivision of papillary proliferations of the endometrium (PPE) only emphasizing the complexity of papillae precisely predicts the neoplastic risk and reflects the neoplasia-related molecular characteristics—a single-centered analysis of 207 cases

AbstractPapillary proliferation of the endometrium (PPE) is subdivided based on the complexity of the papillae and the proliferation of lesions, and the complex group is considered to have an increased risk of concurrent/subsequent endometrial neoplasia. However, the current subdivision criteria fail to prove the equivalence of the quantity of simple papillae and structural complexity. In this study, we divided PPE of 207 cases from 2014 to 2022 into 3 groups according to structural complexity and proliferation degrees: Group 1 equaled to the simple PPE with a simple papillary structure and typical localized proliferation; group 2 had the simple structure similar to group 1 but occupy over 50% of the endometrial polyp or &gt; 2 lesions in the surface of nonpolypoid endometrium; group 3 had the truly complex branching papillae despite of its proportion. Group 3 was implicated with significantly more concurrent endometrial neoplasia (EAH and carcinoma) compared with groups 1 and 2 (P &lt; 0.01), while no difference was found between groups 1 and 2. In 128 cases with no concurrent endometrial abnormalities in the initial biopsy or curettage specimens, 4 cases presented endometrial neoplasia (3 carcinoma and 1 atypical hyperplasia) in the subsequent specimens, all of which presented PPE of group 3 but not group 1 or 2 in the prior tissues (P &lt; 0.01). The immunochemistry of 83 cases showed similar expressions of ER, PTEN, ARID1A, PTEN, p16, β-catenin, and p53 between PPE and the surrounding normal endometrium. Nearly 100% of PPE cases lost expressions of PR. A total of 2/83 cases showing PAX2 expression were all in the group 3 and correlated with endometrial neoplasia (2/17, 11.76%, P &lt; 0.05). 76/83 (91.57%) of PPE lesions had KRAS mutations, and the distributions of which were similar among 3 groups. The frequency of mucinous metaplasia was significantly higher in the PPE lesions with KRAS mutations (72/74, 97.30%, P &lt; 0.01). Group 3 showed higher frequency of single KRAS mutations compared with the combination of groups 1 and 2 (P &lt; 0.01). Finally, the concordance of KRAS mutation profiles between PPE and endometrial neoplasia was significantly higher in group 3 than either group 1 or 2 (P &lt; 0.01), while no difference was found between group 1 and 2. Thus, a new 2-tier subdivision system only emphasizing the complexity of papillae is recommended, which might precisely predict the risk of endometrial neoplasia and neoplasia-related molecular characteristics.

Benign metastasizing fumarate hydratase (FH)-deficient uterine leiomyomas: clinicopathological and molecular study with first documentation of multi-organ metastases

AbstractLeiomyoma is the most prevalent benign tumor of the female reproductive system. Benign metastasizing leiomyoma (BML) is a rare phenomenon that presents at distant sites, typically the lungs, exhibiting histopathological features similar to the primary uterine tumor in the absence of malignancy features in both. Fumarate hydratase-deficient uterine leiomyoma (FH-d UL) is an uncommon subtype among uterine smooth muscle tumors (0.5–2%), showing distinctive histomorphology and FH inactivation. The majority of FH-d ULs are sporadic, caused by somatic FH inactivation, while a minority of cases occur in the context of the hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome caused by germline FH inactivation. Metastasizing FH-d UL has not been well documented and might be under-reported. Here, we present two cases (21- and 34-year-old females) who presented with metastasizing FH-d UL after myomectomy/hysterectomy with histologically proven multiple lung metastases in both, in addition to multi-organ involvement in one case (cervical-thoracic lymph nodes, left kidney, perihepatic region, left zygomatic bone, and soft tissues). Pathological examination confirmed FH-d leiomyomas in the primary/recurrent uterine tumors, multiple lung lesions, and a renal mass. The minimal criteria for diagnosis of leiomyosarcoma were not fulfilled. Genetic testing revealed germline pathogenic FH variants in both cases (c.1256C &gt; T; p.Ser419Leu in Case 1 and c.425A &gt; G; p.Gln142Arg in Case 2). These novel cases highlight a rare but possibly under-recognized presentation of FH-d BML. Our study suggests that FH-d BML cases might be enriched for the HLRCC syndrome.

ESR1::NCOA2/3 fusions in uterine neoplasms with adenosarcoma-like morphology: clinicopathologic and molecular features of 12 cases and review of the literature

Abstract The molecular pathogenesis of uterine adenosarcoma (uAS; synonym: Müllerian adenosarcoma) is confounded by its pathologic heterogeneity, with DICER1 mutations and TP53 pathway alterations reported in most tumors with high-grade morphology, stromal overgrowth and/ or rhabdomyoblastic differentiation. However, a small subset of tumors harbor gene fusions, but their molecular spectrum and clinicopathological correlations have not been defined. We identified 12 uAS-like neoplasms in our files carrying ESR1 gene fusions and reviewed 4 previously reported cases (total: 16). Patient’s age range was 34 – 76 years (median, 54). The tumors originated in the uterus body (5), unspecified uterus segment (5), cervix (1) and isthmus (1). Follow-up was available for 6 patients (median 44 months, range 9 months—20 years). One patient developed lung metastasis 52 months later and one had an abdominopelvic recurrence &gt; 20 years later. Four patients were disease-free at 9—55 months. All tumors harbored in-frame ESR1 fusions (10 with ESR1::NCOA3 , 1 ESR1:NCOA2 and 1 ESR1::MAMLD1 ). Nine tumors were low-grade and 3 high-grade. All lacked heterologous elements. Stromal overgrowth was recorded in three (all high-grade) and sex cord-like elements in 4 tumors. Phyllodiform architecture, admixed Mullerian glands, and periglandular stromal condensation distinguished the 4 tumors with sex cord-like elements from conventional uterine tumors resembling ovarian sex cord tumor (UTROSCT). This study expands the overlapping morphologic and molecular spectrum of uterine neoplasms resembling uAS showing recurrent fusions (mostly ESR1::NCOA3/2 ), associated with mostly low-grade histology, lack of heterologous elements and paucity of stromal overgrowth. The nosological relationship of these uAS-like tumors to UTROSCT (driven similarly by ESR1::NCOA3/2 fusions) and to fusion-negative high-grade uAS remains to be verified in future studies utilizing epigenetics and other tools.

P-cadherin overexpression is associated with early transformation of the Fallopian tube epithelium and aggressiveness of tubo-ovarian high-grade serous carcinoma

Abstract Tubo-ovarian high-grade serous carcinoma (HGSC) with proficient homologous recombination (HR) DNA repair (HRP) accounts for approximately 50% of cases and is associated with platinum-resistance and poor prognosis. We hypothesize that the acquisition of hybrid phenotypes displaying both epithelial and mesenchymal (E/M) features may be involved in the malignant transformation and tumour dissemination in this subgroup. Therefore, we analysed, by digital pathology, the expression and prognostic significance of 3 classic cadherins (E-cadherin, epithelial marker; N-cadherin, mesenchymal marker; and P-cadherin, candidate marker of hybrid E/M) in 577 formalin-fixed paraffin-embedded human samples representing the putative stepwise serous carcinogenesis in the Fallopian tube epithelium (FTE). We observed a non-canonical N-to-P-cadherin switch along the carcinogenic progression, with a statistically significant overexpression of P-cadherin in pre-malignant and malignant samples, compared to the control FTE. Interestingly, this overexpression was most pronounced in precursor lesions and HGSC cells from malignant ascites. Tumours with high P-cadherin expression were significantly associated with worse overall survival, especially in the subgroup without BRCA1/2 mutations. Transient P-cadherin knock-down resulted in in vitro significant reduction of functional hybrid E/M hallmarks, namely decreased anoikis resistance, reduced collective migration and invasion in a representative platinum-resistant HRP cell line. Taken together, our results suggest that P-cadherin overexpression is an early event in the serous carcinogenesis and may be involved in hybrid E/M activation in HRP-HGSC, further supporting this adhesion molecule as a promising biomarker for this poor prognostic subgroup.

KRAS mutation in primary ovarian serous borderline tumors correlates with tumor recurrence

Oncogenic activation of the mitogen-activated protein kinase (MAPK) pathway due to KRAS or BRAF gain-of-function mutation is frequently found in ovarian serous borderline tumor (SBT) and their extraovarian implants. We investigated mutational status of KRAS and BRAF of the primary ovarian SBTs that had a high stage presentation in correlation with clinical outcome. Among 39 consecutive primary SBTs with either invasive implants (20 cases) or non-invasive implants (19 cases), KRAS and BRAF mutational analysis was informative in 34 cases. Sixteen cases (47%) harbored a KRAS mutation, while 5 cases (15%) had a BRAF V600E mutation. High-stage disease (IIIC) was seen in 31% (5/16) of patients with a KRAS mutation and 39% (7/18) of patients without a KRAS mutation (p = 0.64). KRAS mutations were present in 9/16 (56%) tumors with invasive implants/LGSC versus 7/18 (39%) tumors with non-invasive implants (p = 0.31). BRAF mutation was seen in 5 cases with non-invasive implants. Tumor recurrence was seen in 31% (5/16) of patients with a KRAS mutation, compared to 6% (1/18) of patients without a KRAS mutation (p = 0.04). A KRAS mutation predicted an adverse disease-free survival (31% survival at 160 months) compared to those with wild-type KRAS (94% at 160 months; log-rank test, p = 0.037; HR 4.47). In conclusion, KRAS mutation in primary ovarian SBTs is significantly associated with a worse disease-free survival, independent of the high tumor stage or histological subtypes of extraovarian implant. KRAS mutation testing of primary ovarian SBT may servce as a useful biomarker for tumor recurrence.

High-grade serous ovarian carcinoma with a sertoliform pattern associated with BRCA mutation: a clinicopathological and molecular analysis

Herein, we report a clinicopathological and molecular analysis of a case of tubo-ovarian high-grade serous carcinoma (HGSC) with a sertoliform pattern. A 45-year-old woman underwent surgery due to an advanced bilateral adnexal carcinoma with peritoneal and appendiceal metastases. Histological examination revealed an HGSC exhibiting a distinct sertoliform component. Such component showed diffuse PAX8, p53 (mutation-type), and p16 (block-type) expression, increased vimentin and decreased WT1 expression compared to the conventional HGSC component, membrane β-catenin positivity, heterogeneous estrogen, and progesterone positivity, and retained PTEN and mismatch repair expression and negativity for GATA3, TTF1, inhibin, calretinin, CD10, CDX2, chromogranin, and synaptophysin. Molecular analysis showed a germline BRCA2 mutation; no mutations were detected in POLE, POLD1, MLH1, MSH2, MSH6, PMS2, APC, CTNNB1, MUTYH, and EPCAM. In conclusion, a sertoliform pattern can be part of the morphological spectrum of BRCA-related HGSC.

Discordance of PD-L1 expression in primary and metastatic ovarian high-grade serous carcinoma and its correlation with CD8 + tumor-infiltrating lymphocytes and patient prognosis

Differential expression of programmed death-1 ligand (PD-L1) and its clinical significance in primary and metastatic ovarian high-grade serous carcinoma (HGSC) have not been defined. Thus, we investigated the PD-L1 expression of paired ovarian primary and omental metastatic HGSC and its correlation with CD8 + tumor-infiltrating lymphocyte (TILs) and patient survival. A total of 212 cases of ovarian HGSCs with matched primary ovarian and metastatic omental tumors accessioned between 2003 and 2018 were selected for further analysis. Using immunohistochemistry, we evaluated the density of CD8 + TILs and expression of PD-L1 on whole tissue sections. Applying tumor proportion score (TPS, cutoff 1%) and combined positive score (CPS, cutoff 1), the prevalence of PD-L1 expression was similar but with significant discordance in ovarian and omental tumor. Using TPS, patients with PD-L1-positive tumors demonstrated significantly worse recurrence free survival (RFS) and overall survival (OS) than patients with PD-L1-negative tumors. Using CPS, patients with PD-L1-positive ovarian tumors demonstrated significantly worse OS while no significant difference in RFS was found. Patients with PD-L1-positive omental tumors demonstrated significantly worse RFS and OS. Patients with omental PD-L1-positive tumors (TPS) were associated with poorer RFS and OS, while patients with ovarian PD-L1-positive tumors (TPS) were associated with OS not RFS, in COX multivariant analysis. Nonetheless, ovarian and omental high CD8 TILs density was not associated with worse OS in univariant and COX multivariant analysis. PD-L1 expression in ovarian and omental tumor associated with an increased CD8 + TILs density. PD-L1 expression by TPS was better correlated with survival than by CPS, and PD-L1 expression in omental tumors was a stronger prognostic indicator than that in ovarian tumors.

Prognostic value of HPV-PCR, p16 and p53 immunohistochemical status on local recurrence rate and survival in patients with vulvar squamous cell carcinoma

AbstractThe primary aim of this study was to assess the association between human papilloma virus (HPV) and p53 expression and local recurrence (LR), disease specific survival (DSS), and overall survival (OS) in patients with vulvar squamous cell carcinoma (VSCC). Secondary, the accuracy of p16 immunohistochemistry for HPV status was assessed. The tumor tissue of 255 patients, surgically treated for primary unifocal VSCC between 2000 and 2010, was analyzed. HPV-PCR and P16 and p53 immunohistochemical stainings were performed. All histologic slides were independently reviewed by two expert gyneco-pathologists. Time to first LR, DSS, and OS for the variables p16, p53, and HPV-PCR were compared using univariable and multivariable Cox-regression analyses. In 211/255 (83.5%) patients, HPV-PCR was negative. The local recurrence rate was significantly lower in patients positive with HPV-PCR (10-year LR rate 24.6%) versus negative tumors (47.5%), p = 0.004. After multivariable analyses, this difference remained significant (HR 0.23 (95% CI 0.08–0.62) p = 0.004). There was no difference in LR rate correlated to the p53 expression. DSS and OS did not significantly differ after multivariable analyses for all different subgroups. Sensitivity and specificity of p16 staining for presence of HPV detected by HPV-PCR were 86.4% and 93.8%, respectively. In conclusion, patients with HPV-negative VSCCs have significantly more LR compared to patients with HPV-positive VSCCs, and p16 immunohistochemistry is a reliable surrogate marker for HPV status. No relevant subgroup for LR or survival based on HPV/p53 status could be identified. We advise to perform an HPV-PCR or p16 IHC staining in all patients with VSCC.

Endometrial giant cell carcinoma: new insights from a morphological, immunohistochemical, and molecular analysis of three cases

Herein, we present a morphological, immunohistochemical, and molecular analysis of three cases of endometrial giant cell carcinoma (EGCC) with a literature review. Patient age was 55 to 76 years. The tumors were limited to the uterus and showed dyshesive, bizarre giant cells with numerous atypical mitoses. Minor components were low-grade endometrioid, spindled/myxoid (case nos. 1 and 2), serous (case no. 3), and undifferentiated (all cases). The giant cells were e-cadherin-, cytokeratins/EMA + (focal/multifocal), hormone receptors + (focal/multifocal), vimentin + , p16 + (diffuse), CD68-, α-FP-, β-HCG-, muscle markers-, CD10-, and ERG-. Case no. 3 was p53-abnormal. All cases were mismatch repair-proficient and microsatellite-stable. No POLE mutations were detected. Based on our and previous reports, EGCC is often accompanied by a conventional carcinomatous component (mostly endometrioid) and shows partial loss epithelial markers and negativity for specific differentiation markers. EGCC shows evident similarities to both undifferentiated/dedifferentiated carcinoma and carcinosarcoma and should be managed similarly. Unlike the latter two, EGCC might preferentially derive from "no-specific-molecular-profile" carcinomas.

Mature and immature ovarian teratomas share methylation profiles of imprinted genes: a MS-MLPA analysis

Immature teratomas are a subset of ovarian teratomas, and the pathogenic relationship between mature and immature ovarian teratomas is unclear. Mature ovarian teratomas are parthenogenetic tumors that arise from a single oocyte/ovum, whereas the origin of immature ovarian teratomas has not been extensively investigated. Since parthenogenetic tumors contain only maternal genomes, genome imprinting in these tumors usually follows a maternal pattern. DNA methylation is among the most important mechanisms of genome imprinting. Therefore, we analyzed the methylation profile of imprinted genes by performing methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 25 imprinting control regions (ICRs) in 10 imprinted genes/gene clusters from formalin-fixed, paraffin-embedded samples obtained from 4 immature ovarian teratomas, 8 mature ovarian teratomas, and 4 ovarian yolk sac tumors (YSTs). Both the immature and mature components showed similar methylation levels in each ICR in immature teratomas. Overall, immature ovarian teratomas showed maternal methylation patterns of imprinted genes in concordance with their parthenogenetic origin. However, they also showed aberrant methylation levels in a few imprinted genes, suggesting that genome imprinting in immature teratomas may partially differ from that in mature teratomas. Microscopic foci of YST were seen in one immature teratoma; the YST component also showed a maternal methylation pattern, unlike the pure YSTs that showed irregular patterns. Thus, teratoma-associated YST and pure YST may have different pathogenic mechanisms.

Incidental FOXL2 mutated adult granulosa cell tumour of the ovary with thecoma-like foci

Abstract We report on the incidental finding of a FOXL2 mutated adult granulosa cell tumour of the ovary with thecoma-like foci, a rare entity recently described by Jennifer N. Stall and Robert H. Young in a series of sixteen cases in 2019, displaying features differing from conventional adult granulosa cell tumour. Our aim is to specify the morphologic and molecular particularities of this presumably underrecognized finding, with a short presentation of the typical clinical context. Awareness of this rare and challenging neoplasm with indeterminate clinical course is crucial in routine diagnostics.

Aberrant non-canonical WNT pathway as key-driver of high-grade serous ovarian cancer development

Expression of Wnt pathway molecules is associated with disease outcome in metastatic high-grade serous carcinoma

The objective of this study was to analyze the expression and clinical role of Wnt pathway molecules in metastatic high-grade serous carcinoma (HGSC). mRNA expression by qPCR of 20 molecules related to Wnt signaling (WNT1, WNT2, WNT3, WNT4, WNT5A, WNT6, WNT7, WNT11, FZD1, FZD4, FZD5, FZD6, FZD7, FZD8, FZD10, LRP5, LRP6, DKK, CCND, RUNX2) was analyzed in 87 HGSC effusions. Thirty-nine surgical specimens (19 ovarian, 20 from other intra-abdominal sites) were analyzed for comparative purposes. Protein expression of YAP and LRP and their phosphorylated forms by western blotting were analyzed in 52 tumors. Significant differences in mRNA expression as a function of the anatomic site were observed for WNT3 (p = 0.005), WNT5A (p = 0.008), WNT7 (p < 0.001), FRZ5 (p = 0.04), and FRZ6 (p < 0.001). YAP and LRP and their phosphorylated forms were detected in HGSC specimens. FZD10 was overexpressed in effusions from patients who had complete response to chemotherapy compared with those with less favorable response (p = 0.037). WNT4 (p = 0.005), WNT7 (p = 0.047), RUNX2 (p = 0.038), LRP5 (p = 0.022), LRP6 (p = 0.011), FZD6 (p = 0.036), FZD7 (p = 0.004), and FZD10 (p = 0.015) levels were inversely related to primary chemoresistance. High FZD5 levels in pre-chemotherapy effusions tapped at diagnosis and high WNT2 levels in post-chemotherapy disease recurrence effusions were related to shorter overall survival (p = 0.018 and p = 0.011, respectively), whereas high RUNX2 (p = 0.031) and FZD1 (p = 0.029) in post-chemotherapy effusions were associated with longer overall survival. In multivariate analysis of post-chemotherapy cases, WNT2 (p = 0.002), RUNX2 (p = 0.017), FZD1 (p = 0.036), and FZD4 (p = 0.013) were independent prognosticators. In conclusion, expression of Wnt pathway molecules is anatomic site dependent. In HGSC effusions, it is informative of chemoresponse and survival.

Comparing the 2017 ASCO/CAP guideline for gastroesophageal adenocarcinoma surgical specimen to the 2018 ASCO/CAP guideline for breast cancer in assessing the HER2 status in primary mucinous ovarian carcinoma

The successful experiences of HER2 inhibitors in patients with HER2 ( +) breast cancer (BC) and advanced gastroesophageal adenocarcinoma (GEA) have encouraged us to continuously explore the HER2 status and its potential as a therapeutic target in primary mucinous ovarian carcinoma (mOC). Using 49 primary mOC samples, we compared the assay characteristics of HER2 status between both 2017 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for GEA and 2018 ASCO/CAP for BC guideline recommendations. We demonstrated moderate to strong agreement between their HER2 IHC results (Weighted Kappa = 0.78) and perfect agreement between their HER2 FISH results (Kappa = 1.00). The overall concordance of non-equivocal HER2 IHC and HER2 FISH results was 97.56% (kappa = 0.93) by 2017 ASCO/CAP for GEA criteria and 100% (kappa = 1.00) by 2018 ASCO/CAP for BC criteria. The number (n = 8; 16.32%) of HER2 IHC equivocal (score + 2) by 2017 ASCO/CAP for GEA criteria was twofold higher than that (n = 4; 8.16%) by 2018 ASCO/CAP for BC criteria. Additionally, we identified one false-positive (FP) case (n = 1; 2.04%) that was HER2 IHC positive (score + 3), but HER2 FISH non-amplified result by the 2017 ASCO/CAP for GEA criteria. In conclusion, owing to the absence of FP/ FN and fewer equivocal cases of HER2 IHC, we recommend that the 2018 ASCO/CAP for BC are more appropriate than 2017 ASCO/CAP for GEA criteria in appraising the HER2 status in mOC and justifying the inclusion of eligible subjects for basket clinical trials of the newly developmental anti-HER2 treatments.

PD-L1 expression is associated with tumor infiltrating lymphocytes that predict response to NACT in squamous cell cervical cancer

Cancer immunotherapy has significantly improved the management of many malignancies in recent years. Although cervical cancer is the second most common women's cancer in the world, there are still few information about the role of checkpoint inhibitors in this neoplasm, especially in the neoadjuvant setting. In the present study, we retrieved 38 consecutive patients with squamous cell cervical cancer who underwent platinum-based neoadjuvant chemotherapy (NACT) followed by radical surgery. Pre-therapy biopsies were evaluated for the presence of tumor-infiltrating lymphocytes (TILs), including T (both cytotoxic CD8+ and helper CD4+) and B lymphocytes, macrophages, natural-killer cells, and eosinophils. Immunohistochemistry was performed to characterize the inflammatory cells and to evaluate programmed death-ligand 1 (PD-L1) expression on both neoplastic and inflammatory cells. We divided our study population in three groups using three cut-offs (40%), for both TILs and PD-L1 evaluation. Pathological response to NACT was obtained from the histological reports of the post-therapy surgical specimens. We observed that all cases showed stromal TILs, with a predominance of CD3+/CD4+ T helper cells, thus supporting the strong immunogenic potential of cervical cancer. The vast majority of neoplasms expressed PD-L1: 100% on immune cells and 92% on tumor cells. Firstly, we noticed that the percentage of neoplastic cells PD-L1+ was positively associated with high TIL percentage (p = 0.0073) and with increased PD-L1 expression on inflammatory cells (p = 0.0297). Secondly, we observed a significant correlation between both the percentage (p = 0.0105) of TILs and the expression of PD-L1 (p = 0.01045) on inflammatory cells and pathological response to NACT. These results suggest that cervical cancer could be a good target for immunotherapy, also in the neoadjuvant setting. Furthermore, PD-L1 expression was significantly associated with stromal TILs that interestingly may predict pathological response to NACT.

Lymphovascular invasion and p16 expression are independent prognostic factors in stage I vulvar squamous cell carcinoma

AbstractThe objective of this study was to identify clinicopathologic parameters associated with disease outcome in FIGO stage I vulvar squamous cell carcinoma (vSqCC). The cohort consisted of 126 patients diagnosed with vSqCC in the period 2006–2016 who underwent primary vulvar surgery and evaluation of groin lymph node status. Tumors were reviewed by an experienced gynecologic pathologist. p16 and p53 protein expression by immunohistochemistry and HPV status were analyzed in 116 tumors. Clinicopathologic parameters, protein expression and HPV status were analyzed for association with progression-free and overall survival (PFS, OS). p16 expression and aberrant p53 were found in 49 (42%) and 61 (53%) tumors, respectively. Sixty-six tumors were HPV-associated (57%). Relapse was diagnosed in 35/126 (28%) of patients, and 23 (18%) died of disease. Tumor diameter &gt; 4 cm (p = 0.013), lymphovascular space invasion (LVSI; p &lt; 0.001), the presence of lichen sclerosus (p = 0.019), p16 expression (p = 0.007), p53 expression (p = 0.012), HPV status (p = 0.021), lymph node metastasis (p &lt; 0.001) and post-operative radiotherapy (p &lt; 0.001) were significantly related to OS in univariate analysis. Tumor diameter &gt; 4 cm (p = 0.038), LVSI (p = 0.003), the presence of lichen sclerosus (p = 0.004), p16 expression (p = 0.004), HPV status (p = 0.039), lymph node metastasis (p &lt; 0.001) and post-operative treatment (p &lt; 0.001), were significantly related to PFS in univariate analysis. Age, BMI and surgical resection involvement were not significantly associated with OS or PFS. In multivariate Cox analysis, LVSI and p16 expression were independent prognosticators of OS (p &lt; 0.001 and p = 0.02, respectively) and PFS (p = 0.018, p = 0.037). In conclusion, LVSI and p16 expression are independent prognostic factors in stage I vSqCC.

Comprehensive genomic profiling and prognostic analysis of cervical gastric-type mucinous adenocarcinoma

Gastric-type mucinous adenocarcinoma (GAS) is an uncommon cervical adenocarcinoma, which is not associated with human papillomavirus (HPV) infection. Compared with HPV-associated cervical adenocarcinoma, GAS has characteristics of larger volume, deep invasion, and easy to metastasize to distant sites. Also, GAS is typically resistant to chemo/radiotherapy. Few studies have reported the molecular genetic characteristics of GAS. In order to investigate the molecular genetic characteristics of GAS and reveal its possible pathogenesis, 15 GAS patients were enrolled from Peking University People's Hospital (2009-2019) and examined with next-generation sequencing (NGS). Based on the clinicopathologic feature analysis, we found that the presence of lymph node metastasis and extensive lymphovascular invasion were associated with poor survival outcomes of GAS (p = 0.0042 and p = 0.005, respectively). Based on the NGS testing, our results showed that the most frequently mutated gene was TP53 (8/15, 53.3%), followed by STK11, CDKN2A, and ARID1A. STK11 mutations were more frequent in well-differentiated GAS (33.3% vs. 0.0%, p = 0.026) and patients with extensive lymphovascular invasion (33.3% vs. 0.0%, p = 0.044). Survival analysis revealed that STK11 mutations were significantly associated with the poor prognosis of GAS (p = 0.01). Our results also showed that mutations in the target drug were detected in 53.3% of GAS patients. Patients with ERBB2 amplification (13.3%) presented the highest level of evidence according to OncoKB recommendations. These results indicate that the genomic alterations of GAS mainly involved the cell cycle and PI3K/AKT signaling pathways, and some therapeutic candidates were identified in GAS patients.

Prevalence of KDM6A deficiency in human cancer: A tissue microarray study on 18,570 cancers from 153 different tumor types

Abstract KDM6A is a critical part of the COMPASS-like complex. KDM6A deficiency may result in EZH2 dependency. KDM6A deficiency was analyzed by immunohistochemistry on a tissue microarray containing 14,814 samples from 153 different tumor entities and 76 different normal tissue types. In normal tissues, KDM6A staining was ubiquitously seen in nuclei. In tumors, KDM6A deficiency occurred in 58 of 153 tumor categories. KDM6A deficiency predominated in urothelial carcinomas (17.3–42.0%) and was seen in 4–10% of gastric, pancreatic, prostatic, and gallbladder adenocarcinomas, serous endometrial carcinomas, squamous cell carcinomas, papillary renal cell carcinoma, hepatocellular carcinomas, malignant melanomas, and ovarian serous high-grade carcinomas. Reduced or absent KDM6A expression was associated with advanced pT stage (p = 0.0233), high grade (p = 0.0002), and distant metastasis (p = 0.0152) in breast cancer, nodal metastasis in squamous cell carcinomas (p = 0.0498), advanced pT stage (p = 0.0002), nodal metastasis (p = 0.0112), and mismatch repair deficiency (dMMR; p = 0.0147) in colorectal adenocarcinoma, and with dMMR (p = 0.0033) in gastric adenocarcinoma. KDM6A deficiency was significantly more common in tumors from males (4.3%) than from females (1.9%; p &lt; 0.0001). In summary, KDM6A deficiency predominates in urothelial neoplasms but also occurs in many further tumor entities. Whether KDM6A deficient cancers are susceptible to EZH2 inhibitors remains to be seen.

A molecular and immunohistochemical study of 37 cases of ovarian Sertoli–Leydig cell tumor

Abstract     This study provides an analysis of 37 ovarian Sertoli–Leydig cell tumors (SLCT), focusing on their morphological, immunohistochemical, and molecular features. The cohort was comprised of 9 well-differentiated, 25 moderately differentiated, and 3 poorly differentiated tumors. The immunohistochemical analysis was performed with 28 markers, including diagnostic markers and markers with possible predictive significance. The results showed high expression of sex cord markers (FOXL2, SF1, inhibin A, CD99, calretinin, ER, PR, AR), and variable expression of other markers such as CKAE1/3 (83%), CAIX (14%), and MUC4 (1%). Loss of PTEN expression was present in 14% of cases, and CTLA4 expression was seen in 43% of cases. All tumors were MMR proficient and HER2 and PD-L1 negative. The molecular analysis showed DICER1 mutations in 54.5% of cases, and a FOXL2 mutation in 6% of tumors. In addition, we detected 2 cases with TERT promoter mutation. RNA NGS sequencing identified significant differences in mRNA expression between DICER1 MUT and DICER1 WT tumors. The DICER1 WT tumors showed increased expression of PRKCA, HNF1A, LDLR, and MAP2K5 . On the contrary, the DICER1 MUT cases showed increased expression of CDK6, NOTCH2, and FGFR2. The results of our study show that SLCTs exhibit distinct molecular features based on their degree of differentiation. We have confirmed that DICER1 mutations are characteristic of moderately and poorly differentiated SLCTs, while well-differentiated SLCTs may represent a distinct entity. DICER1 MUT and DICER1 WT tumors showed different mRNA expression profiles. The FOXL2 mutation is less common in these tumors and is mutually exclusive with the DICER1 mutation.

ESGO/ESTRO/ESP Guidelines for the management of patients with endometrial carcinoma

A European consensus conference on endometrial carcinoma was held in 2014 to produce multidisciplinary evidence-based guidelines on selected questions. Given the large body of literature on the management of endometrial carcinoma published since 2014, the European Society of Gynaecological Oncology (ESGO), the European SocieTy for Radiotherapy & Oncology (ESTRO) and the European Society of Pathology (ESP) jointly decided to update these evidence-based guidelines and to cover new topics in order to improve the quality of care for women with endometrial carcinoma across Europe and worldwide. ESGO/ESTRO/ESP nominated an international multidisciplinary development group consisting of practicing clinicians and researchers who have demonstrated leadership and expertise in the care and research of endometrial carcinoma (27 experts across Europe). To ensure that the guidelines are evidence-based, the literature published since 2014, identified from a systematic search was reviewed and critically appraised. In the absence of any clear scientific evidence, judgment was based on the professional experience and consensus of the development group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 191 independent international practitioners in cancer care delivery and patient representatives. The guidelines comprehensively cover endometrial carcinoma staging, definition of prognostic risk groups integrating molecular markers, pre- and intra-operative work-up, fertility preservation, management for early, advanced, metastatic, and recurrent disease and palliative treatment. Principles of radiotherapy and pathological evaluation are also defined.

Comparison of the Idylla™ MSI assay with the Promega™ MSI Analysis System and immunohistochemistry on formalin-fixed paraffin-embedded tissue of endometrial carcinoma: results from an international, multicenter study

Determination of microsatellite instability (MSI) and mismatch repair deficiency (MMRD), respectively, in endometrial carcinomas (ECs) is important for diagnostic and prognostic purposes, identification of Lynch syndrome carriers, and selection of patients for immunotherapy. The Idylla™ MSI assay is fully automated, does not require non-tumoral tissue, and can be performed in about 150 min. Two hundred forty-two formalin-fixed paraffin-embedded (FFPE) EC samples from 7 international centers were tested by the Idylla™ MSI assay and compared to the Promega™ MSI Analysis System and immunohistochemistry (IHC) for MMR proteins. The cases were selected with an enrichment of MSI EC to around 40%. Concordance was 87.5% between the Idylla™ MSI assay and IHC and 88.58% between IHC and Promega™ MSI assay. Concordance between Idylla™ and Promega™ MSI assays was 89.91%. Discordant results occurred more frequently in cases with MSH6 or PMS2 deficiency. Invalid cases occurred with the three techniques (IHC, 7.00%; Promega™ MSI assay, 5.37%; and Idylla™ MSI assay, 2.47%). The concordance rate between Idylla™ MSI assay and the other 2 methods increased to 88.83% for IHC and to 91.22% for the Promega™ MSI assay when the cutoff of instability in the scoring system was moved from 0.5 to 0.3. The Idylla™ MSI assay is a rapid and highly concordant test for MSI in EC. Modification of the Idylla™ scoring system could increase the sensitivity and specificity of the MSI assay for EC analysis.