Endocervical gastric-type adenocarcinoma (GAS) is one of the most aggressive subtypes of cervical cancer and is frequently underdiagnosed due to morphological ambiguity, leading to delayed diagnosis. Despite the availability of molecular and genomic assays, their high cost, complexity, and limited reproducibility restrict clinical use. This study therefore proposes a highly sensitive artificial intelligence (AI)-assisted diagnostic system for GAS based exclusively on H&E-stained histopathological images. We included 309 slides from 96 GAS cases collected at Peking University Third Hospital from January 2018 to January 2025, representing the largest GAS cohort reported to date for AI research. In addition, we incorporated other morphologically analogous diseases, encompassing a total of 1,320 slides sourced from four categories: normal cervical mucosa (NORM), benign endocervical lesion entities (BELE), HPV-associated adenocarcinoma (HPVA), and endometrioid carcinoma with mucinous differentiation (ECMD). We developed GASPath, based on a novel multiple instance learning framework that efficiently captures fine-grained morphological variations from H&E-stained images. Beyond internal validation, GASPath was evaluated across 12 independent retrospective cohorts and further subjected to large-scale real-world validation on more than 7,000 samples from March 2024 to April 2025. Across three stages, GASPath demonstrated high performance. In internal validation (Stage I), it achieved an accuracy of 0.980 (95% CI 0.977-0.983) and an ROC-AUC of 0.995 (95% CI 0.994-0.997). In external validation (Stage II), the sensitivity reached 0.902 and improved to 0.968 with proposed strategies. For biopsy samples, GASPath achieved an ROC-AUC of 0.990 (95% CI 0.984-0.997). In large-scale real-world deployment (Stage III, n = 7,056), GASPath achieved a balanced accuracy of 0.953, with 100% sensitivity for GAS (45/45 cases correctly identified). The heatmaps highlight morphological features of GAS that are easily underestimated, such as irregular, angulated glands, subtle loss of nuclear polarity, and mild cytologic atypia, which show substantial morphological overlap with other diagnostic categories. GASPath enables high-sensitivity detection of GAS in routine H&E-stained slides, obviating the need for extensive auxiliary testing while preventing underdiagnosis and misdiagnosis. This advancement addresses a critical gap by streamlining diagnostic workflows without compromising accuracy. Its implementation could enable cost-effective, scalable AI-assisted diagnostics, potentially transforming the early detection and management of this aggressive cancer subtype.
Few studies have evaluated the impact of organ-specific tumor microenvironments (TMEs) on clinical outcomes in diffuse large B-cell lymphoma (DLBCL). This study investigated potential molecular markers, the immune cell composition within the TME, and their associations with clinical outcomes in patients with primary intestinal DLBCL (PI-DLBCL). We initially analyzed RNA expression in tumor cells from 19 patients with PI-DLBCL in the experimental cohort. Candidate biomarkers were then assessed in lymphoma tissue from a total of 48 patients in the same cohort, including the 19 with RNA-expression data, and validated in an independent cohort of 29 patients with PI-DLBCL. Associations between these markers and clinicopathological features, event-free survival (EFS), and overall survival (OS) in the two cohorts were analyzed. In the RNA expression panel, CCL5 expression was higher in patients with advanced-stage PI-DLBCL and was associated with inferior EFS and OS. Its principal receptor, CCR5, expressed in approximately one-third of patients, was associated with lower complete response rates to first-line immunochemotherapy: 50% in the experimental cohort and 10% in the validation cohort and correlated with the 7-year worse OS rate in both the experimental (49.8% versus 71.4%, p = 0.082) and validation (0% versus 76.2%, p < 0.001) cohorts. In both the experimental and validation cohorts, CCR5-positive tumors exhibited decreased CD86-positive (M1-like) and increased CD206-positive (M2-like) macrophage infiltration, consistent with an immunosuppressive TME. Multivariate analyses revealed that CCR5 expression was independently associated with worse EFS (p < 0.001) and OS (p = 0.004) in patients with PI-DLBCL (combined experimental and validation cohorts). CCR5 expression indicates a biologically aggressive subtype of PI-DLBCL with poor clinical outcomes and M2 macrophage-dominant immunosuppression.
B-cell precursor acute lymphoblastic leukemia (preB-ALL) is characterized by pathogenic variants currently used in precision oncology. However, the mutational landscape of Mexican children with preB-ALL has not yet been thoroughly explored and defined in terms of the clinical significance. We used a custom-designed next-generation sequencing exome panel, along with high-resolution chromosome microarrays and gene fusion-targeted assays, to characterize the mutational landscape of 73 Mexican children with preB-ALL, exploring the profile of clinically significant variants. We classified the variants following the AMP-ASCO-CAP 2017 and ACMG-CG 2019 guidelines recommendations. The mutational landscape includes a broad molecular spectrum of variants affecting cell cycle regulation, B-cell development, kinase signaling, and epigenetic regulation genes. Tier 1 diagnostic variants allowed the identification of pre-B ALL genetic subtypes, diminishing the preB-ALL NOS group from 63% to 37%. Furthermore, 37% of cases presented Tier 1 variants conferring intermediate to adverse prognoses (primarily involving CRLF2 gene fusions, as well as PAX5, IKZF1, and TP53 inactivating mutations). Notably, these patients exhibited high-risk clinical features and a lower event free survival rate than patients without these variants (60% versus 41.2%; p = 0.046; 95% CI). Between 19% and 42% of patients had Tier 2 variants targetable with JAK-STAT or RAS-MAPK signaling inhibitors. These patients showed a lower overall survival rate than patients without these variants (64% versus 90%; p = 0.048; 95% CI). Tier 1 potential germline variants in cancer predisposition genes (mainly BRCA1/2 and CHEK2) were observed in 10% of patients, some of whom had a family history of cancer. This highlights the importance of genetic counseling for patients and their families. Finally, 33% of patients had Tier 3 variants that were predicted to be deleterious and potentially upgraded to pathogenic with plausible clinical relevance. In conclusion, the mutational landscape analysis revealed variants useful for oncologic management and genetic counseling of Mexican children with preB-ALL.
Thyroid transcription factor-1 (TTF-1) is a widely used immunohistochemical marker for tumors of thyroid and pulmonary origin; however, its expression profile and diagnostic relevance in primary central nervous system (CNS) tumors remain incompletely characterized, particularly following the revisions in the 2021 WHO Classification of CNS Tumors. We systematically evaluated TTF-1 expression in a cohort of 214 primary CNS tumors diagnosed between 2021 and 2024 at The First Affiliated Hospital of China Medical University and classified according to current WHO criteria. Immunohistochemistry for TTF-1 was performed, and expression patterns were correlated with histological tumor type, molecular classification, and WHO grade. Statistical analyses were conducted to compare expression frequencies across tumor subtypes. TTF-1 showed tumor-specific expression patterns. It was highly sensitive in central neurocytomas (89.47%) and fully specific for subependymal giant cell astrocytoma (SEGA) among circumscribed gliomas. Focal TTF-1 positivity was sporadically detected in high-grade diffuse gliomas, without significant links to molecular alterations, anatomical sites, or other common immunohistochemical indicators. TTF-1 was consistently negative in multiple newly classified and rare CNS tumors, whereas partial intracranial mesenchymal tumors with FET-CREB fusions presented TTF-1 positivity. In conclusion, TTF-1 serves as a reliable sensitive marker for central neurocytomas and a highly specific marker for SEGA. Given its occasional positivity in high-grade gliomas and certain intracranial mesenchymal tumors, pathologists should interpret TTF-1 results prudently to avoid misdiagnosing primary CNS lesions as metastatic tumors. TTF-1 is therefore a valuable auxiliary biomarker for difficult differential diagnosis of CNS tumors.
The B7-H family of immune checkpoint proteins plays a central role in tumor immune regulation, yet their prognostic and predictive significance in metastatic clear cell renal cell carcinoma (ccRCC) remains incompletely defined. We analyzed a retrospective cohort of primary tumor specimens from 145 patients with synchronous or metachronous metastatic ccRCC to evaluate the expression of B7-H3, B7-H4, B7-H5, and B7-H7 and their association with tumor aggressiveness, treatment response, and long-term survival. Immunohistochemistry revealed marked intratumoral heterogeneity and distinct compartmentalized expression patterns, with B7-H3 detected in both tumor cells and stromal compartments, B7-H4 exclusively expressed in tumor cells, B7-H5 localized to tumor cells and tumor-infiltrating lymphocytes (TILs), and B7-H7 detected in both tumor cells and TILs. We did not detect any overlap in expression between B7-H proteins and PD-L1 positivity in TILs. High B7-H3 expression in tumor and stroma was significantly associated with higher histological grade, increased local invasion, lymph node involvement, and advanced stage. B7-H5 expression in TILs correlated with impaired Eastern Cooperative Oncology Group performance status. Elevated B7-H3 predicted unfavorable response to tyrosine kinase inhibitors, mechanistic target of rapamycin inhibitors, and immune checkpoint inhibitors, whereas B7-H5 negativity in tumor cells associated with more favorable treatment outcomes. B7-H4 and B7-H7 expression in tumor cells were associated with features of tumor aggressiveness and immune suppression, further highlighting the potential role of multiple B7-H family checkpoints in metastatic ccRCC. In multivariable Cox regression models, B7-H3 expression in tumor and stroma independently predicted shorter disease-free and overall survival. B7-H5 expression in TILs independently predicted reduced overall survival. These findings identify B7-H3 as a robust biomarker of tumor aggressiveness, therapeutic resistance, and adverse prognosis in metastatic ccRCC, while B7-H5 in TILs provides complementary prognostic information. Integrated profiling of B7-H immune checkpoints in primary tumors may refine clinical risk stratification and support the development of biomarker-guided immunotherapeutic strategies.
Air pollution, such as particulate matter with a diameter of ≤2.5 μm (PM2.5) is a major contributor to lung cancer in the never-smoking population. Anthracotic pigments (black deposits in the lungs) are physical evidence of environmental exposures. While links between anthracosis and lung disease have been established, anthracosis has not been routinely used as a measure of environmental exposure in research, because there is neither a standard quantitative measure of anthracosis nor an efficient method for quantifying it. We developed 'Slide-based methods for High-throughput Anthracosis Detection and Estimation' (SHADE). SHADE is an automated workflow that quantifies anthracotic pigments on scanned images of whole H&E slides. SHADE was optimized to identify anthracosis while avoiding detection of artefacts in images. SHADE scores and manual pathologist rankings of 10-image patches demonstrated high concordance (R = 0.864). Application of SHADE to background lung sections from 140 never-smoking lung cancer patients demonstrated significant associations between high anthracosis and older age, male sex, 30-year residential PM2.5 estimates, and birthplace in Asia (p < 0.05). Low anthracosis was associated with less aggressive adenocarcinomas (p = 0.052). No significant relationship was identified between anthracosis and 3-year residential PM2.5 estimates, lobe sampled, ethnicity, EGFR mutation status or pathologic tumor stage. Anthracosis levels in the never-smoking cohort were similar to that of background lung sections from 59 ever-smoking patients. Exploratory analysis of background lung sections of 15 individuals who had bronchoalveolar lavage specimen gene expression data available, SHADE scores were associated with the expression of several immunoregulatory genes (CEACAM6, CXCL6, IL5RA, LCN2, SPA17), the activation of inflammatory response and cytokine gene sets, and suppression of the antigen processing and presentation gene set (false discovery rate < 0.1). SHADE provides an automated workflow for anthracosis quantification and has demonstrated utility in uncovering insight into anthracosis-associated molecular changes in lung cancer.
Tropomyosin receptor kinase (TRK) fusions are actionable oncogenic drivers, and pan-TRK immunohistochemistry (IHC) serves as a reliable screening tool for NTRK gene aberrations. However, the expression profile and clinical significance of pan-TRK in meningiomas remain unclear. This study aimed to characterize pan-TRK expression, its correlation with clinicopathological features, and underlying NTRK rearrangement status in meningiomas. We retrospectively analyzed 70 primary intracranial tumor specimens, including 50 meningiomas (21 WHO grade 1, 26 grade 2, and 3 grade 3) and 20 non-meningioma CNS tumors (9 solitary fibrous tumors, 6 hemangioblastomas, and 5 schwannomas) from the First Affiliated Hospital of China Medical University (2020-2022). Pan-TRK IHC was performed using the Ventana EPR17341 antibody, with fluorescence in situ hybridization (FISH) validating NTRK gene aberrations in positive cases. Clinicopathological correlations were analyzed using chi-square/Fisher's exact tests and Spearman's rank correlation. Pan-TRK immunoreactivity was detected in 12/50 (24.0%) meningiomas, predominantly with cytoplasmic staining (75.0%). Positivity was significantly higher in high-grade (WHO 2/3) meningiomas (7/29, 46.7% versus 3/21, 14.3% in grade 1; p = 0.031) and tumors with Ki-67 index ≥5% (7/15, 46.7% versus 5/35, 14.3% in Ki-67 <5%; p = 0.027). No pan-TRK expression was observed in non-meningioma tumors (0/20). FISH confirmed NTRK gene aberrations in 2/12 (16.7%) pan-TRK-positive cases (both WHO 3 anaplastic meningiomas), with strong/moderate IHC staining correlating with aberrations. Pan-TRK is frequently expressed in meningiomas, particularly in high-grade and proliferative tumors, and may have preliminary utility in differentiating meningiomas from other CNS tumors. However, NTRK gene aberrations are rare, necessitating FISH or next-generation sequencing confirmation for pan-TRK-positive cases to identify candidates for TRK-targeted therapy. Further studies are needed to clarify the biological role of non-fusion-mediated pan-TRK overexpression in meningioma progression.
Oligodendrogliomas exhibit significant morphological overlap with low-grade glioneuronal tumors/low-grade gliomas with oligodendrocyte-like features, posing challenges in histopathological diagnosis. SOX10, a transcription factor critical for oligodendrocyte maturation and central nervous system myelination, may serve as a diagnostic marker. We evaluated the diagnostic utility of SOX10 immunohistochemical expression in 300 oligodendrogliomas versus 52 low-grade glioneuronal tumors/low-grade gliomas with oligodendrocyte-like features. Notably, SOX10 was negative in 251/300 (83.7%) oligodendrogliomas but diffusely positive in 47/52 (90.4%) low-grade glioneuronal tumors/low-grade gliomas with oligodendrocyte-like features (except polymorphous low-grade neuroepithelial tumors of the young, PLNTY). SOX10 expression was significantly higher in the latter group (p < 0.001) and showed a WHO grade dependent up-regulation in oligodendrogliomas (p < 0.001). The diagnostic utility of SOX10 was evident in IDH1-immunonegative cases, yielding 72% sensitivity, 90.4% specificity, and a 78.3% positive predictive value for oligodendroglioma. Combining H3K27me3 loss with SOX10 negativity markedly improved diagnostic sensitivity for oligodendroglioma to 88.5%, substantially surpassing the 35% sensitivity achieved with H3K27me3 loss alone. Additionally, an immunohistochemical panel comprising SOX10, Olig2, and IDH1 aided in discriminating non-neoplastic oligodendrocyte hyperplasia from oligodendroglioma and evaluating the presence of peritumoral tumor residue, particularly in IDH1-immunonegative oligodendroglioma. In conclusion, SOX10 negativity effectively excludes low-grade glioneuronal tumors/low-grade gliomas with oligodendrocyte-like features (except PLNTY) and non-neoplastic oligodendrocyte hyperplasia from oligodendrogliomas. SOX10 serves as a specific diagnostic marker, enhancing pathological diagnostic accuracy.
Breast cancer (BC) is most prevalent in females but also accounts for <1% of male cancer cases and 0.2% of male cancer-related deaths. Distribution of histological subtypes, receptor status, and age of diagnosis varies based on sex, and a growing body of evidence supports sex-specific molecular differences in BC. However, this is limited by the smaller number of male cases available for study compared to the thousands of cases of female BC. We combined publicly available male BC gene expression datasets for 195 patients from 4 studies and split randomly into discovery and validation sets. Clustering and gene expression analysis were performed. Two stable clusters were identified initially, confirmed in the validation set. Cluster C1 was enriched for genes associated with MAPK signalling and arylesterase activity. Cluster C2 showed enrichment of genes associated with proliferation, invasion, and metastasis, along with enrichment of gene ontology and pathway terms related to ECM regulation, particularly collagen-containing ECM. Of note, when stratified by ERα and PR status, no enrichment was observed with the predicted PAM50 classification. ERα and MAPK signalling were enriched in both clusters, albeit through different gene sets. Since these pathways were enriched, we investigated the signalling regulation of ERα based on immunohistochemical expression of phosphorylated ERα (S104, S118, S167, S294) and their prognostic roles. This analysis also revealed distinctions from female BC, showing a lack of prognostic outcome for any of these biomarkers. We show that male BC does not align with female BC in the same way that intrinsic subtypes of female BC are not identical. As BC heterogeneity is well recognised, we propose that male BC should be considered as a potentially unique clinical subtype of BC.
Bladder cancer is one of the leading causes of cancer-related mortality worldwide. Long-term survival is particularly poor in patients with muscle-invasive bladder cancer (MIBC). Modulation of purinergic signaling through the ectonucleotidases CD39 and CD73 has emerged as a promising therapeutic strategy in cancer medicine. Altered expression patterns of these molecules have been linked to prognosis in various malignancies. In this study, we assessed the value of CD39 and CD73 expression as prognostic markers and potential therapeutic targets in MIBC. CD39 and CD73 expression was determined by immunohistochemistry using tissue microarrays from 180 patients with MIBC. Associations between tumoral and stromal expression and clinicopathological variables, including overall survival (OS), tumor-specific survival (TSS) and disease-free survival (DFS), were analyzed. Tumor cells did not express CD39. High stromal CD39 expression was significantly associated with a lower T category and UICC stage as well as prolonged median OS, TSS, and DFS. High CD73 expression by tumor cells was significantly associated with poorer OS and TSS. Stromal CD73 expression was not significantly correlated with survival outcomes. Our findings indicate distinct and compartment-specific roles for CD39 and CD73 in MIBC. They suggest that high CD73 expression in tumor cells and low CD39 expression in stromal cells are negative prognostic indicators and potential therapeutic targets in MIBC.
Primary signet-ring cell carcinoma and poorly differentiated adenocarcinoma with poorly cohesive morphology in the cervix are rare conditions, and their clinicopathological features remain poorly described. This study defines primary cervical poorly differentiated adenocarcinomas meeting the diagnostic criteria for poorly cohesive carcinoma as outlined in the 2019 WHO Classification of Digestive System Tumors as 'HPV-associated cervical poorly cohesive carcinomas' (HPV-associated CPCC) and describe their clinicopathological and molecular features. Sixteen HPV-associated CPCC cases were analyzed and classified into three histological subtypes: signet-ring cell carcinoma (n = 4), not otherwise specified (n = 6), and mixed types (n = 6). All patients were Chinese (median age: 46 years; range: 30-66). Vaginal bleeding was the primary presenting symptom (100.0%). High-risk human papillomavirus (HPV) was identified in all tumors, with HPV-18 as the predominant genotype (n = 13), HPV-16 in two cases, and a single case exhibiting concurrent infection with HPV-16, -18, and -58. Overall, 56.3% presented with advanced-stage disease (International Federation of Gynecology and Obstetrics [FIGO] IIIB-IVB), frequently involving regional lymph nodes (56.3%) and distant sites (18.8%). Histopathological examination revealed diffuse stromal infiltration (100%), lymphovascular invasion (75.0%), necrosis (75.0%), and desmoplasia. Immunohistochemically, all cases showed p16 block positivity. Variable expression of antibody-drug conjugate targets was observed, with HER2-low expression (33.3%), and positive staining for Trop-2 (85.7%), nectin-4 (42.9%), and tissue factor (92.3%). During follow-up, disease-specific mortality was 50.0%. The 3-year overall survival rate was 56.3%, which was significantly lower in advanced-stage disease (45.0%) than in early-stage disease (75.0%). Whole-exome sequencing revealed low tumor mutational burden (median 1.28 Muts/Mb), recurrent mutations in AK1, ARHGAP39, KRT24, MICAL3, SLC6A9 (27.3%), KRAS, and KMT2C (18.2%), alongside MUC2 copy gain (63.6%) and bidirectional Y_RNA alterations (gain 54.5%/loss 45.5%). Collectively, HPV-associated CPCC represents a distinct and aggressive subtype characterized by distinctive histopathological features, a predominant association with HPV18, frequent presentation at advanced stages, and marked molecular and biomarker heterogeneity.
Desmoplasia, characterized by excessive expansion of cancer-associated fibroblasts (CAFs) and aberrant extracellular matrix (ECM) deposition, is widely present especially in invasive cancers. The extranodal extension (ENE) of nodal metastasis involves the extension of invasive tumor cells through the lymph node capsule into the perinodal adipose tissue, leading to systemic metastasis. Thus, the specific roles of desmoplasia in ENE formation need to be identified. This study aimed to investigate the association between desmoplasia and its role in ENE formation of oral squamous cell carcinoma, with a focus on Yes-associated protein (YAP)-mediated fibroblast activation and ECM remodeling. Transcriptome profiling and histopathology study based on tissue microarrays were performed to explore the association of metastasis-associated CAFs (MAFs) in ECM remodeling with ENE formation. Primary MAFs were isolated from ENE-positive (ENE+) and ENE-negative (ENE-) lymph nodes for functional characterization. The regulatory role of YAP in MAF-mediated ECM remodeling was investigated through immunohistochemistry, gene knockdown, and co-culture assays with HSC-3 tumor cells. A high degree of desmoplasia and rich collagen remodeling in lymph nodes was associated with higher ENE risk. ENE+ MAFs exhibited superior proliferative capacity, enhanced migration, and increased ECM remodeling activity compared to ENE- MAFs. Co-culture experiments demonstrated that ENE+ MAFs significantly promoted HSC-3 cell invasion through Matrigel. Exploratory RNA sequencing revealed enrichment of ECM receptor interaction and Hippo-YAP pathway in ENE+ lymph nodes. Nuclear YAP localization in MAFs correlated with elevated expression of ECM components (collagen type I alpha 1 [COL1A1], fibronectin 1 [FN1], Tenascin C) and matrix metalloproteinase-2. YAP knockdown in ENE+ MAFs significantly attenuated both ECM remodeling capacity and pro-invasion ability in vitro. These findings suggest that YAP-activated MAFs contribute to ECM remodeling that may create a stromal environment permissive for ENE formation, though direct in vivo validation is needed to establish causality.
Recurrent fusions involving FGFR1-4 genes have been previously described in rare subsets of mostly benign chondroid and mesenchymal neoplasms involving bone and soft tissue. However, a more comprehensive analysis of sarcomas associated with FGFR fusions, including their incidence and histotypes, has not been performed. Triggered by an FGFR1-rearranged unclassified high-grade sarcoma with myogenic differentiation, we investigated our molecular database for sarcomas with FGFR gene fusions to assess their recurrent potential and morphologic spectrum. A total of six unclassified sarcomas were identified, occurring in five females and one male, with a median age of 66.5 years. Tumors were located in the uterus and retroperitoneal/trunk soft tissue. Histologically, all tumors were high grade, composed predominantly of spindle cell morphology with variable cytologic atypia, high mitotic activity, and areas of necrosis. By immunohistochemistry, all tumors demonstrated evidence of myogenic differentiation, with focal or diffuse positivity for one or more markers (desmin, h-caldesmon, smooth muscle actin). However, none of the cases displayed diagnostic features of leiomyosarcoma or other known pathologic entities. Molecular studies revealed recurrent fusions involving FGFR1 (n = 3), FGFR2 (n = 1), and FGFR4 (n = 2), retaining the kinase domain. Additional recurrent genomic alterations included TP53 mutations, CDKN2A homozygous deletions, and RB1 alterations. Most patients followed an aggressive clinical course, including metastases and disease-related mortality. These findings expand the spectrum of sarcomas driven by FGFR gene fusions, underscoring the importance of molecular testing for accurate diagnosis and potential targeted therapy in high-grade sarcomas with myogenic features.
Colorectal mucinous adenocarcinomas (MACs) and serrated adenocarcinomas (SACs) demonstrate considerable overlap in morphological and molecular features, suggesting a spectrum rather than two distinct tumour types. This study investigates the presence of serrated morphological features, precursors, and molecular features in a large cohort of MAC. The study includes 698 MAC collected via the Dutch nationwide pathology databank. Histological review and mismatch repair evaluation were performed on all cases. Next generation sequencing was performed on 396 cases. Multiplex nested methylation-specific PCR (MSP) was performed on 260 microsatellite stable (MSS) cases. The majority of tumours were right-sided (62.6%). Precursor lesions were identified in 9.6% of cases, 88% of which were serrated. Serrated morphology was present in 71% of MAC. Of all cases, 33.8% were microsatellite unstable (MSI). Half of the evaluated cases were CpG island methylator phenotype high. Tumours were divided into six molecular subcategories BRAF/MSS (9.8%), BRAF/MSI (24.7%), KRAS/MSS (38.6%), KRAS/MSI (4.3%), double wild-type/MSS (16.7%), and double wild-type/MSI (5.8%). Based on the presence of a serrated precursor, and/or serrated morphology, and/or a serrated molecular profile, 83.1% of MAC in our cohort could be classified as serrated-MAC. Serrated-MAC developed in older patients, was more often right-sided, showed less perineural invasion, and fewer nodal metastases, when compared to MAC-NOS. Survival analysis showed no significant difference between serrated-MAC and MAC-NOS. We have confirmed that a large majority of colorectal MAC have similar characteristics to SAC.
Abstract Mirvetuximab‐soravtansine (MIRV‐S) is an antibody‐drug conjugate targeting folate receptor alpha (FOLR1). MIRV‐S is approved for the treatment of FOLR1‐positive, platinum‐resistant ovarian carcinoma. Patient eligibility is determined by immunohistochemistry (IHC) using a companion diagnostic (CDx) assay (FOLR1‐2.1, Ventana). This assay requires on‐slide positive controls (OPCs) to aid FOLR1 evaluation. The manufacturer recommends normal fallopian tube (NFT) tissue for OPCs. Estrogen receptor signaling represses FOLR1 in cell culture models. It is unknown whether hormonal factors, such as menopausal status, also impact on FOLR1 immunoreactivity in NFTs used as OPCs. To address this question, we studied FOLR1 protein expression in NFTs (n = 51) from women aged 26–83 years. IHC was performed with the FOLR1‐2.1 CDx assay. Immunoreactivity at apical and basolateral cell membranes was assessed using H‐scores (aH‐score and bH‐score respectively). Overall FOLR1 expression was evaluated using a combined H‐score (cH‐score; i.e. aH‐ and bH‐scores added together). Immunoreactivity scores in pre‐, peri‐, and postmenopausal age groups were compared with the chi‐square test for trends. NFTs showed variable FOLR1 protein expression [median aH‐score: 152.5, interquartile range (IQR): 120–175; median bH‐score: 35, IQR: 7–85; median cH‐score: 195, IQR: 140–245]. Apical immunoreactivity was age‐independent (p = 0.619), but low or absent basolateral immunoreactivity (bH‐score <35) was associated with premenopausal age (p = 0.018). Low overall FOLR1 expression (cH‐score <195) was also associated with premenopausal age (p = 0.037). In conclusion, NFTs show an age‐dependent FOLR1 expression pattern, which likely reflects hormonal repression of FOLR1 in premenopausal women. NFT tissue from postmenopausal women is appropriate and meets the requirements for the current FOLR1 CDx assay.
The application of artificial intelligence in computational pathology depends on both robust algorithms and high-quality, clinically reliable data. Progress in this field has been limited by the scarcity of large, diverse, and well-validated whole slide image (WSI) datasets. To address this gap, HISTAI introduced an open-source resource comprising over 112,000 WSIs across multiple organ systems with associated clinical metadata. Here, we present a pathologist-led evaluation of label accuracy, metadata completeness, and dataset composition across 328 selected cases from this resource. Although HISTAI reports 47,279 cases, we identified only 44,564 unique cases after accounting for missing entries and duplicate records. Basic demographic information, including age and sex, was available for only 55% of cases. Dataset composition was uneven, with dermatopathology accounting for 47.1% of cases and gastrointestinal pathology for 24.0%; however, primary specialty was explicitly reported for only 39.6% of cases, obscuring this imbalance within the provided metadata. Notably, clinical ground truth is recorded in the Conclusion column. Concordance between the dataset's Conclusion and Diagnosis fields was observed in only 20.7% of cases, while 27.1% contained conflicting diagnoses. In a focused review of 198 cases, 30.3% were found to contain unclear or ambiguous diagnostic conclusions, including eight cases in which the diagnosis was incorrect. Assessment of molecular annotation revealed that only 18.9% of analyzed lung and colorectal cancer cases included molecular information. Furthermore, among adult-type diffuse gliomas, none of the 55 cases met current World Health Organisation Classification of Tumors of the Central Nervous System 5th Edition (WHO CNS5) diagnostic criteria, with IDH mutation status reported in only 15 cases. Together, these findings highlight substantial ambiguities in ground-truth labeling, incomplete molecular annotation, and limited documentation of dataset provenance and ethical oversight. While HISTAI represents a valuable open-source resource, its effective and responsible use requires careful clinical validation and close collaboration between computational researchers and pathologists.
Abstract MET alterations, including MET fusions and splicing variants (F/SVs), are linked to glioma progression, but the clinical features remain underexplored since the 2021 WHO classification of tumors of the CNS. We aimed to systematically depict the MET F/SVs and patient characteristics in a multicenter cohort focusing on clinical, pathological, and survival features. We studied data from 1,041 patients with MET F/SVs data from the public Chinese Glioma Genome Atlas database and the TruSight Tumor 170 study. Clinical outcomes were evaluated based on the RANO criteria. We used chi‐square and Fisher's exact tests for variable analysis. Kaplan–Meier analysis was used to assess survival trends, while univariate and multivariate analyses revealed the prognostic value of MET F/SVs. Immunohistochemical staining was performed to demonstrate the MET expression level. Among the 1,041 patients, 49 patients had F/SVs (4.70%), and 23 had ZM fusion (PTPRZ1‐MET fusion gene; 2.21%). Among the 67 recurrent grade 4 astrocytomas, the proportions of F/SVs (11.94%, n = 8) and ZMs (5.97%, n = 4) were the highest. MET F/SVs were significantly associated with malignant clinical outcomes in the IDH‐mutant astrocytoma cohort, with a frequency of 5.04% (18/357) across all WHO grades. Multivariate analysis revealed that the MET F/SVs were independently associated with worse survival in astrocytoma patients [overall survival (OS): p = 0.0011; progression‐free survival (PFS): p = 0.004]. ZM fusion was associated with a worse prognosis in both astrocytoma (OS p < 0.001, PFS p < 0.001) and glioblastoma (OS, p = 0.252; PFS, p = 0.010) patients. We highlight the utmost relevance of ZM fusion as an adverse prognostic factor in astrocytoma (11/382, 2.88%) and glioblastoma grade 4 (11/401, 2.74%) patients and suggest that the grading of these tumors should be refined.
Abstract Molecular subtypes are potential prognostic and predictive tools in muscle‐invasive bladder cancer (MIBC). However, subtype concordance between primary tumors and metastases, as well as subtype‐specific differences in metastatic patterns, remain poorly characterized. The present study aimed to evaluate the concordance of molecular subtypes between primary tumors and matched lymph node (LN) metastases and to explore their association with metastatic patterns. Gene expression–based molecular subtypes were determined according to the five‐tiered Lund Taxonomy in 182 primary tumor samples and 34 matched LN metastases from patients with MIBC who underwent upfront radical cystectomy. Subtypes identified in the primary tumors were compared with those in matched positive LNs and patterns of distant metastasis were analyzed. In addition, the association between molecular and histological subtypes was also investigated. We found an overall 62% subtype concordance between primary tumors and corresponding LN metastases, with complete concordance in the basal/squamous subtype, lower concordance in the luminal subtypes (genomically unstable: 67%; urothelial‐like: 57%), and low concordance (33%) in the mesenchymal‐like (Mes) subtype. Luminal subtypes were associated with LN‐only metastases and less frequent distant metastases. In contrast, the Mes subtype was associated with a higher rate of distant metastases (43%), and more frequent multiorgan involvement (≥3 organs: 40%). Higher expression of the mesenchymal gene CDH2 and the neuronal‐differentiation genes GNG4 and ENO2 was associated with a higher number of metastatic sites. Gene expression–based molecular subtypes may change between primary MIBCs and matched LN metastases, and these differences appear to be subtype‐dependent. Mes subtype and the expression of CDH2 as well as GNG4 and ENO2 are associated with more frequent and extensive metastases, indicating highly aggressive forms of MIBC.
The molecular classification of endometrial carcinomas (ECs) is now integrated into clinical practice. However, identification of polymerase-ε (POLE) variants remains reliant on DNA sequencing, which limits broader implementation. Given the strong prognostic value of pathogenic POLE mutations and the established efficacy of immunohistochemistry (IHC) for detecting mismatch repair (MMR) deficiency and p53 abnormalities, there is a clear need for IHC-based screening strategies to identify patients likely to carry POLE variants and prioritize them for confirmatory sequencing. In this study, we analyzed 24 cases with POLE pathogenic mutations (POLEmut ECs), 3 with benign POLE variants, and 32 matched cases with no specific molecular profile (NSMP) from a cohort of 378 ECs. IHC evaluation of the ataxia telangiectasia mutated (ATM) protein revealed that POLE-mutated ECs (with pathogenic or benign POLE variants) exhibited significantly higher frequencies of non-diffuse positive staining patterns, including null, heterogeneous positive, and subclonal loss, compared with NSMP cases. Targeted next-generation sequencing of all exons across 474 cancer-related genes in the 27 POLE-mutated ECs and 20 NSMP cases with ATM non-diffuse positive staining patterns confirmed that POLE-mutated ECs typically had high tumor mutational burden and were enriched for ATM truncating variants. ATM molecular alterations, including various variant subtypes and multisite mutations, also closely correlated with these staining patterns. Based on these findings, we refined the ATM IHC interpretation framework to integrate staining patterns with sequencing data for improved molecular correlation. Specifically, the null and subclonal loss patterns showed high specificity (96.9%), positive predictive value (94.1%), and accuracy (79.7%) for identifying POLE variants. Notably, the null pattern appeared exclusively in ECs with pathogenic POLE mutations. These results suggest that ATM IHC staining is an effective screening tool for identifying patients who may benefit from confirmatory POLE sequencing among those lacking MMR deficiency or p53 abnormalities.
Folate receptor alpha (FRα) is a promising therapeutic target due to its high expression in several tumor types and its rare expression in healthy tissue. Recently, the antibody-drug conjugate mirvetuximab soravtansine has been approved for treatment of advanced platinum-resistant high-grade serous carcinoma (HGSC). Immunohistochemical expression of FRα has been extensively studied in HGSC, but most studies conducted before the clinical studies targeting FRα used variable antibodies and scoring criteria, which makes comparison of older literature data with recent studies difficult. Moreover, the data regarding its expression in other types of ovarian and other female genital tract tumors are limited or absent. In our study, we focused on immunohistochemical expression in 923 tubo-ovarian and endometrial tumors (assessed on tissue microarrays), using standardized scoring criteria and the VENTANA FOLR1 CDx assay. The results of our study showed the highest FRα expression in serous carcinomas, specifically HGSC (45% positive cases), followed by low-grade serous carcinoma (25%), endometrial serous carcinoma (11%), and serous borderline tumor (10%). Endometrioid and clear cell ovarian carcinomas showed rare positivity (2% and 1%, respectively). All other tumors examined were negative, including mucinous ovarian tumors, sex cord-stromal tumors, endometrial endometrioid carcinomas, undifferentiated and dedifferentiated carcinomas, and endometrial clear cell carcinomas. In conclusion, these results confirm that FRα expression in HGSC and LGSC reaches similar values compared to published data, and is present in a minority of endometrial serous carcinomas. In other ovarian and endometrial tumors examined, FRα expression is absent or rare.
