Mucinous ovarian carcinoma (MOC) is an epithelial ovarian cancer subtype that is frequently misclassified as extraovarian mucinous metastasis (EOM) because of overlapping features. To address this diagnostic challenge, we perform genome-wide DNA methylation profiling of 58 MOCs, 38 EOMs, and 18 mucinous borderline ovarian tumors (mBOTs) collected from six institutions. Methylation analysis defines two mBOT groups, one epigenetically similar to normal ovary and one resembling MOC. Unsupervised clustering reveals two distinct MOC methylation subtypes with potential prognostic relevance in the internal cohort. Using these data together with 389 external profiles, we develop and validate a three-step machine-learning classifier that distinguishes MOC from EOM with 95.5% accuracy. External validation of this classifier on 21 MOCs and 24 EOMs yields an accuracy of 91.11% for differentiating MOC from EOM. These findings establish an epigenetic framework for mucinous ovarian tumors and provide a robust clinical classification tool.

Oncogenic alterations in MET represent therapeutically actionable driver alterations in non-small cell lung cancers (NSCLC). Among these, MET fusions are rare, occurring in approximately 0.1%-0.3% of NSCLC. We report the case of a 52-year-old woman with metastatic, TTF1-positive lung adenocarcinoma harboring a KIF5B::MET fusion. After progression on chemotherapy and immunotherapy, she achieved a durable response lasting nearly five years on third-line treatment with the type Ia MET inhibitor crizotinib. At the time of suspected disease progression, two tissue re-biopsies were non-diagnostic due of insufficient tumor cell content. Circulating tumor DNA (ctDNA) analysis identified two newly acquired on-target resistance mutations within the MET kinase domain (L1213V and Y1248C) in addition to the known KIF5B::MET fusion. After re-evaluation by the institutional molecular tumor board, both alterations were considered mediators of resistance to type I MET inhibitors, with available data indicating preserved sensitivity to type II inhibitors. Based on these findings, the patient was switched to cabozantinib, a multikinase type II MET inhibitor, resulting in a radiographic disease stabilization accompanied by a marked decline in tumor marker levels. This case illustrates the clinical utility of liquid biopsy for molecular resistance monitoring, particularly when tissue re-biopsy is not feasible, supports its integration into clinical decision-making, and underscores the therapeutic relevance of MET inhibitor class-switch strategies in MET fusion-positive disease.

BACKGROUND:Antibody-drug conjugates (ADCs) represent a promising therapeutic approach for high-grade serous ovarian carcinoma (HGSOC). Patient selection for ADC therapy depends on tumour target expression, making it essential to characterize molecular, spatial, and temporal heterogeneity. METHODS:We analyzed two HGSOC tissue microarray cohorts: 100 genomically profiled cases (1565 cores) and 64 matched cases with paired adnexal (A), locally advanced (LA), and recurrent (R) samples (2395 cores). Associations between ADC target expression and molecular characteristics, sampling site, and survival were investigated. RESULTS:ADC targets showed no significant associations with homologous recombination deficiency (HRD) or TP53 mutation status; TROP2 was modestly lower in BRCA1/2-mutated tumours. Folate receptor-alpha (FolR1) showed notable spatial heterogeneity: 20.2% switched therapeutic-indication groups between centre and margin at A; 21.7% were reclassified between A and LA. Temporally, all markers showed ≥ 20% switching, reaching 38.4% for FolR1 between A and R. High FolR1 expression in A correlated with poorer survival, a pattern not observed in LA or R samples. CONCLUSIONS:ADC targets in HGSOC display limited molecular but significant spatial and temporal heterogeneity, with expression classifications varying by site and time. FolR1 expression in adnexal tumours associates with aggressive disease.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, with limited therapeutic options, few patients showing targetable molecular changes. New therapeutic strategies are necessary. Antibody-drug conjugates (ADCs) have emerged as alternative therapeutic strategies across various cancer types. Herein, we analyze the expression and spatial heterogeneity (six cores per patients) of three ADC targets (c-MET, NECTIN4, and TROP-2) in a cohort of 62 PDAC patients (1,116 tissue cores) and associate their levels with clinicopathological and genomic parameters, and the expression of immune checkpoints. c-MET exhibited significantly higher expression at the tumor front versus tumor center, along with notable intratumoral heterogeneity. In contrast, NECTIN4 and TROP-2 displayed homogeneous expression patterns, with NECTIN4 being absent in approximately two-thirds of cases, while TROP-2 showed consistently strong positivity across tumor regions (98% 3+). By simulating sampling sufficiency for reliable scoring, we observed that, for c-MET, two tumor samples were sufficient to achieve a maximum score of 1+, while for higher scores (2+ and 3+), four samples were required. For NECTIN4, four samples were necessary to detect scores of 1+ and 2+. For TROP-2, for a 3+ score, just two samples were sufficient to reach the maximum score. c-MET or TROP-2 expression scores were not associated with any clinicopathological parameters. In contrast, NECTIN4 expression showed an association with tumor grade. Correlations with immune checkpoints revealed that high TROP-2 expression was inversely correlated with PD-L1 expression. For all three markers no significant differences in expression were found between SMAD4 wild-type and SMAD4-mutated tumors, nor between TP53 wild-type and TP53-mutated tumors. Furthermore, analysis of lymph node and distant (liver and peritoneal) metastases revealed significantly higher c-MET and NECTIN4 expression in the metastatic setting. In conclusion, TROP-2 is highly expressed in most PDACs, independent of clinicopathological and genomic parameters, and inversely correlating with PD-L1, making TROP-2 an ideal ADC target.

The origin of mucinous cystic neoplasms (MCNs) remains a major challenge in hepato‐pancreato‐biliary pathology. These cystic tumors are defined by their mucinous epithelium and ovarian‐like stroma, with an estimated 10% risk of progression to invasive carcinoma. The origin of the ovarian‐like stroma remains a subject of debate. In this study, we conducted immunohistochemical profiling, targeted DNA sequencing, and genome‐wide DNA methylation analysis on a cohort of 15 pancreatic MCNs (MCN‐P) and six hepatic MCNs (MCN‐L). Using immunohistochemistry and targeted DNA sequencing, we unequivocally established the diagnosis of MCN. Unsupervised DNA methylation profile analysis of reference classes of pancreatic neoplasms (11 entities and normal pancreatic tissue from 224 unique samples) revealed that MCN‐P predominantly forms a distinct group. In the DNA methylation landscape of liver tumors, encompassing five tumor types and normal bile duct tissue from 136 unique samples, MCN‐L demonstrated a specific methylation profile when compared with all other entities. Furthermore, within the DNA methylation landscape of ovarian tumors – featuring five tumor types, normal Fallopian tube, and normal ovarian tissue from 90 unique samples – we found that both MCN‐P and MCN‐L grouped with mucinous ovarian carcinoma and mucinous borderline ovarian tumors (mBOTs). Notably, low‐grade MCNs exhibited greater DNA methylation similarities to mBOTs, while high‐grade or invasive MCNs were primarily associated with mucinous ovarian carcinomas. When analyzing all samples together (19 tumor types and four normal tissue types, n = 430), MCNs similarly grouped with mucinous ovarian tumors and normal ovarian tissue. Additionally, in a network analysis of differentially methylated probes, MCN‐P and MCN‐L share significant methylation traits, closely resembling mucinous ovarian tumors. In conclusion, our findings highlight that MCN‐P and MCN‐L are distinct entities in the landscape of pancreatic and hepatic tumors and show DNA methylation profile similarities with mucinous ovarian tumors, suggesting a potential common origin. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background: ESR1 mutations are biomarkers in breast cancer patients who develop metastatic disease after endocrine therapy (ET). Recently, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Elacestrant, a selective estrogen receptor degrader for patients harboring ESR1 mutations. This has necessitated the establishment of reliable and sensitive NGS- or PCR-based assays to detect these ESR1 resistance mutations in liquid biopsy samples. Methods: We evaluated NGS results of a pan-cancer cohort of almost 6000 patients from two major German institutes of pathology, to show that the occurrence of ESR1 mutations is extremely rare (<1%) in ET-naïve patients. This suggests that ESR1 mutations arise almost exclusively under the pressure of ET. Therefore, we designed a breast cancer-specific hybrid capture-based NGS liquid biopsy assay covering 12 breast cancer-related genes, including ESR1, PIK3CA, AKT1, ERBB2, BRCA1/2, and TP53. We validated the HS2-Mamma-LIQ assay extensively using reference material to detect mutations to 0.1% variant allele frequency (VAF) and compared the performance to a commercially available ESR1 ddPCR assay. Results: We show the results of routine diagnostic analysis of the first consecutive 354 patients with activating ESR1 mutations rate of 43%, with 20% of patients harboring co-mutations in PIK3CA and other genes underlining the relevance of tumor heterogeneity. Our study highlights liquid biopsy as a preferred approach for monitoring ESR1 mutations in breast cancer patients by showing cases where NGS analysis suggests complex tumor heterogeneity with multiple ESR1 as well as PIK3CA mutations at different VAFs. Conclusions: Our findings not only corroborate prior research concerning the rarity of these mutations in unselected patients but also emphasize the importance of robust and broad molecular assays rather than single gene assays in their detection and characterization in the diagnostic setting. Advantages of different approaches are discussed to address the current clinical need.

BACKGROUND:Metastatic uveal melanoma (mUM) is an aggressive cancer predominately affecting the liver. Peritoneal metastases (PM) occur rarely, and there is limited knowledge about this subgroup´s clinical course and biology. METHODS:We analyzed 41 mUM patients with confirmed PM from the Charité-Universitätsmedizin Berlin database, focusing on clinical characteristics, immune cell infiltrates, genetic alterations and tumor mutational burden (TMB). RESULTS:The incidence of PM in mUM was 4.27 %. Metastatic disease was diagnosed 3.6 years after primary UM, with PM developing later (median: 4.7 years). Median overall survival (OS) from mUM diagnosis was 22.4 months. Prognosis correlated with metastatic pattern. Patients presenting with synchronous liver and peritoneal metastases or primary hepatic metastases followed by secondary peritoneal dissemination showed a median OS of 19.7 and 17.7 months, respectively. However, PM patients with exclusive extrahepatic disease at diagnosis of mUM had a significantly longer OS of 48.6 months and this metastatic pattern showed highly significant correlation with low and intermediate genetic risk. Metastasis-free survival and OS upon mUM diagnosis were significantly shorter in patients with high-risk UM tumors. TMB also correlated with metastatic pattern, being lowest in patients presenting with only extrahepatic disease. Higher TMB was generally associated with shorter OS. CONCLUSION:PM in mUM patients is rare and in contrast to other extra-abdominal tumors does not worsen prognosis. Prognosis is greatly influenced by the metastatic pattern, which is determined by tumor biology, as evidenced by its correlation with genetic risk groups and TMB.

A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings.

INTRODUCTION:Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis. METHODS:To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability. RESULTS:The mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences. CONCLUSION:Continuous optimization of bioinformatic workflows and participating in round robin tests are recommended.

PDF file - 54K, Primers and probes used for Quantitative RT-PCR assay.

PDF file - 53K, Selected upregulated canonical Pathways in IL-32 treated MyLa cells.

Supplementary materials and tables

Cytokeratin expression profile NT-3 xenograft

Growth characteristics of BON, AGP and NT-3

Neuroendocrine Phenotype and mutational status NT-3

PDF file - 316K, Selected upregulated and down regulated genes of IL-32 treated MyLa and HH cells.

Liver metastasis model NT-3

PDF file - 240K, IL-32 production is not restricted to already known T-cell subsets.

SSTR expression original tumor

Melanoma brain metastases (MBM) variably respond to therapeutic interventions; thus determining patient's prognosis. However, the mechanisms that govern therapy response are poorly understood. Here, we use a multi-OMICS approach and targeted sequencing (TargetSeq) to unravel the programs that potentially control the development of progressive intracranial disease. Molecularly, the expression of E-cadherin (Ecad) or NGFR, the BRAF mutation state and level of immune cell infiltration subdivides tumors into proliferative/pigmented and invasive/stem-like/therapy-resistant irrespective of the intracranial location. The analysis of MAPK inhibitor-naive and refractory MBM reveals switching from Ecad-associated into NGFR-associated programs during progression. NGFR-associated programs control cell migration and proliferation via downstream transcription factors such as SOX4. Moreover, global methylome profiling uncovers 46 differentially methylated regions that discriminate BRAFmut and wildtype MBM. In summary, we propose that the expression of Ecad and NGFR sub- classifies MBM and suggest that the Ecad-to-NGFR phenotype switch is a rate-limiting process which potentially indicates drug-response and intracranial progression states in melanoma patients.