CIC-rearranged sarcomas represent a distinct rare pathological entity within the spectrum of undifferentiated small round cell sarcomas. This single institution retrospective study investigates the histopathological, molecular, and clinical characteristics of a cohort of 14 CIC-rearranged sarcomas. These tumors were diagnosed in eight male and six female patients, with the age at initial diagnosis ranging from 8 to 67 years and a mean age of 23.7 years. Among the patients, three novel locations of CIC::DUX4 sarcomas were detected in the mesentery, pancreas, and pterygopalatine fossa. Genetically, we identified 12 cases as CIC::DUX4 sarcomas and two as sarcomas with CIC gene rearrangement detected by break-apart fluorescence in situ hybridization only. The most common gene translocation had a breakpoint located in exon 20 of the CIC gene and exon 1 of the DUX4 gene; this alteration was found in 83% of all CIC::DUX4 cases we identified. We also performed DNA methylation analysis on seven cases, which was proven to be a reliable method with high sensitivity for clustering CIC-rearranged sarcomas.

Primary gastric adenosquamous carcinoma (GASC) is a rare tumor that exhibits aggressive behavior and currently lacks standardized therapeutic recommendations. Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) and positive PD-L1 expression confer sensitivity to immune checkpoint inhibitors; however, their statuses in GASC remain uncertain. In this study, clinical features, MMR/MSI status, MLH1 methylation, two T-cell markers, and PD-L1 expression of 30 GASC cases were collected from three institutions. Additionally, 196 gastric adenocarcinomas (GACs) were collected for comparison. The median age of GASC patients was 62 years, with 76.7% being males, and 56.7% at stage III. dMMR/MSI-high with MLH1 hypermethylation was observed in 33.3% GASCs, and was significantly associated with older age, female, distal location, larger size, deeper tumor invasion, and higher CD3 and CD8 densities and PD-L1 expression. Both glandular and squamous components of all dMMR GASCs showed loss of MLH1 and PMS2 expression. No significant difference in overall survival was observed between dMMR and mismatch repair proficiency (pMMR) GASC patients, while inferior overall survival was observed in pMMR GASC treated with surgery alone compared to those receiving chemotherapy. When comparing to GAC, GASC exhibited clinicopathological features indicative of more aggressive behavior (larger size, poorly tumor differentiation, deeper tumor invasion and more lymph node metastases). A significantly higher frequency of dMMR was found in GASC (33.3%) than that in GAC (16.3%). This study offers a comprehensive perspective on the clinicopathological features of GASC, emphasizing a subset of GASC associated with dMMR and MLH1 hypermethylation. Immunotherapy might be a promising strategy for GASC.

Validation of biomarker assays is mandatory not only for their applications in clinical trials but also for their subsequent transfer to clinical laboratories in routine clinical care. There are two critical components relevant to their transfer to clinical practice: regulatory oversight and methodology transfer. Both aspects are simplified where companion diagnostic (CDx) assays relevant to a given indication are being implemented in clinical laboratories. However, when laboratory developed tests (LDTs) are being used either because CDx is not available or because LDT is preferred, both aspects need special consideration from regulatory agencies as well as clinical laboratories. The key component that links these two aspects is evidence of validation of the new LDTs. For predictive and prognostic biomarkers in oncology, clinical validation is feasible only in clinical trials. This approach is not available or feasible to clinical laboratories that develop LDTs. While clinical laboratories routinely perform technical/analytical validation, depending on the type of biomarker, this may not be sufficient to provide evidence of the LDT’s clinical relevance. Laboratories must perform and document their assessment for the need for indirect clinical validation. When indirect clinical validation is required, it must be performed according to existing guidelines for this purpose. This paper provides expert consensus guidance and recommendations on how to assess for the need for indirect clinical validation and how to perform indirect clinical validation where required. This paper also provides a conceptual framework to regulatory agencies for determining requirements for validation of predictive and prognostic biomarkers in oncology.

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.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00428-025-04113-6.

Inguinal lymph node surgery is a standard treatment for penile cancer patients with intermediate or high risk for lymph node metastasis (LNM) according to European Association of Urology (EAU) risk grading. We are proposing a more objective histological prognostic grading system for inguinal LNM in these patients. We assessed worst pattern of invasion, lymphocytic host response, lymphovascular invasion, and perineural invasion in a population-based cohort of 306 penile cancer patients. Patients were classified into low, intermediate, and high risk for inguinal LNM. There was a significant association both between risk groups and pT stage (p < 0.001) and between risk groups and LNM. Univariate logistic regression showed 25.43 times higher odds of LNM for patients in the intermediate risk group compared with the low risk group (odds ratio (OR) 25.43; 95% confidence interval (CI): 5.94–108.97) and a 177.13 times higher odds in the high risk group compared to the low risk group (OR 177.13; 95% CI: 40.09–782.51). When comparing our histological risk grading with the EAU grading, we found a higher specificity, of 53.43% (95% CI: 47.84–59.02) versus 15.20% (95% CI: 11.17–19.22), as well as a higher area under the curve (0.86; 95% CI: 0.81–0.89; versus 0.65; 95% CI: 0.58–0.71) with our grading system. While our grading classified 111 patients as low risk, only 31 were considered low risk for LNM according to the EAU risk classification. The new histological risk grading system shows a higher specificity and includes a higher number of patients in the low risk group in whom lymph node surgery could be avoided, reducing morbidity and costs.

We present the clinicopathological features of 23 cases of the giant cell subtype of urothelial carcinoma, a rare subtype of bladder cancer recognized in the current World Health Organization classification of urological tumors. Histologically, the architectural pattern of the tumor varied from infiltrating to the solid expansile pleomorphic tumor with giant, bizarre, anaplastic cells. Typical or atypical mitotic figures were frequently present in all cases. Between 10 and 30% of the tumor had a giant cell component. All cases were associated with conventional high-grade urothelial carcinoma, with areas of squamous cell divergent differentiation and micropapillary carcinoma present in six and two cases, respectively. In one case each had sarcomatoid, nested, small cell, or glandular divergent differentiation. At diagnosis, 35% of patients had advanced disease and 12% had distant metastases. When comparing giant cell urothelial carcinoma with conventional urothelial carcinoma in a matched analysis, differences in overall and cancer-specific survival were observed, particularly in the T1 stage category. Immunohistochemical staining showed a similar profile of urothelial lineage with frequent positive expression of uroplakin II, GATA3, CK20, CK7, and S100P in both giant cell and conventional urothelial carcinomas. High Ki67 proliferation (range, 60–90%; mean, 71%) and nuclear p53 accumulation (mutant profile; range, 50–90%; mean, 64%) were observed. Using the 22C3 assay, the expression of PD-L1 was found to be variable in two cases, and beta-HCG was negative. In conclusion, giant cell carcinoma is a subtype of urothelial carcinoma associated with advanced clinical stage and a trend to lower survival rates.

Surgical resection for hepatocellular carcinoma (HCC) is burdened with a high recurrence rate and a lack of reliable prognostic factors. The aim of this study was to integrate the HCC pathological features with gene mutations to improve the prognostic role of pathological analysis. This is a monocentric prospective study, including 67 patients resected for HCC. All clinical data and histological features were collected, including tumor grade, architecture, margins, microvascular invasion, and microscopic portal vascular invasion (MPVI). Next-generation sequencing (NGS) was performed using a laboratory-developed multi-gene panel, allowing to amplify 330 amplicons (21.77 kb), covering the relevant targets for solid tumor analysis. The most represented mutations were TERT promoter (n = 41, 61.2%), TP53 (n = 18, 26.9%) and CTNNB1 (n = 17, 25.4%). At follow-up, 13 (19.4%) patients experienced HCC recurrence: at multivariate analysis, tumor dimensions (p = 0.040), MPVI (p = 0.010), and TERT mutation (p = 0.034) correlated with recurrence. Dimensions ≥ 4.5 cm (very close to AJCC stage pT3; 9 recurrences, p = 0.041, odd-ratio = 3.7), MPVI (9 recurrences, p = 0.062, OR = 3.3), and TERT (11 recurrences, p = 0.049, OR = 4.4) correlated with disease-free survival also at univariate analysis. The concomitant occurrence of these three variables was present in 7 cases, among which 5 recurred (p = 0.002, OR = 15.94). In conclusion, NGS analysis in resected HCC could not only be used for future therapies but should be integrated with histopathology to predict the risk of tumor recurrence after surgical resection: TERT mutation is among the strongest predictors of tumor recurrence, together with tumor stage (dimensions) and the occurrence of MPVI, which should always be reported separately from the classic MVI.