BACKGROUND:HER2 represents an actionable target in a subset of biliary tract cancers (BTCs), particularly extrahepatic cholangiocarcinoma (eCCA) and gallbladder carcinoma (GBC). Recent approval of zanidatamab for HER2-positive BTC has further highlighted the clinical relevance of accurate HER2 assessment. METHODS:HER2 status was evaluated in 140 consecutive surgically resected BTCs collected between 2020 and 2025 (87 eCCAs and 53 GBCs) using immunohistochemistry (IHC) and dual chromogenic in situ hybridization (DISH). HER2 expression was scored according to gastric cancer criteria, and HER2 positivity was defined following HERIZON-BTC-01 criteria. Selected cases were additionally analyzed using a combined IHC-DISH approach. RESULTS:HER2 expression was 0 in 72.9% of cases, 1+ in 12.1%, 2+ in 8.6%, and 3+ in 6.4%. HER2 positivity was observed in 9.2% of eCCAs and 9.4% of GBCs. ERBB2 amplification was consistently identified in all IHC 3+ tumors and in a subset of IHC 2+ and, rarely, 1+ cases. The mean ERBB2/CEP17 ratio was significantly higher in HER2 3+ tumors than in amplified HER2 2+/1+ tumors (6.2 vs 3.9; p = 0.001). Combined IHC-DISH analysis confirmed concordance between HER2 overexpression and ERBB2 amplification within the same tumor cells. HER2 expression frequently showed intratumoral heterogeneity, often involving less than 50% of tumor cells. CONCLUSIONS:HER2 alterations occur in a subset of eCCA and GBC and show substantial biological heterogeneity, including lower-level amplification and intratumoral heterogeneity, with potential implications for HER2 assessment and patient selection.

BACKGROUND/OBJECTIVES:Mucinous adenocarcinoma (MAC) is a rare and clinically problematic subtype of rectal cancer, tending to present at an advanced stage and to respond poorly to neoadjuvant therapy. The consistently worse prognosis than that of not-otherwise-specified adenocarcinoma (NOS-AC) is not fully understood, potentially owing to intrinsically more aggressive biology or specific immune evasion mechanisms. We used the IMMUNOREACT multicentre cohort, with external validation in TCGA, to investigate the clinical and immunological features of rectal MAC in detail. METHODS:Two hundred patients with rectal adenocarcinoma (16 MAC, 184 NOS-AC) from the IMMUNOREACT 1 (NCT04915326) and IMMUNOREACT 2 (NCT04917263) prospective cohorts were included. To account for the imbalance in baseline characteristics, propensity score matching (PSM) was performed on age, sex, neoadjuvant treatment and TNM stage. The immune microenvironment was characterised using immunohistochemistry (CD3, CD4, CD8, CD8β, Tbet, FoxP3, PD-L1, MSH6, PMS2, CD80), flow cytometry and NanoString PanCancer IO 360™ transcriptomics of adjacent healthy mucosa. Findings were externally validated against TCGA rectal and colon adenocarcinoma datasets. RESULTS:MAC presented at significantly more advanced stage than NOS-AC across all TNM parameters: higher T stage (p = 0.006), N stage (p < 0.001), M stage (p = 0.039) and overall TNM stage (p < 0.001). In the unmatched cohort, MAC was associated with worse overall survival (HR 2.53; 95% CI 1.03-6.23; p = 0.043) and disease-free survival (HR 2.86; 95% CI 1.25-6.55; p = 0.013), but both differences became non-significant after PSM. MAC patients had higher haemoglobin after adjusting for confounders (mean difference [MD] 1.26 g/dL, 95% CI 0.30-2.31, p = 0.012), consistent with a hypothesis of reduced chronic rectal bleeding as a possible mechanism for late presentation. Transcriptomically, MAC showed suppression of HLA class II antigen presentation genes (HLA-DQA1, HLA-DQB1, HLA-DRB1) and myeloid activation genes (S100A8/A9/A12) in adjacent healthy mucosa. Loss of MMR proteins MSH6 and PMS2 in histologically normal mucosa was significantly more frequent in MAC. These findings were replicated in the TCGA cohort, which also showed lower tumour mutational burden and a distinct mucin-associated transcriptomic profile in MAC. CONCLUSIONS:The worse outcomes of rectal MAC appear to be driven largely by late-stage presentation, possibly owing to later diagnosis. MAC nonetheless carries a distinct immune phenotype, detectable even in histologically normal surrounding mucosa, that likely contributes to its treatment resistance. These observations provide a basis for developing histotype-specific approaches to both early detection and treatment in this uncommon but clinically challenging tumour subtype.

PURPOSE:Comprehensive genomic profiling (CGP) is increasingly adopted in the management of patients affected by GI cancers. However, the applicability, performance, and clinical utility of CGP in the real-world setting are still undefined. METHODS:We retrospectively evaluated CGP performance and clinical benefit in consecutive patients with GI tumor at the Veneto Institute of Oncology-IRCCS, Padua. We assessed CGP success and its advantage over routine diagnostics in detecting actionable molecular targets. A custom list of gain alterations was defined, including targets not classified as ESMO Scale for Clinical Actionability of molecular Targets tier IA at the time of analysis. RESULTS:Of the 1,450 samples, 140 (9.7%) were inadequate for CGP. Failure was mainly due to low quantity of extracted tumor DNA (P = .002). Of the 1,265 metastatic patients, GAs were detected in 355 (28.1%) cases, of whom 55 (15.5%) were treated with targeted therapy. Survival did not differ between patients with no GAs and those with GAs who were not treated accordingly (median overall survival 12.0 v 10.9 months), whereas it was significantly longer for those receiving treatment for actionable GAs (26.4 months). This advantage was confirmed in an exploratory, inverse probability of treatment-weighted analysis (adjusted hazard ratio, 0.72 [95% CI, 0.58 to 0.89]; P = .002). Among 283 (83.7%) untreated patients with complete follow-up, 41.6% did not receive targeted therapy due to lack of clinical trial or failure to meet inclusion criteria. CONCLUSION:Appropriate specimen selection and early molecular assessment at the time of advanced disease diagnosis are essential to detect clinically actionable molecular alterations and therapeutic opportunities in patients with GI cancers. Our results support CGP in comprehensive cancer centers.

The tissue-level processes underpinning metastatic outgrowth remain unclear. We combined single-cell RNA sequencing, spatial transcriptomics, and AI-supported 3D imaging in human breast cancer with functional investigations in mice to uncover a 3D morphogenetic process essential for macrometastatic expansion. Macrometastases pervasively activate a metastatic trabecular morphogenesis (MTM) gene-expression program that redeploys developmental branching morphogenesis to build macrometastases as a 3D trabecular lattice of epithelial cords. MTMHIGH cells pre-exist in primary tumors destined to metastasize, whereas MTMLOW primaries are non-metastatic and display a compact, expansile growth architecture. Chromatin immunoprecipitation sequencing (ChIP-seq) on metastatic organoids identifies ETV1/4/5 as master regulators of MTM and branching cancer morphogenesis, required for metastatic outgrowth but dispensable for primary tumor take, bulk growth, and initial metastatic dissemination. Spatial and functional analyses reveal stromal fibroblast growth factor (FGF)→fibroblast growth factor receptor (FGFR) signaling as an actionable MTM dependency. Thus, we link metastatic outgrowth to a 3D developmental morphogenetic process, exposing therapeutic vulnerabilities specific to the lethal macrometastatic stage.

Aims Gallbladder carcinoma (GBC) is a rare and highly aggressive malignancy associated with poor clinical outcomes. While most GBCs are adenocarcinomas, only a small subset shows squamous differentiation. Tumours containing more than 25% squamous component are classified as gallbladder adenosquamous carcinomas (GBASC), a rare histological subtype characterized by a significantly worse prognosis than conventional adenocarcinomas. Due to their rarity, the molecular landscape and biological basis underlying the aggressive behaviour of GBASCs remain poorly understood.Methods and results In this study, 25 retrospectively collected GBASC cases were comprehensively characterized at both immunohistochemical and molecular levels. Predictive biomarkers including programmed death-ligand 1 (PD-L1), mismatch repair proteins (MMR), human epidermal growth factor receptor 2 (HER2) and claudin 18.2 (CLDN18.2) were evaluated. Nearly all tumours (96%) showed PD-L1 positivity with a combined positive score (CPS) greater than 1. CLDN18.2 expression was identified in 28% of cases, although its confinement to the glandular component may limit its therapeutic applicability. All tumours were mismatch repair proficient (MMRp), and none demonstrated HER2 overexpression, in contrast to a subset of gallbladder adenocarcinomas. Molecular profiling revealed recurrent alterations in TP53 (64%), KRAS (16%) and CDKN2A (20%), partially overlapping with the genomic profile of gallbladder adenocarcinomas. However, GBASCs showed a significant enrichment of alterations involving the PI3K pathway, particularly PIK3CA (44%) and PTEN (24%) mutations, representing their most distinctive molecular feature.Conclusions Overall, these findings support the recognition of GBASC as a distinct molecular entity potentially requiring dedicated therapeutic strategies.

Squamous cell carcinoma of the anal canal (SCAC) is a rare malignancy whose incidence has been steadily increasing worldwide. Persistent infection with high-risk human papillomavirus (HPV), particularly HPV16 and HPV18 genotypes, is the main etiological factor and plays a central role in tumor development. While combined chemoradiotherapy remains the standard treatment for localized disease and achieves high rates of tumor control, a considerable proportion of patients experience recurrence or present with advanced disease. For the latter, therapeutic options remain limited. Over the last decade, advances in genomic profiling have significantly expanded our understanding of SCAC biology. Recurrent alterations affecting the PI3K/AKT/mTOR pathway, especially PIK3CA mutations, have emerged as the most common molecular events, particularly in HPV-positive tumors. Additional alterations involve receptor tyrosine kinase signaling, chromatin remodeling genes, DNA damage response pathways, and components of the MAPK cascade. Moreover, HPV-positive and HPV-negative tumors display distinct molecular features with important prognostic implications. Immunotherapy has recently become an important component of treatment for advanced SCAC, although reliable predictive biomarkers are still lacking. This review summarizes the current evidence on the molecular landscape of SCAC, discusses emerging prognostic and predictive biomarkers, and highlights potential opportunities for the development of more personalized therapeutic strategies.

AIMS:To provide a practical, pathology-centred overview of Claudin 18.2 as a biomarker and therapeutic target, covering biology, assay methods and interpretation, pre-analytical factors, clinicopathological associations and implications for treatment selection. METHODS:We performed a narrative review of the biomedical and pathology literature on CLDN18/Claudin 18.2, including basic science, translational studies, immunohistochemistry (IHC) and in situ assays, and clinical trials of Claudin 18.2-directed therapies. Reference lists were hand-searched to capture additional relevant reports. Emphasis was placed on data informing routine diagnostic practice (expression patterns, scoring, fixation variables, pitfalls). RESULTS:Claudin 18.2 localises to tight junctions of differentiated gastric epithelium and is aberrantly expressed in gastric and gastro-oesophageal junction adenocarcinomas, with variable expression reported in pancreatic, biliary and other tumours. Loss or dysregulation of Claudin 18.2 contributes to tumour progression via disruption of epithelial integrity and activation of oncogenic pathways; infection-related and inflammation-related downregulation is described in gastric mucosa. For IHC, clone selection, tissue handling, fixation time and membrane-dominant scoring critically affect results; common pitfalls include cytoplasmic staining and heterogeneity. Claudin 18.2 expression shows predictive value for targeted agents under clinical use/evaluation, supporting its role as a companion biomarker. Reporting recommendations include membrane intensity/percentage thresholds and clear documentation of pre-analytical conditions. CONCLUSIONS:Claudin 18.2 is a biologically plausible and clinically actionable biomarker. Robust pre-analytical handling, validated IHC protocols and standardised scoring are essential for reliable patient selection. Wider adoption of harmonised methods and further disease-specific studies will refine cut-offs, clarify prognostic value and optimise integration of Claudin 18.2-directed therapies into routine care.

AIM:Sessile serrated lesions with dysplasia (SSLd) are direct precursors of colorectal carcinomas (CRCs) but pose a diagnostic challenge. We aim to explore contemporary practice leading to recommendations to improve detection of dysplasia. METHODS AND RESULTS:(i) The frequency of dysplasia in SSLs was estimated through a nationwide study of individuals undergoing colonoscopy in the Netherlands (2014-2022). Out of 186 427 SSLs, 17 456 showed dysplasia, yielding a frequency of 9.4%. (ii) A national audit evaluating diagnostic practices and interobserver variability revealed diagnostic discrepancies in 11% of SSLd cases, while ancillary immunohistochemistry (IHC) was used by only 20% of participating laboratories. (iii) The additional value of biomarkers (MLH1, p16, p53, beta-catenin and c-myc) was assessed using retrospective (n = 213) and prospective (n = 348) SSL cohorts. MLH1 emerged as the only useful biomarker for identifying dysplasia among SSLs, increasing SSLd diagnoses from 33 to 41 cases in the retrospective cohort (P = 0.008) and from 35 to 43 cases in the prospective cohort (P = 0.013). (iv) Misdiagnosis of SSLd among conventional adenomas was investigated using BRAF IHC in a cohort of 1572 advanced adenomas and was found to be very rare, occurring in only 2 cases. CONCLUSIONS:The diagnosis of SSLd appears to have good reproducibility among pathologists and positive diagnostic trends that are observed in recent years. MLH1 is the only IHC marker with robust clinical utility to identify SSLd that do not meet the morphologic criteria for overt dysplasia but should be used only in selected cases due to its low prevalence.

Introduction: Most rectal cancers are microsatellite-stable (MSS) and derive limited benefit from immune checkpoint inhibition. We assessed whether familial colorectal cancer aggregation defines a distinct biological context within MSS rectal cancer.Materials and Methods: We performed a prespecified analysis of two prospective multicentre cohorts conducted between 2018 and 2024. Patients with MSS rectal adenocarcinoma were classified as familial (FH⁺) or sporadic (FH⁻), with a predefined subgroup of patients with an affected first-degree relative (FDR⁺). Known hereditary colorectal cancer syndromes were excluded. Immune profiling, transcriptomic analysis and targeted sequencing were performed on tumour-adjacent histologically normal rectal mucosa and, when appropriate, tumour tissue. Analyses were stratified by neoadjuvant treatment status.Results: Among 374 patients, 92 were FH⁺ and 65 were FDR⁺. In NAT-naïve patients, FDR⁺ cases showed higher epithelial CD80⁺ cell density than FH⁻ cases. After neoadjuvant therapy, FH⁺ patients had increased activated CD8⁺CD28⁺ T cells and reduced epithelial HLA-ABC expression, whereas FDR⁺ cases showed lower CD3⁺ T-cell density. Transcriptomic analysis indicated a quiescent mucosal phenotype in FH⁺ patients, with reduced DNA repair, proliferative, metabolic, angiogenic, and immune pathway activity. Post-NAT FH⁺ and FDR⁺ patients showed higher mutational ratios. In FH⁻, but not FH⁺, higher mutational ratio was associated with improved disease-free survival and immune activation.Discussion: Familial aggregation may define a biologically distinct MSS rectal cancer subgroup, characterised by altered epithelial–immune coordination and dissociation between therapy-induced genomic stress and immune surveillance.​

Background: Mismatch repair deficiency (MMRd) is a central molecular determinant of colorectal cancer (CRC) biology, prognosis, and treatment response, and Universal Tumour Screening (UTS) is advocated for Lynch syndrome (LS) detection; yet real-world performance across clinical subgroups remains limited. We evaluated MMRd distribution and UTS-based LS detection in a large consecutive surgical cohort. Methods: We retrospectively analyzed 1022 consecutive CRC patients undergoing surgical resection at the University Hospital of Padua (2015-2023). MMR status was assessed by immunohistochemistry; MMRd cases underwent reflex BRAF mutation testing and, when available, MLH1 promoter methylation analysis, followed by germline multigene panel testing for suspected LS. Clinicopathological features were compared by MMR status, age at onset, and tumour location. Results: MMR testing was performed in 875 patients (85.6%), rising from 67.0% (2015-2017) to 97.4% (2021-2023). MMRd was identified in 139 tumors (15.9%) and was independently associated with age ≥ 70 years, colonic location, and stage 0-II. Of 22 patients with confirmed LS, 13 (59.1%) were newly identified through UTS; family history showed no significant univariate association with LS status and was not independently associated with MMRd after multivariable adjustment. MMRd prevalence was numerically higher in early- than late-onset CRC (20.0% vs. 15.4%), approaching significance after multivariable adjustment (OR 1.90, 95% CI 0.99-3.64; p = 0.054); hereditary syndromes were also more frequent in early-onset disease. MMRd was markedly rarer in rectal than colonic cancer (4.1% vs. 22.5%; p < 0.0001), though MMRd rectal cancers arose in younger patients. Conclusions: UTS identified a substantial proportion of LS carriers missed by age- or family-history criteria. The relationship between age and MMRd prevalence proved more nuanced than a simple comparison would suggest, reinforcing the value of universal over selective testing across the age spectrum, while MMRd rectal cancer shows a distinct younger profile relevant to immunotherapy-based organ preservation.

Gastric cancer (GC) represents one of the most common and lethal cancers worldwide. The lack of early detection methods and wide heterogeneity from both histopathological and molecular points of view contribute to the high mortality associated with GC. Nowadays, the growing interest in the epigenetic landscape appears to represent a promising approach for improving early diagnosis, disease monitoring, therapy selection, and drug response. The knowledge of the epigenetic landscape in cancerous and, especially in precancerous lesions, remain scarce and controversial. The limitations must be identified due to the lack of standardization in experimental settings which introduce bias and lead to discrepancies in final conclusions. This review aims to provide an overview of epigenetics in the diagnostic setting of gastric cancer and in preneoplastic conditions, illustrating the available experimental approaches and therapeutic management in the era of epigenetics.

Precision oncology relies on precision diagnostics, and histopathological diagnosis, along with biomarker evaluation, currently represents the cornerstone for personalized treatment. In gastrointestinal neoplasms, diagnostic assessment and molecular profiling are often performed on biopsy tissue, which may be quantitatively/qualitatively limited. Therefore, appropriate sample management is essential to avoid unnecessary waste and to obtain all the information necessary for treatment planning. Several factors may significantly impact biomarker testing: (i) pre-analytical issues; (ii) heterogeneity in biomarker expression; (iii) lack of standardization in biomarker testing and evaluation. Moreover, in the metastatic setting, inadequate/incomplete clinical information can lead to inappropriate sample handling, with negative implications. The application of appropriate guidelines in testing and reporting biomarker status according to clinical context is, therefore, strongly encouraged. In this position paper, the Italian Group of Gastrointestinal Pathologists (GIPAD), a section of the Italian Society of Pathological Anatomy and Cytology (SIAPeC-IAP), aims to summarize all the clinical and pathological requirements for adequate assessment of prognostic and predictive biomarkers in the gastrointestinal oncology patient, from biopsy acquisition to diagnostic reporting.

Background:Mutations in the isocitrate dehydrogenase (IDH) genes are key biomarkers in intrahepatic cholangiocarcinoma (CCA) and adult-type diffuse gliomas, although real-world adoption of comprehensive molecular diagnostics remains uneven. This review aimed to integrate published evidence, clinical experience, and international guidelines to provide pragmatic recommendations that can standardize IDH testing across healthcare systems. Methods:A multidisciplinary panel synthesized data identified through 10 PICO-driven questions, critically appraised guideline statements from ESMO, EANO, NCCN, and WHO-CNS5, and incorporated insights from clinical evidence on IDH molecular profiling in CCA patients. Recommendations were developed through interactive expert discussion. Results:The panel addressed six issues: (i) positioning of next-generation sequencing (NGS) as a first-line assay; (ii) using liquid biopsy to supplement inadequate or uninformative tissue-based molecular analyses; (iii) tumor-adapted workflows combining immunohistochemistry, PCR, or NGS with large genomic panels; (iv) optimizing pre-analytical management of small biopsies in terms of neoplastic cell abundance and nucleic acid fragmentation to safeguard material for integrated testing; (v) evaluating promising biomarkers based on genome-wide methylation profiling and metabolic imaging in specialized centers; and (vi) novel testing strategies including centralized and decentralized algorithms. In addition, emerging approaches based on digital pathology, teleconsultation, and harmonized reimbursement pathways were discussed. These considerations were distilled into a set of "Golden Rules." Conclusions:Optimized molecular profiling is a cornerstone of precision oncology in IDH-mutant tumors, but the lack of harmonized procedures hinders its widespread implementation in the clinical setting. In intrahepatic CCA, upfront NGS should be prioritized to capture the full spectrum of actionable alterations, whereas in diffuse gliomas IHC for IDH1 p.R132H remains recommended, with PCR or NGS reserved for IHC-negative or equivocal cases. Advanced tools such as genome-wide methylation profiling or metabolic imaging may add value in specialized centers. The consensus-based "Golden Rules" pragmatically support harmonization of diagnostic workflows, reducing technical costs and turnaround time, and promoting equitable access to IDH-directed therapies across diverse healthcare settings.