The introduction of next-generation sequencing (NGS) in the molecular diagnostic armamentarium is deeply changing pathology practice and laboratory frameworks. NGS allows for the comprehensive molecular characterization of neoplasms, in order to provide the best treatment to oncologic patients. On the other hand, NGS raises technical issues and poses several challenges in terms of education, infrastructures and costs. The aim of this review is to give an overview of the main NGS sequencing platforms that can be used in current molecular diagnostics and gain insights into the clinical applications of NGS in precision oncology. Hence, we also focus on the preanalytical, analytical and interpretative issues raised by the incorporation of NGS in routine pathology diagnostics.

Background: Anastomotic leaks (ALs) remain a critical complication after rectal cancer surgery. Emerging evidence suggests that local immune dysregulation may play a key role in anastomotic healing. We investigated the immune microenvironment of histologically normal, tumor-adjacent rectal mucosa—a tumor-conditioned field—as a potential substrate for AL predisposition. Methods: IMMUNOREACT 4 is a sub-analysis of the IMMUNOREACT project (clinicaltrials.gov NCT04915326 and NCT04915326), a multicenter translational study evaluating immune features of histologically normal, tumor-adjacent rectal mucosa of patients undergoing colorectal anastomosis. A prospective cohort (n = 121) was analyzed using flow cytometry, in addition to a retrospective cohort (n = 262) using immunohistochemistry. Immune markers of epithelial activation and lymphocyte subsets were compared between patients with and without postoperative ALs. Exploratory predictive models combining immune and clinical variables were developed and evaluated using discrimination, calibration and decision curve analyses. Results: At flow cytometry, the CK⁺HLAabc⁺ MFI (AUC 0.66, 95% CI 0.52–0.80), CD8⁺CD38⁺ cell rate (AUC 0.65, 95% CI 0.52–0.78) and CD3⁺CTLA4⁺ cell rate (AUC 0.65, 95% CI 0.51–0.80) showed moderate predictive potential for ALs. In immunohistochemistry, CD3⁺ (AUC 0.57, 95% CI 0.54–0.60), CD8⁺ (AUC 0.57, 95% CI 0.52–0.62), CD8β⁺ (AUC 0.59, 95% CI 0.53–0.65) and Tbet⁺ (AUC 0.60, 95% CI 0.56–0.64) showed some predictive ability for ALs. The model including CD8β⁺, the BMI, neutrophile/lymphocyte ratio and tumor location had an AUC of 0.67 (95% CI 0.62–0.72). Conclusions: Immune activation within histologically normal, tumor-adjacent rectal mucosa—characterized by epithelial HLA upregulation and cytotoxic or Th1 T cell infiltration—is associated with postoperative ALs. Although predictive accuracy is limited, these findings support the concept that a tumor-conditioned immune microenvironment may predispose patients to impaired anastomotic healing. Integration of mucosal immune profiling with clinical variables represents a promising exploratory approach that warrants further prospective validation.

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.

There is a growing interest in optimizing the therapeutic management of older cancer patients as well as understanding the sex-specific differences in terms of efficacy and safety of cancer treatments. However, limited data are available on the initial therapy of patients with advanced gastro-oesophageal cancer. The ARMANI phase III trial showed progression-free survival (PFS) and overall survival (OS) benefit with paclitaxel plus ramucirumab switch maintenance (arm A) versus the continuation of first-line oxaliplatin and fluoropyrimidine (arm B) in patients with advanced human epidermal growth factor receptor 2-negative gastric or gastro-oesophageal junction cancer. We conducted a subgroup analysis aimed at assessing the differences in efficacy, safety and quality of life (QoL) according to age (<70 versus ≥70 years) and sex. No significant differences in terms of PFS (P = 0.757), OS (P = 0.588) and overall response rates (ORRs) (P = 0.238) were observed between older and younger patients. No significant differences in PFS (P = 0.646), OS (P = 0.858) and ORRs (P = 0.649) were observed between females and males. The effect of treatment arm on survival outcomes was similar across age (P = 0.094) and sex groups (P = 0.469). Looking at specific adverse events, peripheral neuropathy occurred more frequently in younger patients (P = 0.020), anaemia in older patients (P = 0.044) and hand-foot syndrome in women (P = 0.021). Older patients were less likely to receive a post-discontinuation treatment (P = 0.010), especially in arm B (P = 0.026). The impact on QoL was better in arm A, irrespective of age and sex. The ARMANI trial supported the benefit of the investigated switch maintenance strategy irrespective of age and sex.

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.