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Prognosis of gastric cancer is dramatically improved by early diagnosis. Correa’s cascade correlates the expression of some molecular markers with the progression of preneoplastic lesions toward carcinoma. This article reviews the diagnostic and prognostic values of molecular markers in complete (MUC2) and incomplete (MUC2, MUC5AC, and MUC6) intestinal metaplasia, gastric dysplasia/intra-epithelial neoplasia, and early gastric cancer. In particular, considering preinvasive neoplasia and early gastric cancer, some studies have demonstrated a correlation between molecular alterations and prognosis, for example, mucins phenotype in gastric dysplasia, and GATA6, TP53 mutation/LOH and MUC6 in early gastric cancer. Moreover, this review considers novelties from the literature regarding the (immuno)histochemical characterization of diffuse-type/signet ring cell gastric cancer, with particular attention to clinical outcomes of patients. The aim of this review is the evaluation of the state of the art regarding suitable biomarkers used in the pre-surgical phase, which can distinguish patients with different prognoses and help decide the best therapeutic strategy.

Next-Generation Sequencing (NGS) has transformed cancer research and clinical practice, with Whole Exome Sequencing (WES) driving advances in mutational profiling and personalized oncology. Yet, transcriptomic signatures remain essential for understanding disease mechanisms, including therapy resistance pathways. RNA sequencing (RNA-seq), however, faces unique challenges when dealing with low-input or degraded RNA, as often found in archival formalin-fixed paraffin-embedded (FFPE) tissues. Although previous studies have compared library preparation protocols, rapidly evolving technologies call for updated evaluations. Here, we present a direct comparison of two FFPE-compatible stranded RNA-seq library preparation kits: TaKaRa™ SMARTer ® Stranded Total RNA-Seq Kit v2 (Kit A) and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Kit B). Both kits generated high-quality RNA-seq data, yet important differences emerged. Notably, Kit A achieved comparable gene expression quantification to Kit B while requiring 20-fold less RNA input, a crucial advantage for limited samples, albeit with increased sequencing depth. We critically discuss these results in relation to RNA availability, technical performance, cost-effectiveness, processing time, and automation potential, offering practical guidance for selecting optimal RNA-seq strategies in clinical and translational research settings.

Background Dendritic cells (DCs) are the most efficient antigen-presenting cells and play a central role in the immune system, orchestrating immune response against tumors. We previously demonstrated that DC-based vaccination effectively induces anti-tumor immunity, yet at the same time showing a robust safety profile, making this treatment a potential candidate for effective adjuvant immunotherapy. To explore this possibility, we designed a randomized phase II trial (EudraCT no. 2014-005123-27) to provide a complementary autologous DC vaccination to patients (pts) with resected stage III/IV melanoma. Methods Overall, a total of 18 eligible pts were included in this study, 10 of whom received 6 monthly DC vaccination cycles combined with IL-2 administration (arm A), and 8 pts were enrolled in the follow-up observational cohort (arm B). A deep immune biomarkers profiling by multiplex immunoassay, human leukocyte antigens (HLA) typing, multiparametric flow cytometry and in situ tumor microenvironment analysis was performed for the entire pts cohort. The immunological response was assessed in vivo by DTH test and ex vivo against selected melanoma-associated antigens applying the IFN-γ ELISPOT assay. Results Pts receiving DC vaccination showed a better relapse-free survival compared to the observational cohort (median 6.6 months, 95% CI, 2.3–not reached (nr) (arm A) vs 5.2 months, 95% CI, 2.5–nr (arm B), not significant), with a favorable trends for female pts (median 15.5 months, 95% CI, 2.6–nr (female) vs 3.3, 95% CI, 2.3–nr (male)), pts with less than 60 years (median 22.5 months, 95% CI, 2.6–nr (age < 60) vs 4.7 months, 95% CI, 2.3–nr (age ≥ 60), and pts with wild-type BRAF status (median 22.5 months, 95% CI, 8.6–nr (BRAF wt) vs 3.8 months, 95% CI, 2.3–nr (BRAF mutated). The toxicity profile was favourable, with no severe adverse events and only mild, manageable reactions. Moreover, additional immune response data suggested increased immune modulation in vaccinated patients, which may reflect a shift in immune dynamics. Conclusions Our findings support the safety and tolerability of DC vaccination as an adjuvant treatment for melanoma, demonstrating significant immune modulation at both the tumor site and peripherally in relapsed and non-relapsed patients. These results highlight the potential of autologous, personalised DC-based therapies and pave the way for the development of innovative immunotherapy combinations in future treatment strategies. Trial registration ClinicalTrials.gov NCT02718391; EudraCT no. 2014-005123-27.

BackgroundEarly Gastric Cancer (EGC) reaches 25% of the gastric cancers surgically treated in some areas of Northeastern Italy and is usually characterized by a good prognosis. However, among EGCs classified according to Kodama’s criteria, Pen A subgroup is characterized by extensive submucosal invasion, lymph node metastases and worse prognosis, whereas Pen B subgroup by better prognosis. The aim of the study was to characterize the differences between Pen A, Pen B and locally advanced gastric cancer (T3N0) in order to identify biomarkers involved in aggressiveness and clinical outcome.MethodsWe selected 33 Pen A, 34 Pen B and 20 T3N0 tumors and performed immunohistochemistry of mucins, copy number variation analysis of a gene panel, microsatellite instability (MSI), TP53 mutation and loss of heterozygosity (LOH) analyses.ResultsPen A subgroup was characterized by MUC6 overexpression (p = 0.021). Otherwise, the Pen B subgroup was significantly associated with the amplification of GATA6 gene (p = 0.002). The higher percentage of MSI tumors was observed in T3N0 group (p = 0.002), but no significant differences between EGC types were found. Finally, TP53 gene analysis showed that 32.8% of Pen tumors have a mutation in exons 5–8 and 50.0% presented LOH. Co-occurrence of TP53 mutation and LOH mainly characterized Pen A tumors (p = 0.022).ConclusionsOur analyses revealed that clinico-pathological parameters, microsatellite status and frequency of TP53 mutations do not seem to distinguish Pen subgroups. Conversely, the amplification of GATA6 was associated with Pen B, as well as the overexpression of MUC6 and the TP53mut/LOH significantly characterized Pen A.

By examining the B-cell receptor (BCR) repertoire of metastatic melanoma (MM) patients with favorable treatment outcomes, it is now possible to identify unique patterns of immune responses, with the potential of discovering novel antitumor antibodies. Here, we isolated CD27-positive circulating memory B cells from non responders, partial responders, and complete responders MM patients to first-line therapy with anti-PD-1 immune checkpoint inhibitor (ICI) nivolumab, to perform a BCR repertoire sequencing analysis. We looked for complementarity-determining region 3 (CDR3) sequences that were enriched ( formed) following ICI treatment. Fully-human immunoglobulins were then produced in Expi293F™ cells using CDR3-sequencing information and tested for specificity and sensitivity on different MM cell lines and patient-derived xenograft cells by flow cytometry and by immunohistochemistry on human tissue microarrays. As a result of immunotherapy stimulation in responder patients, we observed that some CDR3 clonotypes have emerged . Among the nine candidate antibodies we assessed, two antibodies exhibited encouraging tumor-targeting properties, although they also showed a degree of cross-reactivity with normal skin and melanocytes. Although our study is based on a limited number of individuals, our observations indicate that it may be possible to further investigate the human response to immunotherapy for the identification of rare mature B clonotypes targeting plasma membrane antigens on tumor cells. These preliminary findings could contribute to the future development of fully human-compatible immunotherapies, pending additional validation and studies.

Carcinomas with apocrine differentiation (CAD) of the breast are rare tumours typically presenting high immunohistochemical expression of androgen receptor (AR) which is a target molecule for personalised therapy. To date, no studies have evaluated the genetic changes that are associated with AR immunohistochemical expression in CADs. The present work aims to characterise AR status in CADs. Twenty CAD tumours were studied with immunohistochemistry, in situ fluorescence hybridization and DNA methylation analysis, to evaluate AR expression and its regulator status. All tumours demonstrated high AR immunohistochemical expression, with over 95% of the neoplastic cells showing AR positivity in 19/20 cases. CADs showed AR gene copy loss in a percentage of neoplastic cells ranging from 5 to 84% (mean 48.93%). AR regulator genes, including the MAGE family, UXT and FLNA, presented variable methylation levels, but were mainly hypomethylated and therefore all transcriptionally active. The results of this study indicate that CADs present AR monosomy, paralleled by higher transcriptional activity of the gene with potential to influence response to AR deprivation therapy.

(1) Background: NRAS mutations affect fewer than 1% of lung adenocarcinomas. The aim of this study was to describe the clinicopathological features of lung carcinomas with NRAS mutations. (2) Methods: A series of NRAS-mutated lung carcinomas was collected from a molecular diagnostic unit (from four hospitals). The cases were analyzed with next-generation sequencing. A log-rank test for overall survival (OS) was calculated. (3) Results: NRAS mutations were detected in 14/1948 samples (0.72%) of non-small-cell lung carcinomas from 13 patients (8 males, 5 females). NRAS mutations involved codon 61 in the majority (9/13, 69.2%) of cases. The other NRAS mutations affected codon 12 (2/13, 15.4%), codon 13 (1/13, 7.7%), and codon 142 (1/13, 7.7%). In 7/13 cases, co-alterations in additional genes were also present. Pleomorphic/sarcomatoid features were identified in 3/13 (23.1%) cases, in 2/8 (25.0%) histological specimens, and in 2/5 (40.0%) surgical specimens, respectively. Follow-up data were available in 11/13 cases, with 6 patients deceased. By a log-rank test, patients with NRAS mutations in codon 61 had a better outcome (estimated mean of 32.6 ± 7.1 months) compared to those with other NRAS mutations (estimated mean of 8.7 ± 4.4 months), with a significant difference (p = 0.048 for OS). (4) Conclusions: Lung carcinomas with NRAS mutation may display pleomorphic or sarcomatoid features. Mutations in codon 61 showed a more favorable prognosis than those in other codons.

Breast cancer (BC) is the most diagnosed carcinoma and the leading cause of cancer death in female over 100 countries. Thanks to the advance in therapeutic strategies, patients' survival has improved. However, the lack of response to treatments and drug resistance are still a main concern, demanding for new therapeutic approaches, in particular for the advanced stages of the disease. Androgen receptor (AR) is gaining increasing interest as a fourth targetable receptor in BC, however, its regulation in BC cells is still poorly understood. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Here, we identified miR-9-5p as an inhibitor of AR expression, we validated the inverse correlation between miR-9-5p and AR in primary BC samples and we further identified a feedback loop in which androgen agonists of AR up-regulate miR-9-5p. We also provided evidence that miR-9-5p elicits anti-proliferative effects in BC cell lines regardless of their estrogen receptor status. Finally, we showed that miR-9-5p can revert AR-downstream signaling even in presence of AR-agonists, highlighting the role of this miR in the hormonal response of BC. In conclusion, this study supports the role of miR-9-5p as an anti-proliferative miR in BC and as a central modulator of AR-signaling response to circulating androgens in BC.

Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor ( PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22–8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient.

Immune checkpoint inhibitors have recently been approved for the treatment of advanced head and neck squamous cell carcinoma (HNSCC). The determination of PD-L1 using the combined positive score (CPS) is of utmost importance in the selection of patients. However, it is unclear which material should be examined. This study aimed to compare PD-L1 CPS in the resections of primary tumors and metastatic lymph nodes, and in the biopsies of the primary tumors.We collected 30 resected HNSCCs with lymph node metastases; in 17 of these, preoperative biopsies were retrieved. PD-L1 immunostaining of 75 samples was performed using the Dako 22C3 antibody on the Ventana ULTRA platform. An appropriate internal control was performed on each slide. CPS was calculated for each reaction. Concordance values and k were calculated for each patient. CPS cut-off values were fixed at 0 and 20.Tumors were resected from the oral cavity (4), oropharynx (17), hypopharynx (1), and larynx (8). The overall concordance of CPS between tumor resection and lymph node metastasis was 76.7% (k = 0.593). The overall concordance of CPS between tumor resection and tumor biopsy was 86.7% (k = 0.688). The agreement was moderate to substantial for each comparison.PD-L1 CPS may be correctly determined not only in resected primary tumors, but also in removed lymph node metastases, as well as in preoperative biopsies.