AIM:High-resolution HLA-G typing in selected patients with locally advanced rectal cancer (LARC) enrolled at the National Cancer Institute "Centro di Riferimento Oncologico di Aviano (CRO) IRCCS" (Italy) (CRO-2023-77). METHODS:HLA-G genotyping was conducted in 405 LARC patients with allele identification at the genomic (4-field), exonic (3-field), and protein (2-field) levels. Genomic DNA was extracted from buffy coat samples, and full-length HLA-G amplicons were obtained by long-range PCR. DNA libraries were prepared with the QIAseq FX DNA Library kit (Qiagen) and sequenced on the Illumina MiSeq platform (2x250bp). High-coverage sequencing data (>50x) were analyzed using an updated version (v5) of the hla-mapper workflow. RESULTS:This LARC cohort predominantly included patients from the North-East regions of Italy. Variants in the HLA-G gene were mostly located in intronic (50, 41.0%) and regulatory regions, including promoter, 5'UTR, and 3'UTR (50, 41%), compared to exonic regions (22, 18.0%). Most variants were bi-allelic single nucleotide variants, although insertions/deletions were also detected. Almost half of patients (186, 45.9%) showed at least one missense (rs41551813, rs770412396, rs12722477, or rs12722482), start-lost (rs143732275), or frameshift (rs41557518) variants. Moreover, in 60 (14.8%) patients, 39 different rare or putative novel variants were found, all in heterozygosity. One patient carried the G*01:01:11 rare allele, previously reported only in two Brazilian relatives of Italian ancestry. The most prevalent allotypes were HLA-G*01:01, HLA-G*01:04, HLA-G*01:06, HLA-G*01:03, and the HLA-G*01:05N null allele, consistent with European reference data. Our LARC cohort shows frequencies similar to those reported for the Italian population. The differences observed comparing HLA-G 4-field allele frequencies between this LARC cohort and the European population for G*01:01:01:01 (p = 0.0004, q = 0.0048), G*01:01:02:01 (p = 0.0033, q = 0.0132), G*01:01:01:05 (p = 0.0012, q = 0.0072), G*01:01:01:04 (p = 0.0093, q = 0.0279), G*01:03:01:02 (p = 0.0354, q = 0.0708), and rare 4-field alleles (p = 0.0117, q = 0.0281) reflected the known allele differences across European countries. CONCLUSIONS:This study represents the largest Italian cohort to date with comprehensive high-resolution HLA-G genotyping in LARC patients. Ongoing analyses aim to correlate these genetic findings with clinical parameters and HLA-G expression levels in liquid biopsies. This integrative approach may identify HLA-G-related genetic factors influencing prognosis, therapeutic response, and treatment-related adverse events in patients with LARC.

At present, the gold standard for gastric cancer (GC) confirmation relies mostly on histopathology, an invasive procedure. Noninvasive detection methods using serum for large-scale screening may be useful for the early diagnosis of GC. Helicobacter pylori (HP) infection and chronic atrophic gastritis are major GC risk factors. We recently developed a noninvasive test called the DSC test-based on the patient's age, sex, their serum PGI and PGII, anti-HP immunoglobulin (IgG), and gastrin G17 levels-predicting GC risk as low (score 0, S0) or high (score 2, S2). The comparative investigation at the serum protein level of the two different patient groups detected by our DCS test (S0 and S2) may undoubtedly help to identify gastric disease-dependent proteins, resulting from bacterial infection or gastric mucosa inflammation, as well as get better insight into the molecular scenario associated with pre-cancerous conditions. We used an untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic profiling approach, followed by univariate statistical analysis to compare the different DSC groups across two patient cohorts (exploratory and validation). Significantly differentially abundant proteins differing more than 1.5-fold between S0 and S2 groups were selected and validated, and their putative role(s) in gastritis and GC were discussed. In both the exploratory and the validation cohorts, four proteins (beta-2-microglobulin, EGF-containing fibulin-like extracellular matrix protein 1, complement factor D, and cystatin-C) were more abundant, while two (sex hormone-binding globulin and pregnancy zone protein) were less abundant in the sera of S2 individuals (|fold change| ≥ 0.6, p < 0.05, t-test). The higher presence of beta-2-microglobulin (B2M) and the lower content of pregnancy zone protein (PZP) in S2 sera were validated by immunoblotting. Replacing age and sex in our DSC model with two specific candidate biomarkers can lead to a refined, albeit modest, improvement in classification accuracy. This study identified a proteomic signature that was differentially associated with the sera of patients with a different risk to develop advanced atrophy/GC according to the DSC test. Moving from a demographic model to a proteomic-driven model can better reflect the personalized biology of pathological processes associated with DSC.

Participation in organised colorectal cancer (CRC) screening is influenced by multiple organisational factors; however, existing evidence has mainly focused on isolated interventions, with limited understanding of how these components jointly influence participation within real-world screening settings. This study aimed to identify the organisational determinants of participation in population-based CRC screening in Italy and to quantify their relative contribution within a real-world, decentralised healthcare system, hypothesising that participation is determined by the effect of multiple organisational domains rather than by isolated interventions. We conducted a national cross-sectional survey of Italian CRC screening programmes using a validated web-based questionnaire. Fifty-eight programmes from 17 regions were included. The outcome was the adjusted participation rate in 2022. Associations between organisational characteristics and participation were assessed using multiple fractional logistic regression models. The participating services covered a target population of 5.9 million individuals. Participation varied substantially across settings (range 6.5

BACKGROUND:Studies on surveillance of Intraductal Papillary Mucinous Neoplasms (IPMNs) are mainly retrospective with inherited bias. AIM:To identify factors associated with the development of relevant changes in low risk IPMN under surveillance. METHODS:This study analysed IPMN patients enrolled between 2015-2017 in the prospective observational multicentric registry PANcreatic CYsts (PANCY), focusing on brunch duct (BD). Extension of surveillance until December 31st, 2021 was proposed to the recruited patients. Relevant changes were defined as: worrisome features/high risk stigmata/pancreatic cancer, pancreatectomy, death due to IPMN/pancreatic cancer. RESULTS:At diagnosis, from 647 IPMNs, 547 (60%) were BD, 87 (9%) mixed type, and 13 (1%) main duct. 57 (8.8%) underwent immediate surgery and 590 (91.2%) active surveillance. Of them 34 (5.7%) underwent surgery with 2/3 malignancy. Malignancy rates for BD- and mixed-IPMNs under surveillance were 2.7% and 12.5%. Smoking (OR 2.2) and cyst size >15 mm at diagnosis (OR 7.1) were independent risk factors for relevant changes at multivariate analysis. The combination of cyst size ≤15 mm & age >65 was a protective factor at univariate analysis (OR 0.1), mainly in no smokers (OR 0.2, p < 0.01). CONCLUSIONS:BD-IPMN progression risk is low for lesions <15 mm in non-smokers, >65 years patients. Surgery and follow-up criteria are still imperfect, leading to inappropriate utilization of resources.

Eosinophilic gastroenteritis (EGE) is a rare, chronic inflammatory condition characterized by symptoms and eosinophilic infiltration of the gastrointestinal tract in the absence of secondary causes of gastrointestinal eosinophilia. The advent of probe-based confocal laser endomicroscopy (pCLE) has introduced the possibility of real-time, in vivo microscopic imaging, enabling dynamic assessment of mucosal architecture and barrier function. We report the case of a 32-year-old woman with an atypical manifestation of mucosal EGE, initially presenting with pseudothrombotic microangiopathy secondary to vitamin B12 deficiency. The diagnostic work-up included standard endoscopy, histopathological examination of biopsy samples, and pCLE imaging. pCLE was performed both at baseline and during follow-up to evaluate disease distribution and monitor therapeutic response. Imaging was conducted using the GastroFlex UHD Confocal Miniprobe connected to the Cellvizio system, with images captured within 10 min of intravenous fluorescein administration, digitally stored, and later reviewed by blinded pCLE experts. Initial assessment revealed marked eosinophilic infiltration in the stomach, terminal ileum, and cecum, with corresponding interstitial leakage and inflammatory cell infiltration observed on pCLE. Increased epithelial permeability was also detected in additional intestinal segments lacking histological involvement. After 5 weeks of corticosteroid therapy, the patient achieved clinical and histological remission, accompanied by normalization or significant improvement in pCLE findings. Interestingly, pCLE continued to reveal subtle barrier dysfunction in regions showing histological recovery, highlighting persistent subclinical mucosal alterations. This case underscores the value of pCLE in identifying early intestinal barrier dysfunction and in monitoring therapeutic response in EGE. The concordance between pCLE imaging, histological findings, and clinical outcomes supports its use as a complementary diagnostic and monitoring tool in EGE management.

Ovarian cancer is a leading cause of cancer-related death in women. Cytoreductive surgery is the cornerstone of treatment, but frequently requires extensive surgical procedures.1 Frozen section analysis remains the reference for intraoperative differentiation between malignant and normal tissues; however, complementary real-time optical imaging technologies are being developed to support surgical decision-making through rapid tissue characterization directly at the surgical field.2–5 Probe-based confocal laser endomicroscopy (pCLE) is a novel real-time imaging technique that may help achieve this goal.6–8 This video demonstrates the pCLE procedure step by step through two surgical cases: a 57-year-old woman affected by recurrent FIGO stage IIIB epithelial ovarian cancer (EOC) and a 40-year-old woman with a confirmed pathogenic BRCA2 mutation underwent laparoscopic bilateral adnexectomy as a prophylactic procedure. The real-time microscopic images were acquired using the GastroFlex™ UHD Confocal Miniprobe™ connected to the Cellvizio® system (Mauna Kea Technologies, France), which provided illumination at a wavelength of 488 nm. In vivo pCLE images and subsequent biopsies were acquired from various anatomical sites after injection of fluorescein. Each endomicroscopic sequence was analyzed by dedicated investigators highly experienced in pCLE imaging and compared with the histology of the corresponding specimen. Normal tissues exhibited uniform stromal fibers, consistent cellular architecture, and regular vascularization. In contrast, malignant nodules were identified by fluorescein leakage, reflecting abnormal vessel permeability and highlighting clusters of neoplastic cells. Preliminary data from our observations indicate a substantial concordance between pCLE findings and histology. pCLE is a promising intraoperative tool for real-time cellular analysis.

Colonic polyps are well-recognized precursors to colorectal cancer (CRC), typically detected during colonoscopy. However, the variability in appearance, location, and size of these polyps complicates their detection and removal, leading to challenges in effective surveillance, intervention, and subsequently CRC prevention. The processes of colonoscopy surveillance and polyp removal are highly reliant on the expertise of gastroenterologists and occur within the complexities of the colonic structure. As a result, there is a high rate of missed detections and incomplete removal of colonic polyps, which can adversely impact patient outcomes. Recently, automated methods that use machine learning have been developed to enhance polyps detection and segmentation, thus helping clinical processes and reducing missed rates. These advancements highlight the potential for improving diagnostic accuracy in real-time applications, which ultimately facilitates more effective patient management. Furthermore, integrating sequence data and temporal information could significantly enhance the precision of these methods by capturing the dynamic nature of polyp growth and the changes that occur over time. To rigorously investigate these challenges, data scientists and experts gastroenterologists collaborated to compile a comprehensive dataset that spans multiple centers and diverse populations. This initiative aims to underscore the critical importance of incorporating sequence data and temporal information in the development of robust automated detection and segmentation methods. This study evaluates the applicability of deep learning techniques developed in real-time clinical colonoscopy tasks using sequence data, highlighting the critical role of temporal relationships between frames in improving diagnostic precision.

EMILIN-1 is an extracellular matrix glycoprotein with tumor-suppressive functions. While its loss is implicated in cancer progression, its specific role in the gastric tumor microenvironment and its clinical relevance remain poorly defined. Using in vitro cellular systems and genetically modified mouse models we investigated the consequences of impaired EMILIN-1 function on gastric epithelial transformation, stromal remodeling, and fibroblast reprogramming. Histopathological analysis, gene expression profiling, and functional assays were employed to assess phenotypic changes in epithelial, fibroblastic, and endothelial compartments. We found that gastric cancer (GC) cells downregulate EMILIN-1 in stromal fibroblasts and lymphatic endothelial cells via paracrine signaling, leading to reduced EMILIN-1 deposition and disrupted lymphatic organization. Mechanistically, the interaction between EMILIN-1 and α4β1 integrin, which is absent in GC cells, modulates tumor cell proliferation; loss of this axis allows tumor cells to escape ECM-mediated growth control. Furthermore, EMILIN-1 downregulation reprograms fibroblasts into a pro-tumorigenic phenotype, which enhances GC cell migration and clonogenic potential. EMILIN-1 loss-of-function (E955A) mice showed increased susceptibility to pre-neoplastic lesions, a finding mirrored in human dysplastic tissues where EMILIN-1 was markedly reduced. Our findings establish EMILIN-1 as a master regulator of gastric tissue integrity. Its loss creates a tumor-permissive microenvironment by disrupting epithelial homeostasis, impairing lymphatic structure, and promoting fibroblast activation. The consistent reduction of EMILIN-1 in early human dysplasia highlights its potential as a novel stromal biomarker for early GC risk stratification. Moreover, restoring the EMILIN-1/integrin axis represents a promising therapeutic strategy to re-establish growth control and suppress tumor progression.

10618 Background: International guidelines recommend universal Lynch syndrome (LS) screening in colorectal cancer (CRC) using tumor mismatch repair deficiency (dMMR) testing. However, LS remains underdiagnosed worldwide, largely due to inconsistent referral for genetic counseling and germline testing. We report the final feasibility results of a novel, mainstreamed, oncologist-led diagnostic algorithm for LS. Methods: ItaLynch is a prospective, observational, multicenter Italian study on patients with dMMR CRC (May 2021–Dec 2025; 23 high-volume centers). The pathway includes: (1) universal dMMR screening by immunohistochemistry (IHC) in all CRC; (2) reflex testing and “Lynch alert”: MLH1-deficient (dMLH1) tumors undergo BRAFV600E testing and, if wild-type, MLH1 promoter methylation; a Lynch alert is added to all dMMR pathology reports (positive for high LS risk based on reflex testing or non-MLH1 loss; negative for BRAFV600E-mutated or MLH1-hypermethylated cases); (3) oncologist-led mainstream germline testing for patients with positive alert, followed by post-test genetic counseling for carriers of pathogenic variants (PV) or clinically suspicious cases. Results: Up to Dec. 31 2025, we enrolled 1,482 pts with dMMR CRC. Overall, 1,316 were eligible for analysis (14 not eligible; 152 not yet filled out). Excluding 104 cases with incomplete data, 1,212 patients were evaluable from somatic to germline testing. Overall, 1033 (85%) completed the ItaLynch diagnostic pathway, demonstrating the strong feasibility of this this algorithm. Of patients with IHC data, 975 (78%) were dMLH1 and 281 (22%) had non-MLH1 loss. BRAF testing was performed in 904/975 (93%) dMLH1 cases; 386/904 (43%) were BRAFwt. MLH1 methylation testing was done in 329/386 (86%); 146/329 (44%) were not hypermethylated. Germline testing was performed in 108/146 (74%), identifying 21 LS-associated PV (19%). Among non-MLH1 loss, 232/281 (83%) underwent germline testing and 115 (50%) were diagnosed with LS. Overall, germline testing was performed in 361/1,033 adherent patients (35%), identifying 143 PV (40% of tests) and 24 VUS; PV prevalence was 14% (143/1,033). Mainstreaming reduced genetic pre-test counseling visits by 54% (196/361 avoided) and, compared with universal germline testing, avoided 672/1,033 tests (65%). Post-test counseling uptake was 83% among PV carriers. Median turnaround time from somatic to germline diagnosis was 217 days for dMLH1 and 73 days for non-MLH1. Among non-adherent patients, 67 underwent germline testing (14 PV, 3 VUS). In the full cohort, 157 PV were identified (MLH1 37, MSH2 57, MSH6 43, PMS2 20). Conclusions: This oncologist-led, mainstreamed diagnostic pathway for LS is feasible and optimized use of healthcare resources. Analyses of pathway deviations (15%), cost-effectiveness, and clinical/translational outcomes are ongoing.

Background/Objectives: Gastric cancer (GC) incidence remains high worldwide, and the survival rate is poor. GC develops from atrophic gastritis (AG), associated with Helicobacter pylori (Hp) infection, passing through intestinal metaplasia and dysplasia steps. Since Hp eradication does not exclude GC development, further investigations are needed. New data suggest the possible role of unexplored gastric microbiota beyond Hp in the progression from AG to GC. Aimed to develop a score that could be used in clinical practice to stratify GC progression risk, here was investigate gastric microbiota in AG Hp-negative patients with or without high-grade dysplasia (HGD) or GC. Methods: Consecutive patients undergoing upper endoscopy within an endoscopic follow-up for AG were considered. The antrum and corpus biopsies were used to assess the microbiota composition along the disease progression by sequencing the 16S ribosomal RNA gene. Statistical differences between HGD/GC and AG patients were included in a multivariate analysis. Results: HGD/GC patients had a higher percentage of Bacillus in the antrum and a low abundance of Rhizobiales, Weeksellaceae and Veillonella in the corpus. These data were used to calculate a multiparametric score (Resident Gastric Microbiota Dysbiosis Test, RGM-DT) to predict the risk of progression toward HGD/GC. The performance of RGM-DT in discriminating patients with HGD/GC showed a specificity of 88.9%. Conclusions: The microbiome-based risk prediction model for GC could clarify the role of gastric microbiota as a cancer risk biomarker to be used in clinical practice. The proposed test might be used to personalize follow-up program thanks to a better cancer risk stratification.

10580 Background: International guidelines recommend universal screening for LS through somatic DNA mismatch repair deficiency (dMMR) testing in CRC. However, LS remains largely underdiagnosed, often due to inconsistent referrals from oncologists to genetic counseling and germline testing. We report preliminary results of the ItaLynch study, proposing a mainstream, oncologist-led diagnostic pathway for LS. Methods: ItaLynch is a prospective, observational, multicenter, multidisciplinary Italian study on pts with dMMR CRC. It started in May 2021, and is ongoing in 23 high-volume centers. The ItaLynch diagnostic pathway is based on three key steps. The first step is universal screening through dMMR testing by immunohistochemistry (IHC) in all CRC pts. The second step consists of reflex testing and a Lynch alert: MLH1-deficient (dMLH1) pts undergo reflex testing for BRAF V600E and, if wild-type, for MLH1 promoter methylation. A Lynch alert is added to the pathology report of all dMMR CRC pts. The alert is positive for pts with a high risk of LS as per reflex testing results or loss of non-MLH1 proteins; it is negative for pts not likely to have a hereditary predisposition (i.e. BRAF V600E mut or MLH1 promoter hypermethylated dMLH1). The third and most innovative step is the oncologist-led mainstreaming germline testing for pts with a positive Lynch alert. Carriers of a germline pathogenic variant (PV) as well as those who have non-informative test results but are clinically suspicious are then referred to post-test genetic counselling. Results: Up to Dec. 19 2024, we enrolled 1,146 pts with dMMR CRC. Among the 937 pts eligible for the current analysis, 714 (76%) were dMLH1, and 223 (24%) displayed loss of non-MLH1 proteins. Reflex testing was carried out in 653 (91%) of the dMLH1 pts and 271 (41%) were BRAF wt. Of these, 219 (80%) subsequently underwent MLH1 promoter methylation testing, and 98 (45%) were not hypermethylated. Of these, 60 (61%) underwent oncologist-led germline genetic testing, and 11 (18%) were carriers of an LS-associated PV. Among the 223 cases with loss of non-MLH1 proteins, 157 (70%) underwent genetic testing, and 86 (55%) were diagnosed with LS. At the time of writing, the overall proportion of LS cases is 10% (97/937 pts with data available for the current analysis). These 97 LS cases represent 45% of the 217 pts who were flagged with a positive Lynch alert and underwent oncologist-led genetic testing (60 of which with dMLH1 and 157 with non-MLH1 loss). Conclusions: Overall, our large cohort is representative of the population of pts with dMMR CRC. Our novel diagnostic algorithm, through the implementation of the Lynch Alert flagging system that identifies dMMR CRC pts with a high likelihood of LS and of an oncologist-led germline genetic testing, obtained a high diagnostic yield. Further analyses are ongoing on the entire cohort to evaluate the feasibility of the proposed diagnostic pathway.