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Bile-duct cancers (BDC) are a group of solid tumors arising from the biliary tree. Despite their classification as rare cancers, the incidence of BDC is increasing worldwide. Poor prognosis is a common feature of this type of cancer and is mainly determined by the following factors: late diagnosis, lack of effective therapeutic approaches, and resistance to conventional treatments. In the past few years, next-generation sequencing technologies has allowed us to study the genome, exome, and transcriptome of BDC deeper, revealing a previously underestimated class of RNA: the noncoding RNA (ncRNA). MicroRNAs (miRNAs) are small ncRNAs that play an important regulatory role in gene expression. The aberrant expression of miRNAs and their pivotal role as oncogenes or tumor suppressors in biliary carcinogenesis has been widely described in BDC. Due to their ability to regulate multiple gene networks, miRNAs are involved in all cancer hallmarks, including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing/accessing vasculature, activating invasion and metastasis, reprogramming cellular metabolism, and avoiding immune destruction. Their use as diagnostic, prognostic, and predictive biomarkers has been widely explored in several human cancers, including BDC. Furthermore, miRNA-based therapeutic strategies are currently the subject of numerous clinical trials that are providing evidence of their efficacy as potent anticancer agents. In this review, we will provide a detailed update of miRNAs affecting BDC, discussing their regulatory function in processes underlying the molecular pathology of BDC. Finally, an overview of their potential use as biomarkers or therapeutic tools in BDC will be further addressed.

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Purpose:BRAF mutations are grouped in activating RAS-independent signaling as monomers (class 1–V600E) or as dimers (class 2–codons 597/601), and RAS-dependent with impaired kinase activity (class 3–codons 594/596). Although clinical, pathologic, and molecular features of V600EBRAF-mutated metastatic colorectal cancer (mCRC) are well known, limited data are available from the two other classes.Experimental Design:Data from 117 patients with BRAF (92 class 1, 12 class 2, and 13 class 3)-mutated mCRC were collected. A total of 540 BRAF wt mCRCs were included as control. IHC profiling was performed to determine the consensus molecular subtypes (CMS), cytokeratin 7/20 profiles, tumor-infiltrating lymphocyte infiltration, and BM1/BM2 categorization. Overall survival (OS) and progression-free survival were evaluated by Kaplan–Meier and log-rank test.Results:Class 3 BRAF-mutated mCRC was more frequently left sided (P = 0.0028), pN0 (P = 0.0159), and without peritoneal metastases (P = 0.0176) compared with class 1, whereas class 2 cases were similar to class 1. Hazard ratio for OS, as compared with BRAF wt, was 2.38 [95% confidence interval (CI), 1.61–3.54] for class 1, 1.90 (95% CI, 0.85–4.26) for class 2, and 0.93 (95% CI, 0.51–1.69) for class 3 (P < 0.0001). Class 2 and 3 tumors were all assigned to CMS2-3. A higher median CD3/CD8-positive lymphocyte infiltration was observed in BRAF-mutated class 2 (P = 0.033) compared with class 3 cases.Conclusions:For the first time, different clinical and pathologic features and outcome data were reported according to the three BRAF mutation classes in mCRC. Specific targeted treatment strategies should be identified in the near future for such patients.

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Introduction: Ex-vivo FCM is a novel digital optical technique that provides images of fresh tissues in a real-time fashion with magnification to subcellular details of a flattened unprocessed sample. Digital images are hematoxylin-eosin-like and can be shared and interpreted remotely. In urology, FCM has been successfully applied for prostate tissue interpretation, either during biopsy and radical prostatectomy. Possible applications of FCM may reflect those of frozen section analysis and can be extended to all fields in which the intra-operative microscopical control is advisable. Materials and methods: This is an investigative prospective case series that aims to explore FCM feasibility in novel surgical settings and provide a depiction of FCM digital images in those fields. The definite purpose is to check the accuracy of surgical specimen during the following interventions: (a) trans-urethral resection of bladder tumors, to confirm the presence of muscular layer; (b) biopsy of a retroperitoneal mass, to check for the location and quality of cores; (c) training in robotic radical prostatectomy, to control surgical margins after a nerve sparing performed by a trainee. To this aim, we collected FCM images during seven surgical procedures. FCM findings were compared to those from the final histopathological analysis and the agreement was assessed. Results: In all cases, FCM digital images were obtained in the OR. FCM was able to confirm the presence of muscular layer in TURB specimen, the presence of lymphomatous tissue, surgical margins at prostate specimen. FCM intra-operative interpretation was consistent with final histopathology in all cases. Conclusions: Ex vivo FCM may represent a novel approach to control the quality of specimens, likely to tailor surgical strategy in a real-time fashion. Moreover, digitalization represents a step toward the implementation of telepathology in clinical practice.

Aim: To apply extended ctDNA-based RAS genotyping to clinical criteria for improving the selection of patients eligible for anti-EGFR-based rechallenge in a real-world setting. Methods: ctDNA testing was prospectively applied to RASwt mCRC progressed after a first-line anti-EGFR-containing regimen and at least one other line. The primary endpoint was the objective response rate. Results: Among ten enrolled patients, the anti-EGFR rechallenge resulted in an objective response rate and disease control rate of 70% and 90%. The median progression-free survival was 11.3 months and overall survival was not reached. Compared with a historical cohort retreated with anti-EGFR agents based on clinical criteria, the ctDNA-driven approach resulted in a higher chance of achieving an objective response and longer survival. Conclusions: Blood-based RASwt status may enrich metastatic colorectal cancer more likely to benefit from anti-EGFR-based rechallenge. RAS genotyping in ctDNA represents a feasible, fast, and cost-effective tool to be implemented in the clinic for advancing precision medicine.

Aims: To investigate the influence of various concomitant medications on outcomes in patients with locally advanced rectal cancer undergoing neoadjuvant chemoradiation. Materials & methods: The authors retrospectively identified 246 patients from 2003 to 2018, collecting demographic and clinicopathological data of interest. Odds ratio (OR) was used to assess the association between concomitant drugs and outcomes. Results: The authors found an association between statins and a Dworak regression grade of 3–4 (OR = 8.78; p = 0.01). Furthermore, statins were significantly associated with more frequent chemoradiation-related toxicity (OR = 2.39; p = 0.0098) and chemotherapy dose reduction or discontinuation (OR = 2.26; p = 0.03). Conclusion: Despite higher frequency of radiotherapy and chemotherapy interruption or dose reduction, the concomitant use of statins during neoadjuvant chemoradiation proved to be associated with better tumor regression.

Introduction: Bladder cancer (BC) is one of the most frequent malignancy of the urinary tract. Recent studies demonstrated the role of aquaporins urothelial tumor cells (AQPs) as potential prognostic factor for tumor progression and invasion. In this study we investigated the AQP3 expression levels inside primary superficial (pTa) low grade bladder cancer, correlating with pathological parameters and clinical outcomes. Materials and methods: We retrospectively analyzed tumor samples of 66 patients with diagnosis of superficial urothelial (pTa) bladder cancer between 1997 and 2007. All patients underwent transurethral bladder resection (TURB ) and immediate single instillation of mitomycin C. All tumors samples were blindly reviewed by two expert anatomopathologists and only pTa low grade urothelial bladder cancer were included. Cancer recurrence was defined as the detection of bladder lesions during follow-up cystoscopy. AQP3-immunoreactive areas detected at immunohistochemical analysis were classified as AQP3 positive. Results: Of these 60.6% of patients was detected as negative for AQP3 expression. Forty-two patients develop cancer recurrence during follow-up with a mean progression free survival of 16.44 months. The absence of reaction for AQP3 was observed 56% (9/16) tumor grading G1 and 62% (31/50) tumor grading G2. No correlation was observed with sexual gender, grading of tumor differentiation, and recurrence of cancer disease. Kaplan-Meier curves of disease-free survival (DFS) showed a significant separation ( p = 0.028) between patients AQP3-positive and AQP3-negative. It was observed a mean DFS of 23.83 and 14.43 months respectively in absence and presence of AQP3 expression. Conclusion: AQP3 expression is related to disease-free interval (DFI) and the absence of AQP3 expression correlates with a late relapse. The expression of AQP3 does not provide a reproducible quantitative aspect. AQP3 are not suitable to forecast tumor cell behavior but they perform a role as regulator for tumor cell homeostasis and for additional therapeutic developments.

Summary Inflammatory bowel diseases (IBDs) are lifelong disorders in which an interaction between genetic and environmental factors is involved. IBDs include two entities: Crohn’s disease (CD) and ulcerative colitis (UC); these can be adequately diagnosed and distinguished with a correct methodological approach based on communicating exhaustive clinical, endoscopic and laboratory information to the pathologist and performing adequate bioptic sampling and precise morphological signs including crypt architecture, distribution of inflammation and granulomas, when present. IBD needs to be distinguished from non-IBD colitis, mostly at its onset. Moreover, IBDs are associated with an increased risk of developing colorectal adenocarcinoma. In daily pathological practice, correct diagnosis of IBD and its subclassification as well as a correct detection of dysplasia is imperative to establish the best therapeutic approach.

Summary Non-IBD colitides (NIBDC) are intestinal diseases clinically and endoscopically overlapping with Inflammatory Bowel Diseases (IBD), sometimes with a similar histological picture. NIBDC include entities such as infectious colitis, ischemic colitis, pseudomembranous colitis, eosinophilic colitis, autoimmune enterocolitis, segmental colitis associated with diverticulosis, drug-induced colitis, radiation-induced colitis, diversion colitis, and microscopic colitis, this last including two entities: collagenous and lymphocytic colitis. The knowledge of the most useful histological features and the main clinical data for each entity is mandatory in daily clinical practice, for correct pathological diagnosis and clinical management.

Pancreatic ductal adenocarcinoma (PDAC) is a relatively uncommon malignancy; however, its incidence is rising worldwide, and it is expected to become the second-leading cause of cancer-related death by 2030. From a histological point of view, PDAC is characterized by a prominent desmoplastic reaction that compresses blood vessels, limiting oxygen and nutrient availability in the tumor microenvironment. Despite that, PDAC cells are capable to adapt dynamically to these stress conditions, adopting different strategies that includes the strict regulation of the autophagic flux. Thus, studying these adaptive mechanisms is crucial to understand PDAC progression and to establish new therapeutic modalities to tackle it. Here, we explore the role of the tumor suppressor miR-15a in the regulation of its putative target Fra-2, a transcription factor, commonly activated by cell stress. By employing IPA on PDAC tumors from TCGA dataset, we found that both miR-15a and its target Fra-2 are predicted to regulate the IGF-1 signaling pathway. In an independent cohort of 44 PDAC samples, miR-15a levels inversely correlated with both Fra-2 and IGF1R expression; conversely, Fra-2 significantly correlated with IGF1R. By chromatin immunoprecipitation and luciferase assay, we assessed that miR-15a directly targeted Fra-2 and IGF1R that, in turn, is transcriptionally regulated by Fra-2 activity. Then, we investigated the role of miR-15a/Fra-2 regulation of IGF1R in the response to starvation-induced cell stress. In starved PDAC cell lines, Fra-2 transcriptional activity triggered IGF1R promoter, causing IGF1R overexpression in control cells but not in miR-15a-overexpressing cells. Consistently, the IGF1 release after starvation induced phosphorylation of IGF1R and activation of the downstream mTOR pathway in control cells but not in miR-15a-overexpressing cells. Therefore, TEM and western blot analysis demonstrated that activation of mTOR via IGF1 release reduced the autophagic flux of PDAC control cell lines compared to miR-15a overexpressing cells under starvation. To assess our results in vivo, we injected PDAC cells wild type or Fra-2 knockout into the flank of nude mice. At tumor onset, mice were randomly divided in two groups and fed with control or hypoproteic diet for three weeks. Hypoproteic diet did not interfere with the growth of wild-type tumors, by contrast, significantly impaired Fra-2KO tumors growth rate. Tumor analysis revealed that hypoproteic diet potently induced IGF1R overexpression and mTOR pathway activation in wild-type tumors but not in Fra-2KO tumors. Our findings demonstrate that IGF1R expression is regulated by miR-15a directly and indirectly via Fra-2 in PDAC. This novel miR-15a/Fra-2/IGF1R axis, triggered by starvation, regulates the autophagic flux and growth of PDAC cells in stress condition, and could be targeted by specific small inhibitors. Citation Format: Gian Luca Rampioni Vinciguerra, Marina Capece, Luca Reggiani Bonetti, Paolo Magistri, Federica Calore, Giovanni Nigita, Rosario Distefano, Roberto Ballarin, Fabrizio Di Benedetto, Andrea Vecchione, Barbara Belletti, Gustavo Baldassarre, Francesca Lovat, Carlo M. Croce. The novel miR-15a/Fra-2/IGF1R axis drives response to starvation-induced cell stress in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4823.

606 Background: Despite the improved outcome achieved by the addition of the anti-PD-L1 durvalumab to standard chemotherapy, the vast majority of BTC do not benefit from chemo-immunotherapy. Immunosuppressive microenvironment is a dominant feature of BTC, involved in tumour progression and drug resistance. Here, we investigated the biological role and treatment implications of the adenosine-producing enzyme CD73 in a clinically-annotated cohort of BTC. CD73, an ecto-5’-nucleotidase (NT5E), creates an immunosuppressive tumour-promoting microenvironment by converting ATP to adenosine. Methods: Immunohistochemistry for CD73, CD4/CD8 and FOXP3 as well as whole-exome and transcriptomic sequencing were performed on resected specimens of 80 BTC (Illumina Platform). Spatial Transcriptomics was performed by using Visium Spatial Gene Expression-10x Genomics). Silencing of CD73 was obtained by transient transfection od CD73-siRNA, while genetic Knock-out by using CRISPR-Cas system technology in two BTC cell lines. Tumor growth was assessed in 2D and in 3D cell culture by using MTS assay and spheroid growth analysis before and after treatment with selected drugs. Statistical and Survival, correlation analyses were performed. Results: High CD73 expression (CD73high) was associated with older age (>70 years, p=0.01), gallbladder subsite (p=0.03), and nodal involvement (p=0.04). CD73high tumours were significantly enriched in infiltrating FOXP3+ T lymphocytes (phigh status was independent predictor of poorer prognosis at the multivariate analysis (p=0,03) together with ECOG PS ≥2 (p=0,001) and the pathological stage (p=0,025) and was associated with a remarkably shorter RFS in patients treated with adjuvant chemotherapy (p=0,011). At the transcriptomic level, CD73high tumours were significantly enriched in upregulated EMT, TNF-alfa/NFKB, hypoxia and G2/M checkpoint signaling pathways and p53, BMI1, MEL18, EGFR and K-RAS genes. In in vitro models, siRNA-mediated depletion and CRISPR-CAS9 gene KO of CD73 sensitized both BTC 2D and 3D culture to cisplatin/gemcitabine treatment. The pharmacological inhibition of CD73 by AMCP improved the sensitivity of BTC cell lines to cisplatin/gemcitabine treatment. Finally, Spatially resolved transcriptomics of CD73high revealed a critical role of CD73 in tumor immunity and therapeutic response. Conclusions: We showed that CD73high BTC display aggressive biological features, poorer prognosis and resistance to standard chemotherapy. The therapeutic targeting of this adenosinergic ectonucleotidase by clinically-available compounds has the potential to enhance the efficacy of conventional treatment in BTC.

Background Intraductal papillary neoplasm of the bile duct (IPNB) is a rare variant of bile duct tumors, characterized by an exophytic growth exhibiting a papillary mass within the bile duct lumen and it can be localized anywhere along the biliary tree, with morphological variations and occasional invasion. Case summary We present a patient with obstructive jaundice who was diagnosed with IPNB using cholangioscopy during endoscopic retrograde cholangio-pancreatography. Using the SpyGlass DS II technology, we were able to define tumor extension and obtain targeted Spy-byte biopsies. After multidisciplinary evaluation, the patient was scheduled for surgical resection of the tumor, which was radically removed. Conclusion Cholangioscopy appears to be crucial for the rapid and clear diagnosis of lesions in the bile duct to achieve radical surgical resection.

Summary Celiac disease is a multi-factorial chronic inflammatory intestinal disease, characterized by malabsorption resulting from mucosal injury after ingestion of wheat gluten or related rye and barley proteins. Inappropriate T-cell-mediated immune response against ingested gluten in genetically predisposed people, leads to characteristic histological lesions, as villous atrophy and intraepithelial lymphocytosis. Nevertheless, celiac disease is a comprehensive diagnosis with clinical, serological and genetic characteristics integrated with histological features. Biopsy of duodenal mucosa remains the gold standard in the diagnosis of celiac disease with the recognition of the spectrum of histological changes and classification of mucosa damage based on updated Corazza-Villanacci system. Appropriate differential diagnosis evaluation and clinical context also for the diagnosis of complications is, moreover, needed for correct histological features interpretation and clinical management.

Histopathologic examination of the pathologic specimens using hematoxylin & eosin stains represents the backbone of the modern pathology. It is time-consuming; thus, “real-time” assessment of prostatic and periprostatic tissue has gained special interest in the diagnosis and management of prostate cancer. The current study focuses on the review of the different available techniques for “real-time” evaluation of surgical margins during radical prostatectomy (RP). We performed a comprehensive search of the Medline database to identify all the articles discussing “real-time” or intraoperative assessment of surgical margins during RP. Several filters were applied to the search to include only English articles performed on human subjects and published between January 2000 and March 2019. The search revealed several options for pathologic assessment of surgical margins including intraoperative frozen sections, confocal laser endomicroscopy, optical spectroscopy, photodynamic diagnosis, optical coherence tomography, multiphoton microscopy, structured illumination microscopy, 3D augmented reality, and ex vivo fluorescence confocal microscope. Frozen section represents the gold standard technique for real-time pathologic examinations of surgical margins during RP; however, several other options showed promising results in the initial clinical trials, and considering the rapid development in the field of molecular and cellular imaging, some of these options may serve as an alternative to frozen section.

Objective The aim of the paper is to provide an overview of intraoperative sampling methods for frozen section (FS) analysis and of surgical techniques for a secondary neurovascular bundle (NVB) resection, as the method of surgical margin (SM) sampling and the management of a positive SM (PSM) at the nerve-sparing (NS) area are under evaluated issues. FS analysis during radical prostatectomy (RP) can help to tailor the plane of dissection based on cancer extension and thus extend the indications for NS surgery. Evidence acquisition We performed a PubMed/Medical Literature Analysis and Retrieval System Online (MEDLINE), Web of Science, Cochrane Library, and Elton B. Stephens Co. (EBSCO)host search to include articles published in the last decade, evaluating FS analysis in the NS area and surgical attempts to convert a PSM to a negative status. Evidence synthesis Overall, 19 papers met our inclusion criteria. The ways to collect samples for FS analysis included: systematic (analysing the whole posterolateral aspect of the prostate specimen, i.e., neurovascular structure-adjacent frozen-section examination [NeuroSAFE]); magnetic resonance imaging (MRI)-guided (biopsies from MRI-suspicious areas, retrieved by the surgeon in a cognitive way); and random biopsies from the soft periprostatic tissues. Techniques to address a PSM in the NS area included: full resection of the spared NVB, from its caudal to cranial aspect, often including the rectolateral part of the Denonvilliers' fascia; partial resection of the NVB, in cases where sampling attempts to localise a PSM; incremental approach, meaning a partial or full resection that extends until no prostate tissue is found in the soft periprostatic environment. Conclusions There is no homogeneity in prostate sampling for FS analysis, although most recent evidence is moving toward a systematic sampling of the entire NS area. The management of a PSM is variable and can be affected by the sampling strategy (difficult localisation of the persisting tumour at the NVB). The difficult identification of the exact soft tissue location contiguous to a PSM could be considered as the critical point of FS analysis and of spared-NVB management.

Given the current prolonged life expectancy, various pathologies affect increasingly the aging subjects. Regarding the musculoskeletal apparatus, bone fragility induces more susceptibility to fractures, often not accompanied by good ability of self-repairing, in particular when critical-size defects (CSD) occur. Currently orthopedic surgery makes use of allografting and autografting which, however, have limitations due to the scarce amount of tissue that can be taken from the donor, the possibility of disease transmission and donor site morbidity. The need to develop new solutions has pushed the field of tissue engineering (TE) research to study new scaffolds to be functionalized in order to obtain constructs capable of promoting tissue regeneration and achieve stable bone recovery over time. This investigation focuses on the most important aspect related to bone tissue regeneration: the angiogenic properties of the scaffold to be used. As an innovative solution, scleral ossicles (SOs), previously characterized as natural, biocompatible and spontaneously decellularized scaffolds used for bone repair, were tested for angiogenic potential and biocompatibility. To reach this purpose, in ovo Chorioallantoic Membrane Assay (CAM) was firstly used to test the angiogenic potential; secondly, in vivo subcutaneous implantation of SOs (in a rat model) was performed in order to assess the biocompatibility and the inflammatory response. Finally, thanks to the analysis of mass spectrometry (LCMSQE), the putative proteins responsible for the SO angiogenic properties were identified. Thus, a novel natural biomaterial is proposed, which is (i) able to induce an angiogenic response in vivo by subcutaneous implantation in a non-immunodeficient animal model, (ii) which does not induce any inflammatory response, and (iii) is useful for regenerative medicine application for the healing of bone CSD.

We describe a patient with constitutional mismatch repair-deficiency (CMMR-D) in whom the syndrome started at age 10 with the development of multiple adenomas in the large bowel. In the successive 25 years, four malignancies developed in different organs (rectum, ileum, duodenum, and lymphoid tissue). The patient had biallelic constitutional pathogenic variants in the PMS2 gene. We speculate that besides the PMS2 genotype, alterations of other genes might have contributed to the development of the complex phenotype. In the nuclear family, both parents carried different PMS2 germline mutations. They appeared in good clinical condition and did not develop polyps or cancer. The index case had a brother who died at age three of lymphoblastic leukemia, and a sister who was affected by sarcoidosis. Tumor tissue showed diffuse DNA microsatellite instability. A complete absence of immunoreactivity was observed for the PMS2 protein both in the tumors and normal tissues. Next-generation sequencing and multiple ligation-dependent probe amplification analyses revealed biallelic PMS2 germline pathogenic variants in the proband (genotype c.[137G>T];[(2174+1_2175-1)_(*160_?)del]), and one of the two variants was present in both parents—c.137G>T in the father and c.(2174+1-2175-1)_(*160_?)del in the mother—as well as c.137G>T in the sister. Moreover, Class 3 variants of MSH2 (c.1787A>G), APC (c.1589T>C), and CHEK2 (c.331G>T) genes were also detected in the proband. In conclusion, the recognition of CMMR-D may sometimes be difficult; however, the possible role of constitutional alterations of other genes in the development of the full-blown phenotype should be investigated in more detail.

Massimiliano Salati,1,2 Luigi Marcheselli,1 Carlo Messina,3 Valeria Merz,3,4 Marco Messina,5 Pietro Carotenuto,6 Francesco Caputo,1 Fabio Gelsomino,1 Andrea Spallanzani,1 Luca Reggiani Bonetti,7 Stefania Caramaschi,7 Gabriele Luppi,1 Massimo Dominici,1 Michele Ghidini8 1Division of Oncology, Department of Oncology and Hematology, University Hospital of Modena, Modena, Italy; 2PhD Program Clinical and Experimental Medicine, University of Modena and Reggio Emilia, Modena, Italy; 3Department of Medical Oncology, Santa Chiara Hospital, Trento, Italy; 4Digestive Molecular Clinical Oncology Research Unit, University of Verona, Verona, Italy; 5Oncology Unit, Fondazione San Raffaele-Giglio, Cefalu’, Palermo, Italy; 6Telethon Institute of Genetics and Medicine, Pozzuoli, 80078, Napoli, Italy; 7Pathology Section, University Hospital of Modena, Modena, Italy; 8Medical Oncology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, ItalyCorrespondence: Massimiliano Salati, PhD Program Clinical and Experimental Medicine, University Hospital of Modena, Modena Cancer Centre, via del Pozzo 71, Modena, 41125, Italy, Tel +39/0594223808, Fax +39/0594222647, Email massimiliano.salati@unimore.itBackground: The role of second-line chemotherapy in advanced biliary cancers (ABCs) has only recently been established in phase III randomized trial and the optimal selection of patients most likely to benefit from it remains challenging.Methods: A cohort of 98 ABC treated second-line chemotherapy was used as a developmental dataset to identify covariates independently associated with overall survival (OS). Kaplan–Meier analysis was used to investigate the association between variables and OS and those retaining statistically significance were combined in a multiplexed score.Results: The following pretreatment variables were independently associated with OS: ECOG PS > 0, peritoneal disease, LDH > 430 UI/L, albumin < 3.5 gr/dL, gamma-GT > 100 UI/L, sodium < 140 mEq/L, absolute lymphocyte count < 1000/mmc, and PFS to first-line < 6 months. Based on these results, a scoring system was developed that identified three subgroups with statistically different OS: low-risk (mOS 18 months), intermediate-risk (mOS 9.4 months) and high-risk (mOS 2.9 months) (p < 0.001). The prognostic model was both internally and externally validated in a multicentre cohort of 120 ABCs.Conclusion: The Modena score is a multiplexed scoring system capable of accurately risk-stratified ABCs treated with second-line chemotherapy. Based on its reproducibility, usability and generalizability, it has the potential for assisting therapeutic decision-making in the clinic and risk-stratification in future trials.Graphical Abstract: Keywords: biliary tract cancer, chemotherapy, second-line, prognostic score, survival

In recent years, the consumption of energy drinks (EDs) has increased constantly among young people because of their capacity to enhance alertness and improve mental and physical performance, reducing fatigue. EDs are beverages containing a high and variable amount of caffeine, which exerts effects on many tissues of the cardiovascular system. In addition to caffeine, they contain several other psychoactive substances including the amino acid taurine, the glucose derivative glucuronolactone, as well as herbal extracts such as guaranà (another source of caffeine, with caffeine-like effects) and ginseng, often present in not well-known concentration. In our project we aim to evaluate whether the consumption of EDs causes alterations in the organs involved in their contact, absorption and metabolism in an animal model, i.e. the rat. We used 28 Sprague Dawley adult male rats, weighing 230–250 g and fed with a standard laboratory diet, randomly divided into four groups (N = 7). Every group received a different treatment (ED, soda-cola, sweetened coffee or water-controls-) for 5 days. All animals were anesthetized and underwent histological analysis and blood sampling at the end of the treatment. We observed eosinophilic infiltrates in gastrointestinal tract, but not in cardiovascular system. We quantified various indicators of tissue damage and cytokines in plasma, including ICAM-1, L-selectin, TIMP-1, VEGF, IL-10, IL-2, IL-4, IL-6, IL-1β, IL-13, IL-33, TNF-α, and IFN-γ. In contrast with the eosinophilic infiltration, we did not detect a systemic increase of Th2 cytokines (i.e. IL-4 and IL-13) after treatment: thus, even if the experiments evaluating the possible presence of an unbalanced Th2 response in the tissues featured by eosinophilic infiltration are still lacking, we exclude further deepening on Th2 immune responses. Interestingly, we observed a decrease of TIMP-1 in plasma from rats orally supplemented with ED, soda-cola or sweetened coffee compared to animals treated with water. TIMP-1 is a major player in preserving tissue integrity and controlling wound healing by balancing the enzymatic activity of matrix metalloproteinases (MMPs) and regulating extracellular matrix turnover. Moreover, elevated levels of the adhesion molecules ICAM-1 and L-selectin were found in plasma from rats receiving ED or soda-cola, together with a high concentration of IL-33 in animals assuming the ED. The circulating form of ICAM-1 is associated with inflammation, particularly due to endothelial damage, and L-selectin and IL-33 play an important role in inflammatory conditions as well. These observations suggest that the consumption of EDs could alter the architecture, and hence function, of the organs involved in their contact, absorption and metabolism. Indeed, the alterations we observed in the periphery could reflect the establishment of inflammation and deregulated mechanisms of damage repair locally in the target tissues.

Bone and muscle have been recognized as endocrine organs since they produce and secrete &ldquo, hormone-like factors&rdquo, that can mutually influence each other and other tissues, giving rise to a &ldquo, bone&ndash, muscle crosstalk&rdquo, In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/&beta, catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

The aim of this study was to evaluate splenic eosinophil and mast cell accumulation using pagoda red stain in a series of anaphylaxis-related deaths that underwent medico-legal investigations. Our goal was to assess whether fatal reactions to insect stings, intramuscularly administered antibiotics and intravenously injected contrast media are responsible for specific patterns of eosinophil and mast cell accumulation. Two study groups were prospectively formed, an anaphylaxis-related death group and a control group. Autopsy, histology (haematoxylin-eosin stain, pagoda red stain and immunohistochemistry using anti-tryptase antibodies), toxicology and postmortem biochemistry (beta-tryptase, total IgE and specific IgE) were performed in all cases. All tested parameters (spleen weight, beta-tryptase and total IgE levels as well as eosinophil, mast cell and degranulated mast cell numbers in the spleen) were significantly higher in the anaphylaxis-related death group. No statistically significant differences were observed among the various groups (intramuscular antibiotic injection, intravenous contrast medium administration and stinging insects) in any combination, suggesting that mast cell and eosinophil accumulation in the spleen during anaphylaxis does not have any specific pattern related to the triggering allergen. Despite a lower sensitivity than immunohistochemical staining in discriminating eosinophil and mast cells, pagoda red stain allowed these cells to be identified and could therefore be proposed as a low-cost, first-line diagnostic procedure in those situations where immunohistochemistry is not systematically performed or cannot be carried out.

Poorly differentiated Clusters (PDCs) of tumor cells composed of more than five elements have been recently described in gastrointestinal cancers and correlate with a worse prognosis. Our study aims to investigate PDC occurrence in a series of patients with gastric cancer and correlate it with lymph node status and clinical outcome.50 patients were included in the study; PDCs count was graduated as G1, G2, and G3 according to Ueno classification (PDCs count at 20×5, 5-9 and ≥10 respectively). We collected several clinicopathologic variables such as tumor location, pTNM stage, vascular or perineural invasion, and lymph-node ratio for each case.The presence of PDCs was related to vascular invasion (PDC is a strong prognostic factor in gastric cancer, easily detectable, and feasible. As far as we know, this is the first report in Literature of a strong correlation between PDC and survival in patients with operated gastric cancer.

Cutaneous metastasis from solid tumors is a rare event and usually represents a late occurrence in the natural history of an advanced visceral malignancy. Rarely, cutaneous metastasis has been described in colorectal cancer patients. The most frequent cutaneous site of colorectal metastasis is the surgical scar in the abdomen following the removal of the primary malignancy, followed by the extremities, perineum, head, neck, and penis. Metastases to the thigh and back of the trunk are anecdotical. Dermatological diagnosis of cutaneous metastasis can be quite complex, especially in unusual sites, such as in the facial skin or thorax and in cases of single cutaneous lesions since metastasis from colorectal cancer is not usually the first clinical hypothesis, leading to misdiagnosis. To date, due to the rarity of cutaneous metastasis from colorectal cancer, little evidence, most of which is based on case reports and very small case series, is currently available. Therefore, a better understanding of the clinic-pathological characteristics of this unusual metastatic site represents an unmet clinical need. We present a large series of 29 cutaneous metastases from colorectal cancer with particular concerns regarding anatomic localization and the time of onset with respect to primitive colorectal cancer and visceral metastases.