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Hepatocellular carcinoma (HCC) represents the primary carcinoma of the liver and the fourth leading cause of cancer-related deaths. The World Health Organization estimates an increase in cases in the coming years. The risk factors of HCC are multiple, and the incidence in different countries is closely related to the different risk factors to which the population is exposed. The molecular mechanisms that drive HCC tumorigenesis are extremely complex, but understanding this multistep process is essential for the identification of diagnostic, prognostic, and therapeutic markers. The development of multigenic next-generation sequencing panels through the parallel analysis of multiple markers can provide a landscape of the genomic status of the tumor. Considering the literature and our preliminary data based on 36 HCCs, the most frequently altered genes in HCCs are

Patients with a history of primary brain tumors can be eligible for organ donation under extended criteria. The risk assessment of tumor transmissionTo investigate the role of tumor transmission risk factors in donors with oligodendrogliomas and astrocytomas.We searched PubMed and EMBASE databases for studies reporting extraneural spreading of oligodendrogliomas and astrocytomas and extracted clinical-pathological data on the primary tumor histotype and grade, the elapsed time from the diagnosis to the onset of metastases, sites and number of metastases, prior surgeries, prior radiotherapy and/or chemotherapy, ventriculo-atrial or ventriculo-peritoneal shunt placement, and the presence of isocitrate dehydrogenase 1/2 mutation and 1p/19q codeletion. Statistical analysis was performed using R software. Statistical correlation between chemotherapy or radiotherapy and the presence of multiple extra-central nervous system metastases was analyzed usingData on a total of 157 patients were retrieved. The time from the initial diagnosis to metastatic spread ranged from 0 to 325 mo in patients with oligodendrogliomas and 0 to 267 mo in those with astrocytomas. Respectively, 19% and 39% of patients with oligodendroglioma and astrocytoma did not receive any adjuvant therapy. The most frequent metastatic sites were bone, bone marrow, and lymph nodes. The lungs and the liver were the most commonly involved visceral sites. There was no significant correlation between the occurrence of multiple metastases and the administration of adjuvant chemo-/radiotherapy. Patients who developed intracranial recurrences/metastases had a significantly longer extraneural metastasis-free time compared to those who developed extraneural metastases in the absence of any intra- central nervous system spread.A long follow-up time does not exclude the presence of extraneural metastases. Therefore, targeted imaging of bones and cervical lymph nodes may improve safety in the management of these donors.

SummaryPatients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models summing up CUP features are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96), and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. Genetic testing and FISH analysis identified FGFR2 amplification as therapeutic target in tumor tissues and patient-derived models. Drug-screening assays were performed to test the activity of FGFR2 targeting drug BGJ-398 (infigratinib) and the combination treatment with the MEK inhibitor trametinib, which proved to be synergic and exceptionally active, bothin vitroandin vivo. This study brings personalized therapy closer to CUP patients and paves the way to future applications of personalized medicine for metastatic patients with adverse prognosis.

PRAME (PReferentially expressed Antigen in MElanoma), a cancer-testis antigen expressed in normal and neoplastic tissues with several functions, proved to be a useful diagnostic tool in the differential diagnosis between benign and malignant melanocytic lesions. The current study aims to perform PRAME stain on a retrospective case series of mucosal melanocytic tumors of the head and neck region to compare 3 different scores and evaluate the most reliable one in this diagnostic set. Immunohistochemical analysis for PRAME was performed in 54 benign and malignant mucosal melanocytic tumors of the head and neck region collected from 41 patients. The best-performing cutoff of PRAME-positive cells (nuclear stain) to differentiate benign and malignant mucosal melanocytic tumors of the head and neck region is that proposed by Raghavan and colleagues (

Hepatocellular carcinoma (HCC) is characterized by a low mutation burden and a relatively low number of tumor-infiltrating lymphocytes (TILs), making still difficult to identify targets for specific therapies. The aim of this study was the identification of the prognostic role of TILs in HCC, focusing on their distribution and status of activation. We retrospectively enrolled 41 patients, undergone to liver resection for HCC. A significant increase of CD8 + intratumoral lymphocytes was observed in HCCs with prevalent solid architecture, but with a higher PD-1/TIA1 ratio, suggesting that HCCs with solid architecture have more peri-tumoral lymphocytes, but with minor functionality. At multivariate and univariate analyses, TIA1/CD8 ratio correlated with tumor recurrence, meaning that HCC with more activated TILs are characterized by a higher tumor aggressiveness. The use of a feasible and cheap immunohistochemical panel can help in post-surgical prognostic stratification, focusing not only in the raw number and density of TILs, but more on their state of activation and morphology.

Background The use of extended criteria donors (ECD) in liver transplantation is increasing due to the organ shortage. Histological evaluation of the liver graft in the context of procurement is an important tool for extending the donor pool without affecting the quality of the transplanted organs. Macrovesicular steatosis is widely accepted as predictor of early allograft dysfunction (EAD), while other features, such as portal fibrosis, are poorly studied. Aim To identify morphological features, other than macrovesicular steatosis, that may affect recipients’ outcome. Methods Between 2014 and 2016, 132 donors with extended criteria underwent pre-transplant liver biopsy during procurement. Histological variables of the graft, donors’/recipients’ clinical data, EAD and patient/graft survival were registered. Results The recipients who received a graft with histological-proven portal fibrosis had a significant lower patient and graft survival in comparison to patients without fibrosis (P = 0.044 and P = 0.039, respectively). Donors’ dyslipidemia was significantly associated with the occurrence of EAD (P = 0.021). When dyslipidemia was combined with histological liver fibrosis a 54.5% incidence of EAD was observed (P = 0.012). Conclusions The histological assessment of liver fibrosis in pre-transplant biopsy of ECD grafts, together with donor’s clinical data, provides important information on recipients’ outcome.

We present an unusual case of liver involvement in monoclonal gammopathy with generalized crystal-storing histiocytosis (G-CSH). A bone marrow storage disease was diagnosed in a 79-year-old man with monoclonal gammopathy of uncertain significance (MGUS). The patient presented with pleural effusion, an osteolytic lesion of the humerus, and an increase of aspartate transaminase and cholestatic markers that raised the clinical suspect of liver disease. A second bone marrow biopsy confirmed the diagnosis of MGUS with a histiocytic component suggestive for G-CSH. Liver biopsy showed an unremarkable histology, no significant inflammatory infiltrates, and intrasinusoidal foamy histiocytes. PAS and Masson’s trichrome stains, showed, in the cytoplasm of both histiocytes and hepatocytes, rod-shaped eosinophilic crystals, which were immunoreactive for kappa light chains. Transmission electron microscopy performed on reprocessed histological sections confirmed the presence of crystals in the hepatocyte cytoplasms. Immunogold labeling intensely stained crystals for kappa light chains. To the best of our knowledge, this is the second case in which an intrahepatocellular crystal storage is described during liver involvement in G-CSH. The present case also suggests that an increase in liver serum enzymes may support the clinical diagnosis of liver CSH in a patient with MGUS.

Introduction: Newly discovered thyroid nodules in deceased donors are investigated to rule out cancer that can be transmitted, but there are no established protocols. The aim of the study was to compare fine needle aspiration versus intraoperative frozen section in the donor management with limited time. Methods: Data were extracted only from the records of Italian second opinion consultation service in the years 2016 to 2017 and included donor details, pathology diagnoses, complications, transmission risk profile, and impact on transplantation. Results: Among 31 deceased donors with thyroid nodules, we documented 4 with a clinical history of cancer and 27 with a newly discovered nodule. The latter was evaluated by thyroidectomy with frozen section in 22 and fine needle aspiration in 5. Among all donors, 7 had papillary thyroid carcinoma with negligible transmission risk, whereas 8 with unacceptable risk. Two donors presented major bleeding after thyroidectomy, with organ discard in 1 case. Transplantation was delayed in 4 cases that were evaluated with frozen section. Discussion: There was no uniform approach for the investigation of thyroid nodules. Our results showed that fine needle aspiration was more accurate and useful than frozen section. Fine needle aspiration had minor economic impact and a far less rate of bleeding/hemodynamic complications, potentially delaying and compromising organ recovery. Our results suggested considering fine needle aspiration as a first step in the evaluation of thyroid nodules in donors.

Atherosclerosis is considered a chronic inflammatory disease of arteries associated with the aging process. Many risk factors have been identified and they are mainly related to life-styles, gene-environment interactions and socioeconomic status. Carotid and coronary artery diseases are the two major atherosclerotic conditions, being the primary cause of stroke and heart attack, respectively. Nevertheless, carotid plaque assumes particular aspects not only for the specific molecular mechanisms, but also for the types of atheroma which may be associated with a better or a worst prognosis. The identification of circulating blood biomarkers able to distinguish carotid plaque types (stable or vulnerable) is a crucial step for the improvement of adequate therapeutic approaches avoiding or delaying endarterectomy in the oldest old individuals (> 80 years), a population predicted to growth in the next years. The review highlights the most recent knowledge on carotid plaque molecular mechanisms, focusing on microRNAs (miRs), as a site-specific accelerated aging within the conceptual framework of Geroscience for new affordable therapies.

Key Points Three-day change in angiopoietin-2 levels predicts COVID-19 in-hospital mortality, whereas the 10-day trend is associated with chronic lung disability. Angiopoietin-2 may play an important pathogenic role in patients with COVID-19, and it could be a target for new treatments., This study examined the association between dynamic angiopoietin-2 assessment and COVID-19 short- and long-term clinical course. We included consecutive hospitalized patients from 1 February to 31 May 2020 with laboratory-confirmed COVID-19 from 2 Italian tertiary referral centers (derivation cohort, n = 187 patients; validation cohort, n = 62 patients). Serum biomarker levels were measured by sandwich enzyme-linked immunosorbent assay. Lung tissue from 9 patients was stained for angiopoietin-2, Tie2, CD68, and CD34. Cox model was used to identify risk factors for mortality and nonresolving pulmonary condition. Area under the receiver operating characteristic curve (AUROC) was used to assess the accuracy of 3- and 10-day angiopoietin-2 for in-hospital mortality and nonresolving pulmonary condition, respectively. Three-day angiopoietin-2 increase of at least twofold from baseline was significantly associated with in-hospital mortality by multivariate analysis (hazard ratio [HR], 6.69; 95% confidence interval [CI], 1.85-24.19; P = .004) with AUROC = 0.845 (95% CI, 0.725-0.940). Ten-day angiopoietin-2 of at least twofold from baseline was instead significantly associated with nonresolving pulmonary condition by multivariate analysis (HR, 5.33; 95% CI, 1.34-11.77; P ≤ .0001) with AUROC = 0.969 (95% CI, 0.919-1.000). Patients with persistent elevation of 10-day angiopoietin-2 levels showed severe reticular interstitial thickening and fibrous changes on follow-up computed tomography scans. Angiopoietin-2 and Tie2 were diffusely colocalized in small-vessel endothelia and alveolar new vessels and macrophages. Angiopoietin-2 course is strongly associated with COVID-19 in-hospital mortality and nonresolving pulmonary condition. Angiopoietin-2 may be an early and useful predictor of COVID-19 clinical course, and it could be a relevant part of disease pathogenesis. Angiopoietin-2 blockade may be a COVID-19 treatment option., Visual Abstract

We describe a patient with liver metastases from colorectal cancer treated with chemotherapy and hepatic resection, who developed unresectable multifocal liver recurrence and who received liver transplantation using a novel planned technique: heterotopic transplantation of segment 2-3 in the splenic fossa with splenectomy and delayed hepatectomy after regeneration of the transplanted graft. We transplanted a segmental liver graft after in-situ splitting without any impact on the waiting list, as it was previously rejected for pediatric and adult transplantation. The volume of the graft was insufficient to provide liver function to the recipient, so we performed this novel operation. The graft was anastomosed to the splenic vessels after splenectomy, and the native liver portal flow was modulated to enhance graft regeneration, leaving the native recipient liver intact. The volume of the graft doubled during the next 2weeks and the native liver was removed. After 8months, the patient lives with a functioning liver in the splenic fossa and without abdominal tumor recurrence. This is the first case reported of a segmental graft transplanted replacing the spleen and modulating the portal flow to favor graft growth, with delayed native hepatectomy.

Liver biopsy is crucial for the diagnosis of autoimmune hepatitis (AIH), and new reproducible histological criteria would be highly desirable, especially in acute-on-chronic cases. The aims of the present study were (i) to evaluate the AIH histopathological criteria as a function of the time and modality of AIH onset, and (ii) to validate the count of apoptotic bodies in the portal tracts as a histopathological criterion for AIH diagnosis. Sixty-five patients were retrospectively enrolled: 20 underwent biopsy for the first diagnosis and 45 had a previous histological AIH diagnosis. Biopsies were revised, and all histological variables were collected, including the lymphocytic apoptotic bodies in the portal tracts. Clinical and serological data were revised as well. First-diagnosis patients showed a higher grade of inflammation (p = 0.001), but also worse portal fibrosis (p = 0.001). The apoptotic body count was higher in first-diagnosis patients than in follow-up patients (p = 0.002), and it was strongly correlated to inflammation. Using the apoptotic body count among the simplified AIH score variables, the first-biopsy patients in the “definite” category rose from 42 to 68%. Our results confirm the histopathological criteria proposed by the literature and introduce the count of portal apoptotic bodies for the diagnosis of active AIH, especially in first biopsies without other classic features, as well as in AIH diagnostic score, albeit future studies are required to find a definite cutoff.

INTRODUCTION: Our study aimed at investigating tumor heterogeneity in esophageal adenocarcinoma (EAC) cells regarding clinical outcomes. METHODS: Thirty-eight surgical EAC cases who underwent gastroesophageal resection with lymph node dissection in 3 university centers were included. Archival material was analyzed via high-throughput cell sorting technology and targeted sequencing of 63 cancer-related genes. Low-pass sequencing and immunohistochemistry (IHC) were used to validate the results. Results Thirty-five of 38 EACs carried at least one somatic mutation that was absent in the stromal cells; 73.7%, 10.5%, and 10.5% carried mutations in tumor protein 53, cyclin dependent kinase inhibitor 2A, and SMAD family member 4, respectively. In addition, 2 novel mutations were found for hepatocyte nuclear factor-1 alpha in 2 of 38 cases. Tumor protein 53 gene abnormalities were more informative than p53 IHC. Conversely, loss of SMAD4 was more frequently noted with IHC (53%) and was associated with a higher recurrence rate (P = 0.015). Only through cell sorting we were able to detect the presence of hyperdiploid and pseudodiploid subclones in 7 EACs that exhibited different mutational loads and/or additional copy number amplifications, indicating the high genetic heterogeneity of these cancers. DISCUSSION: Selective cell sorting allowed the characterization of multiple molecular defects in EAC subclones that were missed in a significant number of cases when whole-tumor samples were analyzed. Therefore, this approach can reveal subtle differences in cancer cell subpopulations. Future studies are required to investigate whether these subclones are responsible for treatment response and disease recurrence.

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.

The choice of surgical treatment for hepatocellular carcinoma (HCC) depends on several prognostic variables, among which histological features, like microvascular invasion and tumor grade, are well established. This study aims to identify the tissue miRNAs predictive of recurrence after liver resection in "histologically advanced" HCC. We selected 54 patients: 15 retrospective resected patients without recurrence (group A), 19 retrospective resected patients with HCC recurrence (group B), and 20 prospective patients (group C), with 4 recurrence cases. All selected HCC were "histologically advanced" (high Edmondson grade and/or presence of microvascular invasion). A wide spectrum of miRNAs was studied with TaqMan Human microRNA Arrays; qRT-PCR assays were used to validate results on selected miRNAs; immunohistochemistry for IGF2 was applied to study the mechanism of miR-483-3p. As a result, a significant differential expression between group A and B was found for 255 miRNAs. Among them we selected miR-483-3p and miR-548e (P < 0.001). As a single variable (group C), HCC with miR-483-3p downregulation (mean fold increase 0.21) had 44.4% of recurrence cases; HCC with miR-483-3p upregulation (mean fold increase 5.94) showed no recurrence cases (P=0.011). At immunohistochemistry (group C), the HCC with loss of cytoplasmic IGF2 expression showed a down-regulation of miR-483-3p (fold increase 0.57). In conclusion, in patients with "histologically advanced" HCC, the analysis of specific tissue miRNAs (particularly miR-483-3p) could help identify the recurrence risk and choose which treatment algorithm to implement (follow-up, resection or transplantation). This could have an important impact on patient survival and transplantation outcome, improving organ allocation.

Telepathology has been an important application for second opinion consultation ever since the introduction of digital pathology. However, little is known regarding teleconsultation for second opinion in transplantation. There is also limited literature on telepathology during organ donor procurement, typically utilized when general pathologists on-call request back-up to help assess donor biopsies for organ suitability or to diagnose newly discovered tumors with urgent time constraints. In this review, we searched Pubmed/Embase and websites of transplant organizations to collect and analyze published evidence on teleconsultation for donor evaluation and organ procurement. Of 2725 records retrieved using the key terms 'telepathology', 'second opinion' and 'transplantation', 26 suitable studies were included. Most records were from North America and included validation studies of telepathology being used for remote frozen section interpretation of donor biopsies with whole slide imaging. The data from these published studies supports the transition towards digital teleconsultation in transplant settings where consultations among pathologists are still handled by pathologists being called on site, via telephone and/or email.

Background : We report the findings of a single Italian center in the evaluation of renal lesions in deceased donors from 2001 to 2017. In risk evaluation, we applied the current Italian guidelines, which include donors with small (< 4 cm, stage pT1a) renal carcinomas in the category of non-standard donors with a negligible risk of cancer transmission. Methods : From the revision of our registries, 2,406 donors were considered in the Emilia Romagna region of Italy; organs were accepted from 1,321 individuals for a total of 3,406 organs. Results : The evaluation of donor safety required frozen section analysis for 51 donors, in which a renal suspicious lesion was detected by ultrasound. Thirty-two primary renal tumors were finally diagnosed: 26 identified by frozen sections and 6 in discarded kidneys. The 32 tumors included 13 clear cell renal cell carcinomas (RCCs), 6 papillary RCCs, 6 angiomyolipomas, 5 oncocytomas, 1 chromophobe RCC, and 1 papillary adenoma. No cases of tumor transmission were recorded in follow-up of the recipients. Conclusion : Donors with small RCCs can be accepted to increase the donor pool. Collaboration in a multidisciplinary setting is fundamental to accurately evaluate donor candidate risk assessment and to improve standardized protocols for surgeons and pathologists.

Background: Evidence about the reliability of pre-implantation biopsy is still conflicting, depending on both biopsy type and pathologist’s expertise. Aim of the study is to evaluate the agreement of general v specialist pathologists and to compare scores on biopsy and whole organs in a set of discarded kidneys. Methods: 46 discarded kidneys were identified with their corresponding biopsies. The biopsies were reviewed by three general and two specialist pathologists, blinded to the original report, according to Remuzzi score. The intraclass correlation coefficient (ICC) was calculated for both groups. Discarded kidneys were scored according to Remuzzi score by a single specialist pathologist. Biopsies and organs were compared by Wilcoxon signed rank test. Weighted κ coefficients between biopsy and organ scores were also calculated. Results: Specialist pathologists achieved higher values of ICC, reaching excellent or good agreement in most of the parameters, while general pathologists values were mainly fair or good. On whole organs, scores were consistently lower than biopsies, with a significant difference in most of the parameters. Weighted κ coefficient was slight or fair for most of the parameters. Conclusions: Our data suggests that the creation of a pool of specialist pathologists would improve organ utilization. Moreover, biopsies are not representative of the whole organ. As the Remuzzi score on biopsy is a major reasons for discard, a quota of transplantable kidneys may be erroneously discarded. Refinement in Remuzzi cut-offs based on expert reporting and recognition of sampling error of biopsies in correlation with clinical outcome data should be undertaken.

Background Extended criteria donors (ECD) are widely utilized due to organ shortage, but they may increase the risk of graft dysfunction and poorer outcomes. Hypothermic oxygenated perfusion (HOPE) is a recent organ preservation strategy for marginal kidney and liver grafts, allowing a redirect from anaerobic metabolism to aerobic metabolism under hypothermic conditions and protecting grafts from oxidative species–related damage. These mechanisms may improve graft function and survival. Objective With this study, we will evaluate the benefit of end-ischemic HOPE on ECD grafts for livers and kidneys as compared to static cold storage (SCS). The aim of the study is to demonstrate the ability of HOPE to improve graft function and postoperative outcomes of ECD kidney and liver recipients. Methods This is an open-label, single-center randomized clinical trial with the aim of comparing HOPE with SCS in ECD kidney and liver transplantation. In the study protocol, which has been approved by the ethics committee, 220 patients (110 liver recipients and 110 kidney recipients) will be enrolled. Livers and kidneys assigned to the HOPE group undergo machine perfusion with cold Belzer solution (4-10°C) and continuous oxygenation (partial pressure of oxygen of 500-600 mm Hg). In the control group, livers and kidneys undergoing SCS are steeped in Celsior solution and stored on ice. Using the same perfusion machine for both liver and kidney grafts, organs are perfused from the start of the back-table procedure until implantation, without increasing the cold ischemia time. For each group, we will evaluate clinical outcomes, graft function tests, histologic findings, perfusate, and the number of allocated organs. Publication of the results is expected to begin in 2021. Results Dynamic preservation methods for organs from high-risk donors should improve graft dysfunction after transplantation. To date, we have recruited 108 participants. The study is ongoing, and recruitment of participants will continue until January 2020. Conclusions The proposed preservation method should improve ECD graft function and consequently the postoperative patient outcomes. Trial Registration ClinicalTrials.gov NCT03837197; https://clinicaltrials.gov/ct2/show/NCT03837197 ; Archived by WebCite® at http://www.webcitation.org/76fSutT3R International Registered Report Identifier (IRRID) DERR1-10.2196/13922

BACKGROUND Extended criteria donors (ECD) are widely utilized due to organ shortage, but they may increase the risk of graft dysfunction and poorer outcomes. Hypothermic oxygenated perfusion (HOPE) is a recent organ preservation strategy for marginal kidney and liver grafts, allowing a redirect from anaerobic metabolism to aerobic metabolism under hypothermic conditions and protecting grafts from oxidative species–related damage. These mechanisms may improve graft function and survival. OBJECTIVE With this study, we will evaluate the benefit of end-ischemic HOPE on ECD grafts for livers and kidneys as compared to static cold storage (SCS). The aim of the study is to demonstrate the ability of HOPE to improve graft function and postoperative outcomes of ECD kidney and liver recipients. METHODS This is an open-label, single-center randomized clinical trial with the aim of comparing HOPE with SCS in ECD kidney and liver transplantation. In the study protocol, which has been approved by the ethics committee, 220 patients (110 liver recipients and 110 kidney recipients) will be enrolled. Livers and kidneys assigned to the HOPE group undergo machine perfusion with cold Belzer solution (4-10°C) and continuous oxygenation (partial pressure of oxygen of 500-600 mm Hg). In the control group, livers and kidneys undergoing SCS are steeped in Celsior solution and stored on ice. Using the same perfusion machine for both liver and kidney grafts, organs are perfused from the start of the back-table procedure until implantation, without increasing the cold ischemia time. For each group, we will evaluate clinical outcomes, graft function tests, histologic findings, perfusate, and the number of allocated organs. Publication of the results is expected to begin in 2021. RESULTS Dynamic preservation methods for organs from high-risk donors should improve graft dysfunction after transplantation. To date, we have recruited 108 participants. The study is ongoing, and recruitment of participants will continue until January 2020. CONCLUSIONS The proposed preservation method should improve ECD graft function and consequently the postoperative patient outcomes. CLINICALTRIAL ClinicalTrials.gov NCT03837197; https://clinicaltrials.gov/ct2/show/NCT03837197 ; Archived by WebCite® at http://www.webcitation.org/76fSutT3R INTERNATIONAL REGISTERED REPORT DERR1-10.2196/13922

Background Cancer of Unknown Primary is a rare syndrome of metastatic cancers for which the primary site cannot be recognized after detailed physical examinations, blood analyses, imaging and immunohistochemical (IHC) testing. CUPs make up 3-5% of newly diagnosed cancers worldwide and represent an important diagnostic and clinical problem. Treatment failure to empirical chemotherapy is common, resulting in progressive disease and poor prognosis. We and others proposed that a molecular inference of the tissue of origin could be feasible for most CUP tumors (Ferracin et al. J Pathol, 2011), thus giving access to more effective therapeutic agents and potentially extending CUP patient life-expectancy. Methods We optimized a cancer-type classifier based on microRNA (miRNA) expression for the prediction of CUP primary tumor site (Laprovitera et al., Front Oncol. 2018). Specifically, we designed a molecular assay to quantify the absolute copy number of 89 miRNAs in formalin-fixed paraffin-embedded (FFPE) samples using EvaGreen-based Droplet Digital PCR (BioRad). We tested 153 samples from three groups: primary tumors (N=95), metastases of known origin (N=10) and cancers of unknown origin (N=48). CUP diagnosis was obtained after a detailed histo-pathological investigation as per CUP diagnostic guidelines (NCCN occult primary v.2-2019 and ESMO guidelines). The study was approved by the local ethical committee. Results We investigated a pre-determined set of 89 miRNAs to infer the site-of origin of metastatic cancers of unknown or uncertain primary. Primary tumors from 16 different sites (lung, pancreas, liver, bile duct, kidney, bowel, testis, ovary, endometrium, stomach, bladder, breast, prostate, melanoma, gastrointestinal neuroendocrine, head and neck) were tested for absolute miRNA expression and used to train our classifier (Tibshirani et al. PNAS, 2002). We used the metastases of known origin as a test-set for our molecular prediction. We applied the shrunken centroids approach as predictive algorithm to obtain the most probable CUP site(s)-of- origin. Our results were judged against the hypothesized primary site suggested by standard diagnostic workup and pathological assessment. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification (with a probability > 50%) for all samples, potentially within a one-week time frame from sample inclusion. Conclusions Our study demonstrated that miRNA expression profiling in CUP samples have the potential to be employed in a clinical setting. Our molecular analysis can be performed on-request, concomitantly with the standard diagnostic workup and in association with genetic profiling, to offer valuable indication about the possible primary site thereby supporting treatment decisions. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to MF (IG 18464). Citation Format: Noemi Laprovitera, Elisa Porcellini, Mattia Riefolo, Elisabetta Broseghini, Ingrid Garajova, Silvia Sabbioni, Martin Pichler, Andrea Ardizzoni, Antonia D'Errico, Manuela Ferracin. Cancer of unknown primary site-of-origin: An enigma ready to be miR-solved [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5432.

Background Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers whose primary site cannot be determined after standard clinical and pathological evaluation. CUP patients are generally treated with empirical chemotherapy and have poor prognosis. As reported in recent studies, CUPs present an average of 4 genetic alterations per tumor and these genetic alterations can be detected in circulating tumor DNA (Kato 2017). Thus, a potential improvement in CUP outcomes could derive from targeted therapies directed toward actionable mutations, and Circulating tumor cells (CTCs) can provide valuable information. Here, we describe a case of a 51-yr-old Caucasian female (Pt#71) with metastatic CUP. CTCs were detectable in her blood and analyzed for genetic alterations. Methods Using CellSearch and DEPArray NxT, n=3 CTCs and n=1 leukocyte as a control were isolated from patient's peripheral blood. Single-cell whole genome amplification was obtained with Ampli1 WGA Kit and libraries were generated with Ampli1 OncoSeek Panel and Ampli1 LowPass kits. In parallel, formalin-fixed paraffin embedded (FFPE) tissue derived from a lymph node biopsy was analyzed with two different methods. One curl was sent for comprehensive genomic profile to FoundationOne assay testing (Roche). From another curl, DNA was extracted and directly processed with DEPArray LibPrep and DEPArray OncoSeek Panel kits. After sequencing on Illumina MiSeq platform, raw data were analyzed on MSBiosuite platform (Menarini Silicon Biosystems). Results CTCs genome-wide characterization allowed the detection of sub-chromosomal losses, including a LOH region comprising the APC gene, and patterns of extensive gains. Moreover, amplification signals were detected in correspondence of FGFR2 and CCNE1 genes. Targeted sequencing on both CTCs and bulk tumor DNA confirmed the FGFR2 amplification in all the samples and detected a somatic variant in APC gene (APC:p.T1556Nfs*3). FoundationOne assay on FFPE biopsy reported the same amplification on FGFR2 and CCNE1 genes, along with a somatic variant in ARID1A gene (ARID1A:p.R1276*), a gene not present in the OncoSeek Panel. On the other hand, APC somatic variant was not identified by FoundationOne, probably due to contamination by normal cells and/or tumor heterogeneity. Conclusions The comprehensive genomic profile of tumor FFPE tissue and CTCs was highly overlapping and allowed the characterization of genetic alterations in this CUP case, revealing potentially actionable mutations and copy number alterations. The advantage of isolating and analyzing single CTCs is clearly shown by the non-invasive procedure combined with a precise detection of druggable alterations due to cell purity. Citation Format: Elisa Porcellini, Francesco Gelsomino, Noemi Laprovitera, Mattia Riefolo, Marianna Garonzi, Paola Tononi, Francesca Fontana, Antonia D'Errico, Maria Pantaleo, Nicolò Manaresi, Andrea Ardizzoni, Manuela Ferracin. Isolation and genetic characterization of circulating tumor cells from cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5361.

An appraisal of recent data highlighting aspects inspired by the new Geroscience perspective are here discussed. The main findings are summarized as follows: i) liver has to be considered an immunological organ, and new studies suggest a role for the recently described cells named telocytes; ii) the liver-gut axis represents a crucial connection with environment and life style habits and may influence liver diseases onset; iii) the physiological aging of liver shows relatively modest alterations. Nevertheless, several molecular changes appear to be relevant: a) an increase of microRNA-31-5p; -141-3p; -200c-3p expressions after 60 years of age; b) a remodeling of genome-wide DNA methylation profile evident until 60 years of age and then plateauing; c) changes in transcriptome including the metabolic zones of hepatocyte lobules; d) liver undergoes an accelerated aging in presence of chronic inflammation/liver diseases in a sort of continuum, largely as a consequence of unhealthy life styles and exposure to environmental noxious agents. We argue that chronic liver inflammation has all the major characteristics of "inflammaging" and likely sustains the onset and progression of liver diseases. Finally, we propose to use a combination of parameters, mostly obtained by omics such as transcriptomics and epigenomics, to evaluate in deep both the biological age of liver (in comparison with the chronological age) and the effects of donor-recipient age-mismatches in the context of liver transplants.

Background: Sorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results. Methods: 2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers. Results: Using a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo. Conclusion: We have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

Guidelines for donor selection have changed to expand the donor pool, considering potential donors affected by a neoplasm. Aim of this retrospective study is to look at the use of organs from donors with a current or history of neoplasm within the Italian Transplant Network. Data, collected and validated by Italian National Health Institute for the time interval 2006–2015, have been reviewed retrospectively by mean of multivariable pivot tables. Donors with neoplasia represented about 5% of all donors, resulting in about 4% of all transplants. Donors presented a benign neoplasm in 29.08% of cases, a malignancy with variable risk of transmission in 69.75% while in 1.34% the nature of neoplasm could not be assessed. Considering all procedures, rate of transmission of a malignancy was 0.03% (10 cases) of all 29858 transplants of the time interval. Notably, cases of transmission were not from donors of this pool, but from donors that, according to our protocols, had no elements of suspect at time of donation. As recipient safety is always the priority and as guidelines have set exclusion criteria for donors with some specific types of malignancy, these results show that use of this type of donors is safe and improve organ pool. Furthermore represent basis for improvement and standardization of donor assessment protocols suggesting that efforts in data collection systems, to produce complete and homogeneous data, are mandatory.

We describe three cases of liver lesions, characterized by a discrepancy between presurgical imaging and histological features, in which the final histological diagnosis was quite different from what the surgeons expected. We present (1) a case of primary liver angiomyolipoma associated with focal nodular hyperplasia, (2) a case of perivascular epithelioid cell tumor, and (3) a case of liver splenosis associated with focal nodular hyperplasia. In all cases, a presurgical diagnosis of hepatocellular adenoma was made. Due to nonspecific clinical and radiological features, these rare liver lesions are often presurgically misdiagnosed, especially in young noncirrhotic patients. The association among different lesions represents one additional diagnostic challenge.

The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic. Non‑synonymous SNVs were analyzed with SNPs&GO and PROVEAN. A total of 13 samples were analyzed by high resolution copy number analysis on an Affymetrix SNP array 6.0. RNAseq resulted in an average of 264 coding non‑synonymous novel SNVs (ranging from 146‑374) and 16 novel insertions or deletions (In/Dels) (ranging from 6‑24) for each sample, of which a mean of 11.2% were disease‑associated and somatic events, while 34.7% were frameshift somatic In/Dels. From this analysis, alterations in the known oncogenes associated with PDAC were observed, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations (93.7%) and inactivation of cyclin‑dependent kinase inhibitor 2A (CDKN2A) (50%), mothers against decapentaplegic homolog 4 (SMAD4) (50%), and tumor protein 53 (TP53) (56%). One case that was negative for KRAS exhibited a G13D neuroblastoma RAS viral oncogene homolog mutation. In addition, gene fusions were detected in 10 samples for a total of 23 different intra‑ or inter‑chromosomal rearrangements, however, a recurrent fusion transcript remains to be identified. SNP arrays identified macroscopic and cryptic cytogenetic alterations in 85% of patients. Gains were observed in the chromosome arms 6p, 12p, 18q and 19q which contain KRAS, GATA binding protein 6, protein kinase B and cyclin D3. Deletions were identified on chromosome arms 1p, 9p, 6p, 18q, 10q, 15q, 17p, 21q and 19q which involve TP53, CDKN2A/B, SMAD4, runt‑related transcription factor 2, AT‑rich interactive domain‑containing protein 1A, phosphatase and tensin homolog and serine/threonine kinase 11. In conclusion, genetic alterations in PDCA were observed to involve numerous pathways including cell migration, transforming growth factor‑β signaling, apoptosis, cell proliferation and DNA damage repair. However, signaling alterations were not observed in all tumors and key mutations appeared to differ between PDAC cases.

Aims: The aims of this study were to: validate the use of the immunohistochemical (IHC) markers glutamine synthetase (GS), glypican-3 (GPC3), heat shock protein-70 (HSP70) and enhancer of zeste homologue 2 (EZH2) in liver biopsies for the differential diagnosis between small hepatocellular carcinoma (HCC) and non-neoplastic liver nodules, with special attention to

Hepatocellular carcinoma (HCC) is the most common primary liver malignant neoplasia. HCC is characterized by a poor prognosis. The need to find new molecular markers for its diagnosis and prognosis has led to a progressive increase in the number of scientific studies on this topic. MicroRNAs (miRNAs) are small non-coding RNA that play a role in almost all main cellular pathways. miRNAs are involved in the regulation of expression of the major tumor-related genes in carcinogenesis, acting as oncogenes or tumor suppressor genes. The aim of this review was to identify papers published in 2017 investigating the role of miRNAs in HCC tumorigenesis. miRNAs were classified according to their role in the main molecular pathways involved in HCC tumorigenesis: (1) mTOR; (2) Wnt; (3) JAK/STAT; (4) apoptosis; and (5) MAPK. The role of miRNAs in prognosis/response prediction was taken into consideration. Bearing in mind that the analysis of miRNAs in serum and other body fluids would be crucial for clinical management, the role of circulating miRNAs in HCC patients was also investigated. The most represented miRNA-regulated pathway in HCC is mTOR, but apoptosis, Wnt, JAK/STAT or MAPK pathways are also influenced by miRNA expression levels. These miRNAs could thus be used in clinical practice as diagnostic, prognostic or therapeutic targets for HCC treatment.

Nowadays, the major limit to the immunohistochemical (IHC) analysis of tissue PD-L1 is the high variability of the monoclonal antibodies commercially available. Aims of the present paper are to assess the best clone and the most suitable scoring for PD-L1 IHC determination on human hepatocellular carcinoma (HCC) among three commercially available clones, and to evaluate which PD-L1 clone is the best in predicting HCC aggressiveness in vivo. We built a tissue microarray (TMA) with 60 retrospective HCC cases, including the correspondent non-tumoral tissue. IHC was automatically performed using the following anti-PD-L1 clones: 28.8, SP142, and SP263. As results, we did not find any immunoreactivity for PD-L1 in both neoplastic and normal hepatocytes included in the TMA using the three antibodies. Positivity for PD-L1 was exclusively seen in inflammatory cells within the HCC tissue and in cirrhotic parenchyma. When a gold standard was assessed, the sensitivity of SP142, 28.8 and SP263 was 46 %, 54 % and 85 % respectively. Using the SP263 clone, the absolute number of PD-L1-positive inflammatory cells in the HCC cores was paired with the number of PD-L1-positive inflammatory cells in the corresponding non-tumoral tissue (P = 0.001). Finally, using SP263, the mean number of PD-L1-positive cells was 11.3 ± 12.6 in HCC from deceased patients, versus 4.7 ± 5.2 in alive patients (p = 0.039). SP263 is the most sensitive clone for PD-L1 IHC tissue determination in HCC, as well as the best antibody for the assessment of its biological behavior.

Summary The aim of the present study is to describe the histological and mutational characteristics of a series of both large and small bowel adenocarcinomas in patients with Crohn’s disease from a tertiary referral centre of inflammatory bowel disease. Bowel adenocarcinoma was diagnosed in 11 (1.7%) of 660 consecutive patients submitted to surgery for histologically proven Crohn’s disease in 5 years. The following data were collected: tumour site, stage and grade, intracellular/extra-cellular mucin, lymphovascular invasion, immunohistochem-istry for keratin 7, keratin 20 and CDX-2, mutation analyses of KRAS, B-RAF, PI3K and microsatellite instability. A strong predominance of male gender was observed (10/11). Four (36.4%) adenocarcinomas arose in the small bowel, five (45.4%) in the anus/rectum, and two (18.2%) in anastomosis. Furthermore, all cases of anorectal adenocarcinoma showed >50% of extracellular mucin, with associated KRAS mutations in three of five. No influence in cancer incidence by infliximab therapy was observed. Our series, one of the largest on the topic with immunomorphological and molecular deepening, showed that bowel adenocarcinomas in Crohn’s disease have an aggressive behaviour and a strong predominance of extracellular mucin. In surgical specimens from Crohn’s disease patients, mucinous-looking anal fistulas and ileal areas of adhesion/retraction should always be extensively sampled.

Background aims Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Methods Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. Results We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. Discussion We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.

Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133We demonstrated that the interaction between CD133

The assessment of hepatitis C virus (HCV) RNA in liver tissues is clinically relevant in cases where histology, liver function tests, and HCV serology are not sufficient for a definitive diagnosis of HCV-related hepatitis. We analyzed 215 formalin-fixed, paraffin-embedded liver needle biopsies from patients infected with HCV genotypes 1b and 2. HCV RNA extracted from paraffin sections were quantified by means of a TaqMan real-time reverse transcription-polymerase chain reaction method. The quantification of HCV RNA in liver tissue was correlated with the amount of HCV detected by immunohistochemistry (IHC) on paired frozen biopsies, the HCV RNA load in the serum, and the main serum tests of liver function and cholestasis. HCV RNA was detected by real-time reverse transcription-polymerase chain reaction in 169 liver biopsies (78.6%) with a mean value of 13.59+/-37.25 IU/ng. Tissue HCV RNA levels strongly correlated with the IHC results (P

Histological quality assessment of donated livers is a key factor for extending the cadaveric donor pool for liver transplantation. We retrospectively compared frozen-section analysis with routine histological permanent slides and the outcomes of grafts in liver biopsies from 294 candidate donors. The kappa concordance coefficient of agreement between frozen-section analysis and routine histological analysis was very good for macrosteatosis (kappa = 0.934), microsteatosis (kappa = 0.828), and total steatosis (kappa = 0.814). The correlation between the mean amounts of macrosteatosis, microsteatosis, and total steatosis in frozen and permanent sections was also significant (P 30% in 4 of 32 cases (12.5%) and in 23 of 62 cases (37.1%), respectively. The only 2 histological parameters of frozen sections able to predict graft dysfunction within 7 days of transplantation were macrosteatosis and total steatosis (P = 0.018 and P = 0.015, respectively, Mann-Whitney test). None of the other histopathological features evaluated in frozen sections, including portal inflammation, lobular necrosis, myointimal thickening, biliocyte regression, cholestasis, hepatocellular polymorphism, lipofuscin storage, and fibrous septa, were significantly correlated with the graft outcome. The frozen-section histological evaluation of biopsies from cadaveric liver donors is an accurate, time-effective, and predictive method for the assessment of graft suitability.

Although the morphological features of hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT) have been well established in the last decades, the differential diagnosis still represents a challenge for the pathologist, especially early recurrent hepatitis C vs mild acute cellular rejection. The present review focuses on the role of the pathologist and the pathology laboratory in the management of recipients with recurrent hepatitis C, the usefulness of early and late post-OLT liver biopsies, and the potential role of ancillary techniques (immunohistochemistry and reverse transcription-polymerase chain reaction, RT-PCR). The English literature on the topic is reviewed, focusing on the histopathology, the immunohistochemistry and the use of RT-PCR on HCV-positive post-OLT biopsies. The different histopathological illustrations of early and chronic recurrent hepatitis C are presented, with special focus on the main differential diagnoses and those features with prognostic relevance (cholestasis above all). The usefulness of ancillary techniques are discussed, especially HCV RNA quantitation by RT-PCR. Finally, the usefulness of long-term protocol biopsies is addressed: their usefulness for the study of allograft disease progression is clear, but their meaning in the long term is still debated. The significance of plasma cell infiltrate in HCV-positive allografts, the prognostic weight of graft steatosis, and the impact of donor age in recurrent hepatitis C also represent additional open issues.