Deletion of TNF in Winnie-APCMin/+ Mice Reveals Its Dual Role in the Onset and Progression of Colitis-Associated Colorectal Cancer.

Colorectal cancer (CRC) is among the best examples for depicting the relationship between inflammation and cancer. The introduction of new therapeutics targeting inflammatory mediators showed a marked decrease in the overall risk of CRC, although their chemopreventive potential is still debated. Specifically, a monoclonal antibody that blocks tumor necrosis factor (TNF), infliximab, increases CRC risk in inflammatory bowel disease patients. To address the axis between TNF and CRC development and progression, we depleted the Tnf from our previously established murine model of colitis-associated cancer (CAC), the Winnie-ApcMin/+ line. We characterized the new Winnie-APCMin/+-TNF-KO line through macroscopical and microscopical analyses. Surprisingly, the latter demonstrated that the deletion of Tnf in Winnie-ApcMin/+ mice resulted in an initial reduction in dysplastic lesion incidence in 5-week-old mice followed by a faster disease progression at 8 weeks. Histological data were confirmed by the molecular profiling obtained from both the real-time PCR analysis of the whole tissue and the RNA sequencing of the macrodissected tumoral lesions from Winnie-APCMin/+-TNF-KO distal colon at 8 weeks. Our results highlight that TNF could exert a dual role in CAC, supporting the promotion of neoplastic lesions onset in the early stage of the disease while inducing their reduction during disease progression.

Assessment and management of undifferentiated carcinoma with osteoclastic like giant cells of the pancreas: a case report and revision of literature.

BACKGROUND: Undifferentiated carcinoma with osteoclast-like giant cells (UCOGCs) is a rare and aggressive non endocrine pancreatic carcinoma characterized by the presence of osteoclastic giant cells mixed with mononuclear cell. Very few cases have been reported in the literature and the histogenesis is controversial as, at the time of diagnosis, the tumor is often of advanced size and stage and it is difficult to pathologically observe its relationship with the pancreatic duct. CASE PRESENTATION: We present a case of 65-year-old male patient presenting with abdominal pain, nausea, and weight loss, which was treated with surgical resection. Histological examination revealed an undifferentiated pancreatic carcinoma with osteoclast-like giant cells. The patient underwent to a routine pylorus preserving pancreatoduodenectomy. Actually, the patient was in good performance status and disease-free five months. CONCLUSIONS: Based on the present case and limited previous data, further researches preferably with large cohorts are necessary to clarify the pathogenesis of the neoplasm. However, as show in this case, histopathological and immunohistochemically studies are the gold standard for the diagnosis of UCPOGC. Investigation of the genomic alterations in UPOGCs could help to explain the histologic diversity of variant tumor and could provide a genetic basis for prognosis and treatment.

Quercetin Administration Suppresses the Cytokine Storm in Myeloid and Plasmacytoid Dendritic Cells.

Dendritic cells (DCs) can be divided by lineage into myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). They both are present in mucosal tissues and regulate the immune response by secreting chemokines and cytokines. Inflammatory bowel diseases (IBDs) are characterized by a leaky intestinal barrier and the consequent translocation of bacterial lipopolysaccharide (LPS) to the basolateral side. This results in DCs activation, but the response of pDCs is still poorly characterized. In the present study, we compared mDCs and pDCs responses to LPS administration. We present a broad panel of DCs secreted factors, including cytokines, chemokines, and growth factors. Our recent studies demonstrated the anti-inflammatory effects of quercetin administration, but to date, there is no evidence about quercetin's effects on pDCs. The results of the present study demonstrate that pDCs can respond to LPS and that quercetin exposure modulates soluble factors release through the same molecular pathway used by mDCs (Slpi, Hmox1, and AP-1).

Primary lymph node gastrinoma: a case report and review of the literature.

BACKGROUND: Gastrinoma is a rare form of neuroendocrine neoplasm. The presence of a primary lymph node localization of gastrinoma is a much debated and controversial topic in the literature, as regards whether these cases represent metastatic disease from an as yet unidentified primary tumor, or the de novo occurrence of a gastrinoma in a lymph node. CASE PRESENTATION: We report the case of a 24-year-old male with intense epigastric pain treated at the beginning with high dose proton pump inhibitors. Further workup with CT and subsequent laparotomy revealed a single peripancreatic lymph node. Histological examination highlighted a well-differentiated neuroendocrine tumor. CONCLUSION: This case underlines that the primitive lymph node gastrinoma is a distinct nosological entity with a precise location in the context of rare neuroendocrine tumors that should be considered when specific symptoms are associated with the identification of isolated lymph nodes, after excluding any possible primitive locations of neoplastic localization.

Iron Overload Mimicking Conditions Skews Bone Marrow Dendritic Cells Differentiation into MHCIIlowCD11c+CD11b+F4/80+ Cells.

Iron overload is an undesired effect of frequent blood transfusions or genetic diseases. Myelodysplastic syndrome (MDS) patients become transfusion dependent, but due to the combination of ineffective haematopoiesis and repeated blood transfusions they are often subject to iron overload. In this study, we demonstrate that iron-overload mimicking condition alters bone marrow progenitor differentiation towards dendritic cells (DCs). Cells cultured in iron-enriched culture medium for seven days fail to differentiate into conventional CD11c+MHCIIhi DCs and fail to efficiently respond to LPS (Lipopolysaccharides). Cells appear smaller than control DCs but vital and able to perform FITC-dextran (Fluorescein isothiocyanate-dextran) endocytosis. At molecular level, cells cultured in iron-enriched conditions show increased ARG1 and PU.1, and decreased IRF8 expression.

Winnie-APCMin/+ Mice: A Spontaneous Model of Colitis-Associated Colorectal Cancer Combining Genetics and Inflammation.

(1) Background: Colorectal cancer (CRC) is among the best examples of the relationship between inflammation and increased cancer risk. (2) Methods: To examine the effects of spontaneous low-grade chronic inflammation on the pathogenesis of CRC, we developed a new murine model of colitis-associated cancer (CAC) by crossing Mucin 2 mutated mice (Winnie) with ApcMin/+ mice. (3) Results: The resulting Winnie-ApcMin/+ model combines an inflammatory background with a genetic predisposition to small intestinal polyposis. Winnie-ApcMin/+ mice show an early occurrence of inflammatory signs and dysplastic lesions in the distal colon with a specific molecular signature. (4) Conclusion: The Winnie-ApcMin/+ model is a perfect model to demonstrate that chronic inflammation represents a crucial risk factor for the onset and progression of tumoral lesions in individuals genetically predisposed to CRC.

Quercetin Exposure Suppresses the Inflammatory Pathway in Intestinal Organoids from Winnie Mice.

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses.

Galunisertib modifies the liver fibrotic composition in the Abcb4Ko mouse model.

Transforming growth factor (TGF)-ß stimulates extracellular matrix (ECM) deposition during development of liver fibrosis and cirrhosis, the most important risk factor for the onset of hepatocellular carcinoma. In liver cancer, TGF-ß is responsible for a more aggressive and invasive phenotype, orchestrating remodeling of the tumor microenvironment and triggering epithelial-mesenchymal transition of cancer cells. This is the scientific rationale for targeting the TGF-ß pathway via a small molecule, galunisertib (intracellular inhibitor of ALK5) in clinical trials to treat liver cancer patients at an advanced disease stage. In this study, the hypothesis that galunisertib modifies the tissue microenvironment via inhibition of the TGF-ß pathway is tested in an experimental preclinical model. At the age of 6 months, Abcb4ko mice-a well-established model for chronic liver disease development and progression-are treated twice daily with galunisertib (150 mg/kg) via oral gavage for 14 consecutive days. Two days after the last treatment, blood plasma and livers are harvested for further assessment, including fibrosis scoring and ECM components. The reduction of Smad2 phosphorylation in both parenchymal and non-parenchymal liver cells following galunisertib administration confirms the treatment effectiveness. Damage-related galunisertib does not change cell proliferation, macrophage numbers and leucocyte recruitment. Furthermore, no clear impact on the amount of fibrosis is evident, as documented by PicroSirius red and Gomori-trichome scoring. On the other hand, several fibrogenic genes, e.g., collagens (Col1α1 and Col1α2), Tgf-ß1 and Timp1, mRNA levels are significantly downregulated by galunisertib administration when compared to controls. Most interestingly, ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic ß-catenin pathway, are remarkably reduced by galunisertib-treated Abcb5ko mice. In conclusion, TGF-ß inhibition by galunisertib interferes, to some extent, with chronic liver progression, not by reducing the stage of liver fibrosis as measured by different scoring systems, but rather by modulating the biochemical composition of the deposited ECM, likely affecting the fate of non-parenchymal cells.

Polyphenol administration impairs T-cell proliferation by imprinting a distinct dendritic cell maturational profile.

Currently little is known as to how nutritionally derived compounds may affect dendritic cell (DC) maturation and potentially prevent inappropriate inflammatory responses that are characteristic of chronic inflammatory syndromes. Previous observations have demonstrated that two polyphenols quercetin and piperine delivered through reconstituted oil bodies (ROBs-QP) can influence DC maturation in response to LPS leading to a modulated inflammatory response. In the present study, we examined the molecular effects of ROBs-QP exposure on DC differentiation in mice and identified a unique molecular signature in response to LPS administration that potentially modulates DC maturation and activity in inflammatory conditions. Following LPS administration, ROBs-QP-exposed DCs expressed an altered molecular profile as compared with control DCs, including cytokine and chemokine production, chemokine receptor repertoire, and antigen presentation ability. In vivo ROBs-QP administration suppresses antigen-specific T-cell division in the draining lymph nodes resulting from a reduced ability to create stable immunological synapse. Our data demonstrate that polyphenols exposure can drive DCs toward a new anti-inflammatory molecular profile capable of dampening the inflammatory response, highlighting their potential as complementary nutritional approaches in the treatment of chronic inflammatory syndromes.

miR-29b and miR-198 overexpression in CD8+ T cells of renal cell carcinoma patients down-modulates JAK3 and MCL-1 leading to immune dysfunction.

BACKGROUND: Mammalian microRNAs (miR) regulate the expression of genes relevant for the development of adaptive and innate immunity against cancer. Since T cell dysfunction has previously been reported in patients with renal cell carcinoma (RCC; clear cell type), we aimed to analyze these immune cells for genetic and protein differences when compared to normal donor T cells freshly after isolation and 35 days after in vitro stimulation (IVS) with HLA-matched RCC tumor cells. METHODS: We investigated gene expression profiles of tumor-reactive CD8(+) T cells obtained from RCC patient and compared with their HLA-matched healthy sibling donors using a microarray approach. In addition, miRNAs analysis was performed in a validation cohort of peripheral blood CD8(+) T cells from 25 RCC patients compared to 15 healthy volunteers. RESULTS: We observed that CD8(+) T cells from RCC patients expressed reduced levels of anti-apoptotic and proliferation-associated gene products when compared with normal donor T cells both pre- and post-IVS. In particular, JAK3 and MCL-1 were down-regulated in patient CD8(+) T cells versus their normal counterparts, likely due to defective suppressor activity of miR-29b and miR-198 in RCC CD8(+) T cells. Indeed, specific inhibition of miR-29b or miR-198 in peripheral blood mononuclear cells (PBMCs) isolated from RCC patients, resulted in the up-regulation of JAK3 and MCL-1 proteins and significant improvement of cell survival in vitro. CONCLUSIONS: Our results suggest that miR-29b and miR-198 dysregulation in RCC patient CD8(+) T cells is associated with dysfunctional immunity and foreshadow the development of miR-targeted therapeutics to correct such T cell defects in vivo.

Pyoderma gangrenosum in ulcerative colitis patient treated with vedolizumab: adsorptive granulocyte/monocyte apheresis as a new therapeutic option refractory cases - a case report and literature review.

Extraintestinal manifestations occur rather frequently in ulcerative colitis (UC) and Crohn's disease patients and are usually related to an exacerbation of the underlying intestinal bowel disease but sometimes may run a course independent of the inflammatory bowel diseases (IBD). About one-third of patients with IBD develop extraintestinal manifestations, such as pyoderma gangrenosum (PG). PG is an uncommon inflammatory skin disorder of unknown pathogenesis. There are no specific serological or histological markers, and diagnosis is predominantly clinical. Topical and systemic therapies are both vital aspects of treatment and immune modulators have been used with increasing success in recent years, although immunosuppressive drugs raise some concerns due to an increased risk of serious and opportunistic infections and cancer, particularly in elderly and comorbid patients, underlining the unmet need for safer alternative therapies. Thus, in this case report, we highlighted an adsorptive granulocyte/monocyte apheresis (GMA) as a new therapeutic possibility in IBD patients with extraintestinal manifestations. We report a case of a 60-year woman with a history of UC with a Mayo grade 3 score which was associated with a PG. Given that the patients maintained clinical remission with vedolizumab, we preferred not to perform a combined treatment with other antitumor necrosis factor-alpha or ciclosporin, thus avoiding an increased risk of serious infections in the patient. Therefore, we performed the extracorporeal leukocyte apheresis. The patient progressed favorably, with progressive improvement of skin and bowel disease. Therefore, adsorptive GMA has a very favorable safety profile and has been confirmed in numerous studies. In this study, we underlined that an intensive regimen of GMA paves the way to an ideal option for patients with severe and refractory PG complicated with UC.

A new reliable method for tissue preservation.

Studying and developing preanalytical tools and technologies for preserving high-quality tissues for histological assays is a constantly growing field because preserving biological samples always poses a challenge. Formalin fixation and paraffin embedding (FFPE) remains a key component of long-term tissue preservation in the histopathology and pathology laboratory, despite its known carcinogenic effect and poor preservation of nucleic acids. Over the years, some other methods have been described to preserve biological tissues such as plastination. However, the current techniques do not consent further dissection or histopathologically based studies. The present study describes the applicability of a new method for tissues preservation after surgery or endoscopic removal, to preserve specimens for subsequent histopathological examination and tissue banking as well as for immunohistochemistry, preserving the nucleic acids and protein integrity. This method, protected by patent (EP 18185364.9A) is able to restore tissues to the state prior to drying, thereby allowing them to be further processed for histologic, cytological, immunologic, and/or genetic analyses. This new technique offers an alternative to classic tissue preservation, featuring an innovative and cost-effective process, available locally, to obtain fully preserved samples for longer periods without toxic procedures. In conclusion this new method can be successfully applied in any pathological institute, responding to the increasing demands for reliable tissue preservation in the expanding field of histological, molecular diagnostic and forensic medicine.

Interleukin 1ß Blockade Reduces Intestinal Inflammation in a Murine Model of Tumor Necrosis Factor-Independent Ulcerative Colitis.

BACKGROUND & AIMS: Inflammatory bowel diseases are multifactorial diseases commonly treated with either immunomodulatory drugs or anti-tumor necrosis factor (TNF). Currently, failure to respond to anti-TNF therapy (assessed no earlier than 8-12 weeks after starting treatment) occurs in 20%-40% of patients enrolled in clinical trials and in 10%-20% in clinical practice. Murine models of inflammatory bowel disease provide important tools to better understand disease mechanism(s). In this context and among the numerous models available, Winnie-TNF-knockout (KO) mice recently were reported to show characteristics of ulcerative colitis (UC) that are independent of TNF, and with increased interleukin (IL)1ß production. METHODS: Herein, the efficacy of recombinant IL1-receptor antagonist (anakinra) administration was evaluated in Winnie-TNF-KO mice, used as a UC model of primary anti-TNF nonresponders. RESULTS: We analyzed gut mucosal biopsy specimens and circulating cytokine profiles of a cohort of 30 UC patients; approximately 75% of primary nonresponders were characterized by abundant IL1ß in both the serum and local intestinal tissues. In Winnie-TNF-KO mice, administration of anakinra efficiently reduced the histologic score of the distal colon, which represents the most common site of inflammation in Winnie mice. Furthermore, among lamina propria and mesenteric lymph node-derived T cells, interferon γ-expressing CD8+ T cells were reduced significantly after anakinra administration. CONCLUSIONS: Our study provides new insight and alternative approaches to treat UC patients, and points to anti-IL1 strategies (ie, anakinra) that may be a more effective therapeutic option for primary nonresponders to anti-TNF therapy.

Nutritional Regimes Enriched with Antioxidants as an Efficient Adjuvant for IBD Patients under Infliximab Administration, a Pilot Study.

Antioxidants are privileged candidates for the development of adjuvants able to improve the efficiency of pharmacological therapies, particularly for chronic inflammatory syndromes. During the last 20 years, anti-TNFα (tumor necrosis factor alpha) monoclonal antibodies infusion has been the biological therapy most frequently administered but there is still large space for improvement in disease remission rates and maintenance. In this context, nutritional bioactive compounds contained in dietary patterns or included as supplements, may act as adjuvants for the induction and maintenance of IBD (inflammatory bowel diseases) remission. To verify this possibility, a single-center preliminary study (SI-CURA, Soluzioni Innovative per la gestione del paziente e il follow up terapeutico della Colite UlceRosA) was designed and carried out to evaluate whether a daily administration of purple corn supplement could improve the response to Infliximab (IFX) infusion of IBD patients with both Crohn's disease (CD) and ulcerative colitis (UC). A cohort of 47 patients was enrolled in the study. Biological samples were collected before the first and the third IFX infusion. All patients received nutritional guidelines, 27 of them received commercial red fruit tea with low anthocyanins content, while 20 received a purple corn supplement with a high anthocyanin content. Results show that the administration of an antioxidant-enriched purple corn supplement could improve IFX-mediated disease remission in terms of circulating inflammatory markers. Comparison between CD and UC patients revealed that, at this anthocyanin dosage, the purple corn extract administration improved the IFX response in CD but not in UC patients. Our results may pave the way for a new metacentric study of CD patients, recruiting a wider cohort and followed-up over a longer observational time.

Association of Alpha B-Crystallin Expression with Tumor Differentiation Grade in Colorectal Cancer Patients.

Alpha B-crystallin (CRYAB, HSPB5) belongs to the small heat shock protein (HSP) family and is highly expressed in various human cancers, suggesting a crucial role in tumor progression. However, few studies have examined CRYAB expression in colorectal cancer (CRC). In the present study, we investigated the relationship between CRYAB expression and the clinicopathological features of CRC samples. We comparatively analyzed CRYAB protein expression in 111 CRC tissues and normal adjacent colonic tissue, observing that it was significantly lower in CRC tissues than in corresponding non-cancerous tissues. Moreover, immunohistochemical analysis showed a significant correlation between CRYAB expression and high histological grade G3 (p = 0.033). In summary, our results point to its possible application as a prognostic biomarker in CRC patients.

Dysbiosis Triggers ACF Development in Genetically Predisposed Subjects.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide, characterized by a multifactorial etiology including genetics, lifestyle, and environmental factors including microbiota composition. To address the role of microbial modulation in CRC, we used our recently established mouse model (the Winnie-APCMin/+) combining inflammation and genetics. METHODS: Gut microbiota profiling was performed on 8-week-old Winnie-APCMin/+ mice and their littermates by 16S rDNA gene amplicon sequencing. Moreover, to study the impact of dysbiosis induced by the mother's genetics in ACF development, the large intestines of APCMin/+ mice born from wild type mice were investigated by histological analysis at 8 weeks. RESULTS: ACF development in 8-week-old Winnie-APCMin/+ mice was triggered by dysbiosis. Specifically, the onset of ACF in genetically predisposed mice may result from dysbiotic signatures in the gastrointestinal tract of the breeders. Additionally, fecal transplant from Winnie donors to APCMin/+ hosts leads to an increased rate of ACF development. CONCLUSIONS: The characterization of microbiota profiling supporting CRC development in genetically predisposed mice could help to design therapeutic strategies to prevent dysbiosis. The application of these strategies in mothers during pregnancy and lactation could also reduce the CRC risk in the offspring.

Polyphenol Enriched Diet Administration During Pregnancy and Lactation Prevents Dysbiosis in Ulcerative Colitis Predisposed Littermates.

Neonatal colonization of the gastrointestinal tract depends on mother microbiome, thus mother microbiota dysbiosis is transmitted to the offspring during the delivery and shaped by breastmilk characteristics. Here we used a murine model of UC predisposition (Winnie-/-) to evaluate the effects of maternal diet during pregnancy and lactation. Using heterozygous breeders, we obtained both Winnie-/- and C57BL/6 littermates from the same mother and compared their microbiota at weaning and adult age, using a diet enriched with 1% tomato fruit of a line - named Bronze - highly enriched in bioactive polyphenols, or Control tomato. Females received enriched diets two weeks before the beginning of the breeding and never stopped for the following six months. No significant effect was observed in regard to the percentage of Winnie-/- offspring, as with both diets the percentage was about 25% as expected. Winnie littermates from breeders fed with the Bronze-enriched diet showed reduced dysbiosis at 4 weeks of age if compared with Winnie under the Control tomato diet. This effect was then reduced when mice reached adult age. Conversely, the microbiota of C57BL/6 does not change significantly, indicating that fortified mothers-diet significantly contribute to preventing dysbiosis in genetically predisposed offspring, but has mild effects on healthy littermates and adult mice. An overall tendency towards reduced inflammation was underlined by the colon weight and the percentage of Foxp3+ cells reduction in Winnie mice fed with Bronze diet. Control diet did not show similar tendency.

JAK3/STAT5/6 pathway alterations are associated with immune deviation in CD8 T cells in renal cell carcinoma patients.

To investigate the molecular mechanisms underlying altered T cell response in renal cell carcinoma (RCC) patients, we compared autologous and allogeneic CD8(+) T cell responses against RCC line from RCC patients and their HLA-matched donors, using mixed lymphocyte/tumor cell cultures (MLTCs). In addition, we analyzed the expression of molecules associated with cell cycle regulation. Autologous MLTC responder CD8(+) T cells showed cytotoxic activity against RCC cell lines; however the analysis of the distribution of CD8(+) T-cell subsets revealed that allogenic counterparts mediate superior antitumor efficacy. In RCC patients, a decreased proliferative response to tumor, associated with defects in JAK3/STAT5/6 expression that led to increased p27KIP1 expression and alterations in the cell cycle, was observed. These data define a molecular pathway involved in cell cycle regulation that is associated with the dysfunction of tumor-specific CD8(+) effector cells. If validated, this may define a therapeutic target in the setting of patients with RCC.

Crohn's Disease Following Rituximab Treatment for Follicular Lymphoma in a Patient with Synchronous Gastric Signet Ring Cells Carcinoma: A Case Report and Literature Review.

Recently, there have been a few reports of rituximab (RTX)-induced Crohn's disease, but there is no literature available on successful long-term treatment and the clinical outcome of this condition. We retrospectively analyzed the clinical data of a rare case of Crohn's disease induced by RTX administered as induction and prolonged maintenance therapy of a follicular lymphoma, diagnosed synchronously with a gastric signet ring cells carcinoma, treated at our hospital.

Altered miRNAs Expression Correlates With Gastroenteropancreatic Neuroendocrine Tumors Grades.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare and heterogeneous tumors that present a wide spectrum of different clinical and biological characteristics. Currently, tumor grading, determined by Ki-67 staining and mitotic counts, represents the most reliable predictor of prognosis. This time-consuming approach fails to reach high reproducibility standards thus requiring novel approaches to support histological evaluation and prognosis. In this study, starting from a microarray analysis of paraffin-embedded tissue specimens, we defined the miRNAs signature for poorly differentiated NETs (G3) compared to well-differentiated NETs (G1 and G2) consisting of 56 deregulated miRNAs. We identified 8 miRNAs that were expressed in all GEP-NETs grades but at different level. Among these miRNAs, miR-96-5p expression level was progressively higher from grade 1 to grade 3; inversely, its target FoxO1 expression decreased from grade 1 to grade 3. Our results reveal that the miRNAs expression profile of GEP-NET is correlated with the tumor grade, showing a potential advantage of miRNA quantification that could aid clinicians in the classification of common GEP-NETs subtypes. These findings could reliably support the histological evaluation of GEP-NETs paving the way toward personalized treatment approaches.