Long-Term Survivor of Intrahepatic Cholangiocarcinoma for over 18 Years: Case Study with Longitudinal Histo-molecular and Tumor Immune Microenvironment Characterization and Systematic Review of the Literature.

BACKGROUND: Intrahepatic cholangiocarcinoma is a biliary neoplasm usually showing a dismal prognosis. In early stages, surgical resection is the best treatment option, significantly increasing the overall survival. This approach is also recommended in the case of relapsing disease. In this study, we report the case of a patient affected by intrahepatic cholangiocarcinoma with multiple relapses and still alive for over 18 years. We also provide a systematic review regarding long-survivor (> 60 months) of intrahepatic cholangiocarcinoma. CASE PRESENTATION: A 41-year-old woman with no pathological history was diagnosed with localized intrahepatic cholangiocarcinoma and surgically treated with left hepatectomy. After the first intervention, the patients underwent three further surgical resections because of locoregional recurrences. Histologically, there were some significant similarities among all neoplasms, including the tubule-glandular architecture, but also morphological heterogeneity. The tumor immune microenvironment remained stable across the different lesions. The molecular analysis with next-generation sequencing demonstrated that all neoplasms shared the same genomic profile, including NBN and NOTCH3 mutations and chromosomes 1 and 3 alterations. CONCLUSIONS: This case study highlights the essential role of a stringent follow-up after resection of intrahepatic cholangiocarcinoma for detecting early relapsing tumors. Moreover, it shows the importance of the molecular characterization of multiple tumors for understanding their real nature. The accurate study of long-surviving patients highlights the features that are critical for outcome improvement.

Glucagon-Producing Pancreatic Neuroendocrine Tumors (Glucagonomas) are Enriched in Aggressive Neoplasms with ARX and PDX1 Co-expression, DAXX/ATRX Mutations, and ALT (Alternative Lengthening of Telomeres).

Glucagonomas are functioning pancreatic neuroendocrine tumors (PanNETs) responsible for glucagonoma syndrome. This study aims to shed light on the clinicopathological and molecular features of these neoplasms. Six patients with glucagonomas were identified. All neoplasms were investigated with immunohistochemistry for neuroendocrine markers (Synaptophysin, Chromogranin-A), ATRX, DAXX, ARX, and PDX1 transcription factors. Fluorescent in situ hybridization (FISH) for assessing alternative lengthening of telomeres (ALT), and next-generation sequencing (NGS) for molecular profiling were performed. All cases were large single masses (mean size of 8.2 cm), with necrolytic migratory erythema as the most common symptom (6/6 cases, 100%). All neoplasms were well-differentiated G1 tumors, except one case that was G2. The tumors consistently showed classic/conventional histomorphology, with solid-trabecular and nested architecture. Lymphatic and vascular invasion (6/6, 100%), perineural infiltration (4/6, 66.6%), and nodal metastasis (4/6, 66.6%) were frequently observed. Transcription factors expression showed strong ARX expression in all tumors, and PDX1 expression in 5/6 cases (83.3%), indicating co-occurring alpha- and beta-cell differentiation. NGS showed recurrent somatic MEN1 and ATRX/DAXX biallelic inactivation. Cases with ATRX or DAXX mutations also showed matched loss of ATRX or DAXX protein expression and ALT. One case harbored somatic MUTYH inactivation and showed a high tumor mutational burden (TMB, 41.0 mut/Mb). During follow-up, one patient died of the disease, and four patients developed distant metastasis. Pancreatic glucagonomas are distinct PanNETs with specific clinicopathological and molecular features, including histological aspects of biological aggressiveness, co-occurring alpha- and beta-cell differentiation, MEN1 and DAXX/ATRX mutations enrichment, and the possible presence of high-TMB as an actionable marker.

Comprehensive Characterization of Intraductal Oncocytic Papillary Neoplasm of the Pancreas: A Systematic and Critical Review.

Intraductal oncocytic papillary neoplasm (IOPN) of the pancreas is a recently recognized pancreatic tumor. Here, we aimed to determine its most essential features with the systematic review tool. PubMed, Scopus, and Embase were searched for studies reporting data on pancreatic IOPN. The clinicopathologic, immunohistochemical, and molecular data were extracted and summarized. Then, a comparative analysis of the molecular alterations of IOPN with those of pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasm from reference cohorts (including The Cancer Genome Atlas) was conducted. The key findings from 414 IOPNs were as follows: 1) The male-to-female ratio was 1.5:1. Pancreatic head was the most common site (131/237; 55.3%), but a diffuse tumor extension involving more than one pancreatic segment was described in about 1 out of 5 cases (49/237; 20.6%). The mean size was 45.5 mm. An associated invasive carcinoma was present in 50% of cases (168/336). In those cases, most tumors were pT1 or pT2 and pN0 (>80%), and vascular invasion was uncommon (20.6%). Regarding survival, more than 90% of patients were alive after surgical resection. 2) Immunohistochemical and molecular features were as follows. The most commonly expressed mucins were MUC5AC (110/112; 98.2%) and MUC6 (78/84; 92.8%). Compared with pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasm, the classic pancreatic drivers KRAS, TP53, CDKN2A, SMAD4, and GNAS were less altered in IOPN (P < .01). Moreover, fusions involving PRKACA or PRKACB gene were detected in all of the 68 cases examined, with PRKACB::ATP1B1 being the most common (27/68 cases; 39.7%). These genomic events emerged as an entity-defining molecular alteration of IOPN (P < .01). Thus, such fusions represent a promising biomarker for diagnostic purposes. Recent evidence also suggests their role in influencing the acquisition of oncocytic morphology. IOPN is a distinct pancreatic neoplasm with specific clinicopathologic and molecular features. Considering the clinical or prognostic implications, its recognition is essential for pathologists and, ultimately, patients' management.

Cellular collusion: cracking the code of immunosuppression and chemo resistance in PDAC.

Despite the efforts, pancreatic ductal adenocarcinoma (PDAC) is still highly lethal. Therapeutic challenges reside in late diagnosis and establishment of peculiar tumor microenvironment (TME) supporting tumor outgrowth. This stromal landscape is highly heterogeneous between patients and even in the same patient. The organization of functional sub-TME with different cellular compositions provides evolutive advantages and sustains therapeutic resistance. Tumor progressively establishes a TME that can suit its own needs, including proliferation, stemness and invasion. Cancer-associated fibroblasts and immune cells, the main non-neoplastic cellular TME components, follow soluble factors-mediated neoplastic instructions and synergize to promote chemoresistance and immune surveillance destruction. Unveiling heterotypic stromal-neoplastic interactions is thus pivotal to breaking this synergism and promoting the reprogramming of the TME toward an anti-tumor milieu, improving thus the efficacy of conventional and immune-based therapies. We underscore recent advances in the characterization of immune and fibroblast stromal components supporting or dampening pancreatic cancer progression, as well as novel multi-omic technologies improving the current knowledge of PDAC biology. Finally, we put into context how the clinic will translate the acquired knowledge to design new-generation clinical trials with the final aim of improving the outcome of PDAC patients.

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC.

OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

Integrated procedures for accelerating, deepening, and leading genetic inquiry: A first application on human muscle secretome.

PURPOSE: Beyond classical procedures, bioinformatic-assisted approaches and computational biology offer unprecedented opportunities for scholars. However, these amazing possibilities still need epistemological criticism, as well as standardized procedures. Especially those topics with a huge body of data may benefit from data science (DS)-assisted methods. Therefore, the current study dealt with the combined expert-assisted and DS-assisted approaches to address the broad field of muscle secretome. We aimed to apply DS tools to fix the literature research, suggest investigation targets with a data-driven approach, predict possible scenarios, and define a workflow. METHODS: Recognized scholars with expertise on myokines were invited to provide a list of the most important myokines. GeneRecommender, GeneMANIA, HumanNet, and STRING were selected as DS tools. Networks were built on STRING and GeneMANIA. The outcomes of DS tools included the top 5 recommendations. Each expert-led discussion has been then integrated with an DS-led approach to provide further perspectives. RESULTS: Among the results, 11 molecules had already been described as bona-fide myokines in literature, and 11 molecules were putative myokines. Most of the myokines and the putative myokines recommended by the DS tools were described as present in the cargo of extracellular vesicles. CONCLUSIONS: Including both supervised and unsupervised learning methods, as well as encompassing algorithms focused on both protein interaction and gene represent a comprehensive approach to tackle complex biomedical topics. DS-assisted methods for reviewing existent evidence, recommending targets of interest, and predicting original scenarios are worth exploring as in silico recommendations to be integrated with experts' ideas for optimizing molecular studies.

An Overview of Circulating Biomarkers in Neuroendocrine Neoplasms: A Clinical Guide.

Neuroendocrine neoplasms (NENs) are a heterogeneous group of diseases that are characterized by different behavior and clinical manifestations. The diagnosis and management of this group of tumors are challenging due to tumor complexity and lack of precise and widely validated biomarkers. Indeed, the current circulating mono-analyte biomarkers (such as chromogranin A) are ineffective in describing such complex tumors due to their poor sensitivity and specificity. In contrast, multi-analytical circulating biomarkers (including NETest) are emerging as more effective tools to determine the real-time profile of the disease, both in terms of accurate diagnosis and effective treatment. In this review, we will analyze the capabilities and limitations of different circulating biomarkers focusing on three relevant questions: (1) accurate and early diagnosis; (2) monitoring of disease progression and response to therapy; and (3) detection of early relapse.

Transcriptome analysis of primary sporadic neuroendocrine tumours of the intestine identified three different molecular subgroups.

BACKGROUND: Intestinal neuroendocrine tumours (I-NETs) represent a non-negligible entity among intestinal neoplasms, with metastatic spreading usually present at the time of diagnosis. In this context, effective molecular actionable targets are still lacking. Through transcriptome analysis, we aim at refining the molecular taxonomy of I-NETs, also providing insights towards the identification of new therapeutic vulnerabilities. MATERIALS AND METHODS: A retrospective series of 38 primary sporadic, surgically-resected I-NETs were assessed for transcriptome profiling of 20,815 genes. RESULTS: Transcriptome analysis detected 643 highly expressed genes. Unsupervised hierarchical clustering, differential expression analysis and gene set enriched analysis identified three different tumour clusters (CL): CL-A, CL-B, CL-C. CL-A showed the overexpression of ARGFX, BIRC8, NANOS2, and SSTR4 genes. Its most characterizing signatures were those related to cell-junctions, and activation of mTOR and WNT pathway. CL-A was also enriched in T CD8 + lymphocytes. CL-B showed the overexpression of PCSK1, QPCT, ST18, and TPH1 genes. Its most characterizing signatures were those related to adipogenesis, neuroendocrine metabolism, and splice site machinery-related processes. CL-B was also enriched in T CD4 + lymphocytes. CL-C showed the overexpression of ALB, ANG, ARG1, and HP genes. Its most characterizing signatures were complement/coagulation and xenobiotic metabolism. CL-C was also enriched in M1/2 macrophages. These CL-based differences may have therapeutic implications in refining the management of I-NET patients. At last, we described a specific gene-set for differentiating I-NET from pancreatic NET. DISCUSSION: Our data represent an additional step for refining the molecular taxonomy of I-NET, identifying novel transcriptome subgroups with different biology and therapeutic opportunities.

Extrahepatic Distal Cholangiocarcinoma vs. Pancreatic Ductal Adenocarcinoma: Histology and Molecular Profiling for Differential Diagnosis and Treatment.

Pancreatic ductal adenocarcinoma (PDAC) and distal cholangiocarcinoma (dCCA) are very aggressive tumors with a high mortality rate. Pancreas and distal bile ducts share a common embryonic development. Hence, PDAC and dCCA exhibit similar histological features that make a differential diagnosis during routine diagnostic practice challenging. However, there are also significant differences, with potential clinical implications. Even if PDAC and dCCA are generally associated with poor survival, patients with dCCA seem to present a better prognosis. Moreover, although precision oncology-based approaches are still limited in both entities, their most important targets are different and include alterations affecting BRCA1/2 and related genes in PDAC, as well as HER2 amplification in dCCA. Along this line, microsatellite instability represents a potential contact point in terms of tailored treatments, but its prevalence is very low in both tumor types. This review aims at defining the most important similarities and differences in terms of clinicopathological and molecular features between these two entities, also discussing the main theranostic implications derived from this challenging differential diagnosis.

An Insight on Functioning Pancreatic Neuroendocrine Neoplasms.

Pancreatic neuroendocrine neoplasms (PanNENs) are rare neoplasms arising from islets of the Langerhans in the pancreas. They can be divided into two groups, based on peptide hormone secretion, functioning and nonfunctioning PanNENs. The first group is characterized by different secreted peptides causing specific syndromes and is further classified into subgroups: insulinoma, gastrinoma, glucagonoma, somatostatinoma, VIPoma and tumors producing serotonin and adrenocorticotrophic hormone. Conversely, the second group does not release peptides and is usually associated with a worse prognosis. Today, although the efforts to improve the therapeutic approaches, surgery remains the only curative treatment for patients with PanNENs. The development of high-throughput techniques has increased the molecular knowledge of PanNENs, thereby allowing us to understand better the molecular biology and potential therapeutic vulnerabilities of PanNENs. Although enormous advancements in therapeutic and molecular aspects of PanNENs have been achieved, there is poor knowledge about each subgroup of functioning PanNENs.Therefore, we believe that combining high-throughput platforms with new diagnostic tools will allow for the efficient characterization of the main differences among the subgroups of functioning PanNENs. In this narrative review, we summarize the current landscape regarding diagnosis, molecular profiling and treatment, and we discuss the future perspectives of functioning PanNENs.

Redox-based Disruption of Cellular Hormesis and Promotion of Degenerative Pathways: Perspectives on Aging Processes.

The present work aims to link the redox and cell-centric theories of chronic processes in human biology, focusing on aging. A synthetic overview of cellular redox pathways will be integrated by the concept of hormesis, which disruption leads to several physiopathological processes. The onset of age-related diseases due to the restriction of homeodynamic capacity will be herein considered in a redox fashion. Up-to-date arguments on hormetic agents, such as geroprotectors, dietary interventions, and physical exercise are refining the presented theoretical framework, integrated by insights from extracellular vesicles, microbiota, pollutants, and timing mechanisms. The broad concepts of exposome encompass the redox-based alteration of cellular hormesis for providing meaningful perspectives on redox biogerontology.

Mice lacking growth-associated protein 43 develop cardiac remodeling and hypertrophy.

Growth-associated protein 43 (GAP43) is found in skeletal muscle, localized near the calcium release units. In interaction with calmodulin (CaM), it indirectly modulates the activity of dihydropyridine and ryanodine Ca2+ channels. GAP43-CaM interaction plays a key role in intracellular Ca2+ homeostasis and, consequently, in skeletal muscle activity. The control of intracellular Ca2+ signaling is also an important functional requisite in cardiac physiology. The aim of this study is to define the impact of GAP43 on cardiac tissue at macroscopic and cellular levels, using GAP43 knockout (GAP43-/-) newborn C57/BL6 mice. Hearts from newborn GAP43-/- mice were heavier than hearts from wild-type (WT) ones. In these GAP43-/- hearts, histological section analyses revealed a thicker ventricular wall and interventricular septum with a reduced ventricular chamber area. In addition, increased collagen deposits between fibers and increased expression levels of myosin were observed in hearts from GAP43-/- mice. Cardiac tropism and rhythm are controlled by multiple intrinsic and extrinsic factors, including cellular events such those linked to intracellular Ca2+ dynamics, in which GAP43 plays a role. Our data revealed that, in the absence of GAP43, there were cardiac morphological alterations and signs of hypertrophy, suggesting that GAP43 could play a role in the functional processes of the whole cardiac muscle. This paves the way for further studies investigating GAP43 involvement in signaling dynamics at the cellular level.

The Oxidative Balance Orchestrates the Main Keystones of the Functional Activity of Cardiomyocytes.

This review is aimed at providing an overview of the key hallmarks of cardiomyocytes in physiological and pathological conditions. The main feature of cardiac tissue is the force generation through contraction. This process requires a conspicuous energy demand and therefore an active metabolism. The cardiac tissue is rich of mitochondria, the powerhouses in cells. These organelles, producing ATP, are also the main sources of ROS whose altered handling can cause their accumulation and therefore triggers detrimental effects on mitochondria themselves and other cell components thus leading to apoptosis and cardiac diseases. This review highlights the metabolic aspects of cardiomyocytes and wanders through the main systems of these cells: (a) the unique structural organization (such as different protein complexes represented by contractile, regulatory, and structural proteins); (b) the homeostasis of intracellular Ca2+ that represents a crucial ion for cardiac functions and E-C coupling; and (c) the balance of Zn2+, an ion with a crucial impact on the cardiovascular system. Although each system seems to be independent and finely controlled, the contractile proteins, intracellular Ca2+ homeostasis, and intracellular Zn2+ signals are strongly linked to each other by the intracellular ROS management in a fascinating way to form a "functional tetrad" which ensures the proper functioning of the myocardium. Nevertheless, if ROS balance is not properly handled, one or more of these components could be altered resulting in deleterious effects leading to an unbalance of this "tetrad" and promoting cardiovascular diseases. In conclusion, this "functional tetrad" is proposed as a complex network that communicates continuously in the cardiomyocytes and can drive the switch from physiological to pathological conditions in the heart.

A Protective Strategy to Counteract the Oxidative Stress Induced by Simulated Microgravity on H9C2 Cardiomyocytes.

Microgravity affects human cardiovascular function inducing heart rhythm disturbances and even cardiac atrophy. The mechanisms triggered by microgravity and the search for protection strategies are difficult to be investigated in vivo. This study is aimed at investigating the effects induced by simulated microgravity on a cardiomyocyte-like phenotype. The Random Positioning Machine (RPM), set in a CO2 incubator, was used to simulate microgravity, and H9C2 cell line was used as the cardiomyocyte-like model. H9C2 cells were exposed to simulated microgravity up to 96 h, showing a slower cell proliferation rate and lower metabolic activity in comparison to cell grown at earth gravity. In exposed cells, these effects were accompanied by increased levels of intracellular reactive oxygen species (ROS), cytosolic Ca2+, and mitochondrial superoxide anion. Protein carbonyls, markers of protein oxidation, were significantly increased after the first 48 h of exposition in the RPM. In these conditions, the presence of an antioxidant, the N-acetylcysteine (NAC), counteracted the effects induced by the simulated microgravity. In conclusion, these data suggest that simulated microgravity triggers a concomitant increase of intracellular ROS and Ca2+ levels and affects cell metabolic activity which in turn could be responsible for the slower proliferative rate. Nevertheless, the very low number of detectable dead cells and, more interestingly, the protective effect of NA, demonstrate that simulated microgravity does not have "an irreversible toxic effect" but, affecting the oxidative balance, results in a transient slowdown of proliferation.