Targeted molecular characterization of adult midline and circumscribed gliomas for the identification of new potential targets for personalized therapy

Diffuse midline gliomas (MLG) are primary brain tumours arising from thalamus, hypothalamus, brainstem, cerebellum or spinal cord, mainly occurring in children. In adults, less than 10% of diffuse gliomas arises in midline structures and recent works suggested that this subset of tumours may present with phenotypic and molecular characteristics differing from both pediatric MLG and adult supratentorial gliomas. Circumscribed gliomas (CG) are low-grade tumours but may progress to anaplasia. They have lower genetic complexity than diffuse gliomas and could be better candidate for targeted therapies, when complete surgical resection is not feasible. Unravelling the genomic landscape of MLG and CG will better define the prognostic value of molecular biomarkers and identify new therapeutic strategies that could improve patient care. Adult patients with diagnosis of MLG and CG were retrospectively identified from \uabMaggiore della Carit\ue0\ubb Hospital and GH Piti\ue9-Salp\ueatri\ue8re (Paris). Mutation analysis was performed by Sanger sequencing of the major hot-spots: IDH1, IDH2, H3F3A, HIST1H3B, FGFR1, TERT promoter. FISH analyses of NTRK1-2-3 rearrangements were performed by break-apart probes on tissue microarray of MLG cases. We identified 116 (French) and 47 (Italian) patients. The two cohorts showed a lower percentage of H3F3A mutations (20% vs 33%), the mutation was not associated to a worse prognosis. FGFR1 mutations were identified in 18% of cases and are restricted to MLG. NTRKs analysis in the Italian cohort showed NTRK1 translocations in 15% of cases. We reported a high rate of FGFR1 mutations in optic nerve pilocytic astrocitomas and the presence of alternative BRAF activating mutations (Thr599_Val600insThr and Val600_Lys601>Glu). Our finding of frequent and potentially targetable FGFR1 and BRAF mutations and NTRK1 translocations have important therapeutical implications in the current context of clinical trials, and further reinforces the need for molecular analyses

Frequent detection of Merkel cell polyomavirus DNA in tissues from 10 consecutive autopsies

Merkel cell polyomavirus (MCPyV) has been identified in samples of Merkel cell carcinoma (MCC), an aggressive skin cancer. Seroepidemiologic studies indicated a high frequency of MCPyV infection in humans, suggesting respiratory and faecal–oral routes, or transmission by skin contact. Since MCC is more frequent in immunocompromised patients, a reactivation of MCPyV latently infecting target cells has been proposed. However, neither definite ways of transmission nor specific target organs have been identified with certainty. Ten autopsies with an extensive organ sampling for a total of 121 specimens (tissue and blood samples) were collected. All tissue specimens were fixed in formalin and embedded in paraffin. Real-time PCR was performed to quantify the copy number of the large T antigen (LT) gene and the capsid VP1 gene of MCPyV. MCPyV LT and/or VP genes were detected in all of the collected specimens. A high prevalence of MCPyV was found in the blood (six cases) and lung (five cases); the brain was positive in three cases. The highest viral copy number was detected in blood from two autopsies (21 610 570.09 copies per 105 cells and 380 413.25 copies per 105 cells), whereas the viral copy number in the other organs was low. Our data confirm the high frequency of MCPyV infection in the general population, which seems to indicate that the respiratory tract is a possible route for viral transmission and viral persistence in the brain. The frequent detection of MCPyV DNA in blood suggests that circulating leukocytes could be one of the reservoirs of MCPyV, whereas the high viral copy number also seems to indicate the possibility of viral reactivation in immunocompetent adults

Immunogenicity of SARS-CoV-2 vaccination in patients undergoing autologous stem cell transplantation. A multicentric experience

COVID-19 disease has a strong impact on hematological patients; those receiving autologous hematopoietic stem cell transplantation (aHSCT) represent a particularly vulnerable group, in which the effectiveness of vaccination is very variable. Chiarucci et al. showed that patients affected by non-Hodgkin lymphoma (NHL) and treated with rituximab experienced a lower rate of immunization against SARS-CoV-2 (54%), as well as significantly lower IgG antibody titers. In our multicenter retrospective observational study, we included 82 patients who underwent aHSCT, divided into two groups: 58 patients vaccinated after aHSCT (group A) and 24 vaccinated before getting transplantation (group B). In group A, 39 (67%) patients had positive serology, and the rate of positivity increased with time after aHSCT. In the subgroup of patients with NHL, the administration of rituximab predicted negative serology, particularly when administered in the 6 months before vaccination (13% response rate). Patients affected by plasma cells had a higher rate of positivity (83% overall), independently of the time to aHSCT. In group B, no patient who initially showed positive serology became negative after transplantation, so the aHSCT did not affect the response to the vaccination. Our study confirmed the role of rituximab as a negative predictor of response to SARS-CoV-2 vaccination, whereas the conditioning and transplantation procedure itself seemed to be less important

β-HPV infection correlates with early stages of carcinogenesis in skin tumors and patient-derived xenografts from a kidney transplant recipient cohort

Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active \u3b2-HPV infection in skin tumors from KTRs through the detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the \u3b2-genotypes, we were able to visualize infection in five tumors [one keratoacanthoma (KA), three actinic keratosis (AK) and one seborrheic keratosis (SK)] that were all removed from two patients who had been both transplanted twice, had developed multiple KC, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the \u394Np63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed \u3b2-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both \u3b2-HPV infection and \u394Np63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study represent the first proof-of-concept that \u3b2-HPV+ and \u394Np63+ intraepidermal hyperplasia can indeed progress to an aggressive SCC able to metastasize. Consistent with a series of reports attributing a causative role of \u3b2-HPV at early stages of skin carcinogenesis through \u394Np63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these \u3b2-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression

Structured reporting for fibrosing lung disease: a model shared by radiologist and pulmonologist

Objectives: To apply the Delphi exercise with iterative involvement of radiologists and pulmonologists with the aim of defining a structured reporting template for high-resolution computed tomography (HRCT) of patients with fibrosing lung disease (FLD). Methods: The writing committee selected the HRCT criteria\ue2\u80\u94the Delphi items\ue2\u80\u94for rating from both radiology panelists (RP) and pulmonology panelists (PP). The Delphi items were first rated by RPs as \ue2\u80\u9cessential\ue2\u80\u9d, \ue2\u80\u9coptional\ue2\u80\u9d, or \ue2\u80\u9cnot relevant\ue2\u80\u9d. The items rated \ue2\u80\u9cessential\ue2\u80\u9d by < 80% of the RP were selected for the PP rating. The format of reporting was rated by both RP and PP. Results: A total of 42 RPs and 12 PPs participated to the survey. In both Delphi round 1 and 2, 10/27 (37.7%) items were rated \ue2\u80\u9cessential\ue2\u80\u9d by more than 80% of RP. The remaining 17/27 (63.3%) items were rated by the PP in round 3, with 2/17 items (11.7%) rated \ue2\u80\u9cessential\ue2\u80\u9d by the PP. PP proposed additional items for conclusion domain, which were rated by RPs in the fourth round. Poor consensus was observed for the format of reporting. Conclusions: This study provides a template for structured report of FLD that features essential items as agreed by expert thoracic radiologists and pulmonologists

Lipid-induced ASK1 activation in hepatocytes and Kupffer cells mediates the increased vulnerability of fatty liver to Ischemia/Reperfusion injury in mice

Lipid-induced ASK1 activation in hepatocytes and Kupffer cells mediates the increased vulnerability of fatty liver to ischemia/reperfusion injury in mice Chiara Imarisio1*, Elisa Alchera1*, Bangalore R Chandrashekar1*, Guido Valente2, Antonia Follenzi1, Elena Trisolini1, Renzo Boldorini1 and Rita Carini1 [email protected], [email protected], [email protected], [email protected], [email protected] [email protected], [email protected], [email protected] 1Department of Health Science, University of Piemonte Orientale, 28100 Novara, Italy. 2Department of Translational Medicine, University of Piemonte Orientale, 28100 Novara, Italy. * These Authors equally contributed to the study ABSTRACT Background & Aims: Steatosis enhances parenchymal injury and inflammation in liver exposed to ischemia/reperfusion (I/R). Several alterations, such as ER stress and increased ROS production are associated to such effects but a final and targetable pathogenic mechanism is still undetermined. This study investigates “in vitro” and “in vivo” a molecular mediator of the interplay among ER stress, ROS and cytotoxic/survival pathways and its role in the susceptibility of steatotic hepatocytes (HP) and Kupffer cells (KC) to hypoxia/reoxygenation (H/R) and of fatty liver to I/R injury and inflammation. Methods: Control or steatotic (treated with palmitic acid, PA) primary mouse HP and KC were exposed to H/R to “in vitro” simulate I/R exposure. C57BL/6 mice fed 9 weeks with control or High Fat diet underwent to a non-lethal partial hepatic I/R. Results: In HP, PA increases H/R damage, induces ROS production and enhances the stimulation of the ASK1/JNK cytotoxic axis activated by the ER stress mediator TRAF2 during H/R. Prevention of ROS production nullifies the increased susceptibility of HP to H/R and the enhanced ASK1/JNK activation. ASK1 inhibition also completely protects JNK activation and HP damage. In KC, PA alone induces TRAF2 and a consequent ASK1 and p38 MAPK activation. PA also increases KC damage induced by H/R, but oppositely to HP, ASK1 inhibition enhances H/R damage by preventing the stimulation of the survival mediator p38 MAPK. In mice liver, steatosis induces the expression of activated ASK1 in KC, whereas upon I/R exposure, activated ASK1 expression is evident in both in KC and HP. “In vivo”, ASK1 inhibition prevents ASK1, JNK and p38 MAPK activation and protects fatty mice liver from I/R-induced transaminases release and from the increase of TNF-alpha and iNOS. Conclusions: Our results show that: 1) Lipids increase ASK1/JNK activation induced by ER in HP by rising cellular ROS; 2) Lipids activate ASK1/p38 MAPK in KC by promoting ER stress; 3) ASK1 is cytoxic for HP and protective for KC; 4) ASK1 inhibition protects I/R injury and inflammation of fatty liver. These observations indicate that steatosis, by stimulating ASK1, contextually promotes I/R induced liver injury and inflammation by increasing HP damage and protecting the resident hepatic macrophages (KC) and evidence the potentiality of ASK1 inhibitors as novel therapeutic agents to prevent hepatic damage and reduce inflammatory reactions consequent to fatty liver surgery

Lipid-induced ASK1 activation in hepatocytes and Kupffer cells mediates the increased vulnerability of fatty liver to ischemia/reperfusion injury in mice

Lipid-induced ASK1 activation in hepatocytes and Kupffer cells mediates the increased vulnerability of fatty liver to ischemia/reperfusion injury in mice Chiara Imarisio1*, Elisa Alchera1*, Bangalore R Chandrashekar1*, Guido Valente2, Antonia Follenzi1, Elena Trisolini1, Renzo Boldorini1 and Rita Carini1 [email protected], [email protected], [email protected], [email protected], [email protected] [email protected], [email protected], [email protected] 1Department of Health Science, University of Piemonte Orientale, 28100 Novara, Italy. 2Department of Translational Medicine, University of Piemonte Orientale, 28100 Novara, Italy. * These Authors equally contributed to the study ABSTRACT Background & Aims: Steatosis enhances parenchymal injury and inflammation in liver exposed to ischemia/reperfusion (I/R). Several alterations, such as ER stress and increased ROS production are associated to such effects but a final and targetable pathogenic mechanism is still undetermined. This study investigates “in vitro” and “in vivo” a molecular mediator of the interplay among ER stress, ROS and cytotoxic/survival pathways and its role in the susceptibility of steatotic hepatocytes (HP) and Kupffer cells (KC) to hypoxia/reoxygenation (H/R) and of fatty liver to I/R injury and inflammation. Methods: Control or steatotic (treated with palmitic acid, PA) primary mouse HP and KC were exposed to H/R to “in vitro” simulate I/R exposure. C57BL/6 mice fed 9 weeks with control or High Fat diet underwent to a non-lethal partial hepatic I/R. Results: In HP, PA increases H/R damage, induces ROS production and enhances the stimulation of the ASK1/JNK cytotoxic axis activated by the ER stress mediator TRAF2 during H/R. Prevention of ROS production nullifies the increased susceptibility of HP to H/R and the enhanced ASK1/JNK activation. ASK1 inhibition also completely protects JNK activation and HP damage. In KC, PA alone induces TRAF2 and a consequent ASK1 and p38 MAPK activation. PA also increases KC damage induced by H/R, but oppositely to HP, ASK1 inhibition enhances H/R damage by preventing the stimulation of the survival mediator p38 MAPK. In mice liver, steatosis induces the expression of activated ASK1 in KC, whereas upon I/R exposure, activated ASK1 expression is evident in both in KC and HP. “In vivo”, ASK1 inhibition prevents ASK1, JNK and p38 MAPK activation and protects fatty mice liver from I/R-induced transaminases release and from the increase of TNF-alpha and iNOS. Conclusions: Our results show that: 1) Lipids increase ASK1/JNK activation induced by ER in HP by rising cellular ROS; 2) Lipids activate ASK1/p38 MAPK in KC by promoting ER stress; 3) ASK1 is cytoxic for HP and protective for KC; 4) ASK1 inhibition protects I/R injury and inflammation of fatty liver. These observations indicate that steatosis, by stimulating ASK1, contextually promotes I/R induced liver injury and inflammation by increasing HP damage and protecting the resident hepatic macrophages (KC) and evidence the potentiality of ASK1 inhibitors as novel therapeutic agents to prevent hepatic damage and reduce inflammatory reactions consequent to fatty liver surgery

Oxidative and ER stress-dependent ASK1 activation in steatotic hepatocytes and Kupffer cells sensitizes mice fatty liver to ischemia/reperfusion injury.

Steatosis intensifies hepatic ischemia/reperfusion (I/R) injury increasing hepatocyte damage and hepatic inflammation. This study evaluates if this process is associated to a differential response of steatotic hepatocytes (HP) and Kupffer cells (KC) to I/R injury and investigates the molecular mechanisms involved. Control or steatotic (treated with 50 \u3bcmol palmitic acid, PA) mouse HP or KC were exposed to hypoxia/reoxygenation (H/R). C57BL/6 mice fed 9 week with control or High Fat diet underwent to partial hepatic IR. PA increased H/R damage of HP and further activated the ASK1-JNK axis stimulated by ER stress during H/R. PA also induced the production of oxidant species (OS), and OS prevention nullified the capacity of PA to increase H/R damage and ASK1/JNK stimulation. ASK1 inhibition prevented JNK activation and entirely protected HP damage. In KC, PA directly activated ER stress, ASK1 and p38 MAPK and increased H/R damage. However, in contrast to HP, ASK1 inhibition further increased H/R damage by preventing p38 MAPK activation. In mice liver, steatosis induced the expression of activated ASK1 in only KC, whereas I/R exposure of steatotic liver activated ASK1 expression also in HP. "In vivo", ASK1 inhibition prevented ASK1, JNK and p38 MAPK activation and protected I/R damage and expression of inflammatory markers. CONCLUSIONS: Lipids-induced ASK1 stimulation differentially affects HP and KC by promoting cytotoxic or protective signals. ASK1 increases H/R damage of HP by stimulating JNK and protects KC activating p38MAPK. These data support the potentiality of the therapeutic employment of ASK1 inhibitors that can antagonize the damaging effects of I/R upon fatty liver surgery by the contextual reduction of HP death and of KC-mediated reactions