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Anti-glioma activity of MFF peptidomimetic

MFF1 synthetic peptides used in this study

Morphology of MFF targeting in tumor cells

MFF-VDAC1 complex in cancer

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non–small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF–VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF–VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer.Significance:These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF–VDAC complex and displays anticancer activity in multiple tumor models.See related commentary by Rao, p. 6074

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A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

Healthcare workers experienced high degree of stress during COVID-19. Purpose of the present article is to compare mental health (depressive and Post-Traumatic-Stress-Disorders—PTSD—symptoms) and epigenetics aspects (degree of methylation of stress-related genes) in front-line healthcare professionals versus healthcare working in non-COVID-19 wards. Sixty-eight healthcare workers were included in the study: 39 were working in COVID-19 wards (cases) and 29 in non-COVID wards (controls). From all participants, demographic and clinical information were collected by an ad-hoc questionnaire. Depressive and PTSD symptoms were evaluated by the Patient Health Questionnaire-9 (PHQ-9) and the Impact of Event Scale—Revised (IES-R), respectively. Methylation analyses of 9 promoter/regulatory regions of genes known to be implicated in depression/PTSD (ADCYAP1, BDNF, CRHR1, DRD2, IGF2, LSD1/KDM1A, NR3C1, OXTR, SLC6A4) were performed on DNA from blood samples by the MassARRAY EpiTYPER platform, with MassCleave settings. Controls showed more frequent lifetime history of anxiety/depression with respect to cases (χ2 = 5.72, p = 0.03). On the contrary, cases versus controls presented higher PHQ-9 (t = 2.13, p = 0.04), PHQ-9 sleep item (t = 2.26, p = 0.03), IES-R total (t = 2.17, p = 0.03), IES-R intrusion (t = 2.46, p = 0.02), IES-R avoidance (t = 1.99, p = 0.05) mean total scores. Methylation levels at CRHR1, DRD2 and LSD1 genes was significantly higher in cases with respect to controls (p p = 0.03 and p = 0.03, respectively). Frontline health professionals experienced more negative effects on mental health during COVID-19 pandemic than non-frontline healthcare workers. Methylation levels were increased in genes regulating HPA axis (CRHR1) and dopamine neurotransmission (DRD2 and LSD1), thus supporting the involvement of these biological processes in depression/PTSD and indicating that methylation of these genes can be modulated by stress conditions, such as working as healthcare front-line during COVID-19 pandemic.

Purpose: The identification of factors predicting the benefit of treatments is a major goal for medical oncologists. Methods We retrospectively investigated in metastatic tumor tissues of women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed ESR1 and PI3KCA mutations and miRNA profiles.Results: Fifty-nine patients were evaluable (median age 67 yrs, range 32-92). Overall mPFS was 10.7 months (range 2.2-14) while 18-month PFS rate was 27%; only the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months (OR=0.25 95%CI 0.07-0.9 p=.035). PI3KCA mutations were found in 36% of patients and were associated with a numerically shorter mPFS but not with a lower likelihood of being progression-free at 18 months. As of miRNAs, miR-549a, miR-644a, miR-16-5p were negatively associated with 18-month PFS while let-7c-5p was positively associated with outcome. In addition, miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of predicted targets suggested that miR-520d-3p and miR-548g-3p decrease might induce Hippo and Wnt signaling, while high levels of miR-603, mir-181a-5p,miR-199a-miR-199b-3p might repress endocrine resistance thus potentially promoting the response to treatment. Conclusions: Our clinical findings are consistent with literature data, while the mutation and miRNA results provide some clues on the molecular mechanisms involved in fulvestrant activity and resistance to be confirmed in larger cohorts of breast cancer patients.

Despite the approval of pembrolizumab in all tumors showing mismatch-repair (MMR) deficiency and/or microsatellite instability (MSI), there are currently no companion diagnostics for MMR status assessment in breast cancer. Here, we sought to define the diagnostic and prognostic role of MMR and MSI testing in breast cancer patients. We subjected 444 breast cancers to MMR immunohistochemistry (IHC) and MSI analysis. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and MMR-heterogeneous (hMMR) based on the loss of immunoreactivity; MSI was defined by the instability in the five indicators recommended by the National Cancer Institute for endometrial and colorectal cancers. Correlation of MMR status with patients' survival was assessed using the Kaplan-Meier estimator. In 75 patients (17%) the loss of MMR proteins was homogeneous, classified as dMMR, while 55 cases (12%) were hMMR. The prevalence of cancers with loss of the MMR proteins was homogeneous across ER+ breast cancers (15-19% for dMMR and 10-18% for hMMR tumors). The level of overlap between IHC and MSI analysis was 9% (p Our study documents the clinical impact of MMR testing in a large series of breast cancers, using the most commonly adopted diagnostic tools and criteria. We show that MMR protein loss is a rather common event in breast cancer and has a remarkable degree of intra-tumor heterogeneity, therefore making the analysis of a small area of the tumor, or a small biopsy, of little clinical value. Our investigation supports the concept that MSI occurs rarely in breast cancer and demonstrate that this condition is restricted to a minority of tumors with MMR protein loss. These data suggest that MMR IHC and MSI analysis should not be considered as interchangeable tests in the diagnostic workup of breast carcinomas. Finally, our observations indicate that the complete loss of at least one of the MMR proteins assessed by IHC is able to identify high-risk ER+/HER2- breast cancers that can potentially benefit from pembrolizumab therapy, whereas first-line chemotherapy shows comparatively good results in dMMR ER-/HER2- breast cancers. Citation Format: Fusco N, Lopez G, Corti C, Pesenti C, Colapietro P, Ercoli G, Gaudioso G, Faversani A, Gambini D, Despini L, Blundo C, Vaira V, Miozzo M, Ferrero S, Bosari S. Mismatch repair protein loss is a prognostic and predictive biomarker in breast cancers regardless of microsatellite instability [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-08-21.

Background: The Anaplastic Lymphoma Kinase (ALK) gene is known to be affected by several genetic alterations, such as rearrangement, amplification and point mutation. The main goal of this study was to comprehensively analyze ALK amplification (ALK-A) and ALK gene copy number gain (ALK-CNG) in a large cohort of non-small-cell lung cancer (NSCLC) patients in order to evaluate the effects on mRNA and protein expression. Methods: ALK locus number status was evaluated in 578 NSCLC cases by fluorescence in situ hybridization (FISH). In addition, ALK immunohistochemistry and ALK mRNA in situ hybridization were performed. Results: Out of 578 cases, 17 cases showed ALK-A. In addition, 14 cases presented ALK-CNG and 72 cases presented chromosome 2 polyploidy. None of those carrying ALK-A and -CNG showed either ALK immunohistochemical expression or ALK mRNA expression through in situ hybridization. We observed a high frequency of extra copies of the ALK gene. Conclusions: Our findings demonstrated that ALK-A is not involved in mRNA production and consequently is not involved in protein production, these findings support the hypothesis that ALK-A might not play a role in the pathogenesis of NSCLC, underlining the absence of a specific clinical application.

Alterations in the tumor suppressor phosphatase and tensin homolog (PTEN) occur in a substantial proportion of solid tumors. These events drive tumorigenesis and tumor progression. Given its central role as a downregulator of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, PTEN is deeply involved in cell growth, proliferation, and survival. This gene is also implicated in the modulation of the DNA damage response and in tumor immune microenvironment modeling. Despite the actionability of PTEN alterations, their role as biomarkers remains controversial in clinical practice. To date, there is still a substantial lack of validated guidelines and/or recommendations for PTEN testing. Here, we provide an update on the current state of knowledge on biologic and genetic alterations of PTEN across the most frequent solid tumors, as well as on their actual and/or possible clinical applications. We focus on possible tailored schemes for cancer patients’ clinical management, including risk assessment, diagnosis, prognostication, and treatment.

Background Mitochondrial functions are exploited in cancer and provide a validated therapeutic target. However, how this process is regulated has remained mostly elusive and the identification of new pathways that control mitochondrial integrity in cancer is an urgent priority. Methods We studied clinically-annotated patient series of primary and metastatic prostate cancer, representative cases of multiple myeloma (MM) and publicly available genetic databases. Gene regulation studies involved chromatin immunoprecipitation, PCR amplification and Western blotting of conditional Myc-expressing cell lines. Transient or stable gene silencing was used to quantify mitochondrial functions in bioenergetics, outer membrane permeability, Ca2+ homeostasis, redox balance and cell death. Tumorigenicity was assessed by cell proliferation, colony formation and xenograft tumour growth. Findings We identified Mitochondrial Fission Factor (MFF) as a novel transcriptional target of oncogenic Myc overexpressed in primary and metastatic cancer, compared to normal tissues. Biochemically, MFF isoforms, MFF1 and MFF2 associate with the Voltage-Dependent Anion Channel-1 (VDAC1) at the mitochondrial outer membrane, in vivo. Disruption of this complex by MFF silencing induces general collapse of mitochondrial functions with increased outer membrane permeability, loss of inner membrane potential, Ca2+ unbalance, bioenergetics defects and activation of cell death pathways. In turn, this inhibits tumour cell proliferation, suppresses colony formation and reduces xenograft tumour growth in mice. Interpretation An MFF-VDAC1 complex is a novel regulator of mitochondrial integrity and actionable therapeutic target in cancer.

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non–small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF–VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF–VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer. Significance: These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF–VDAC complex and displays anticancer activity in multiple tumor models. See related commentary by Rao, p. 6074

The role of microRNAs (miRNAs) in celiac disease (CD) is unclear. We evaluated inflammation-related miRNA-146a, miRNA-155, miRNA-21, and miRNA-125b expression in peripheral blood and intestinal mucosa of CD adults. Thirty patients with CD were included: patients with active CD on a gluten-containing diet (CD-active, n = 10), patients on a gluten-free diet (for at least 1 year), and patients with negative blood antibodies (CD-inactivePE, n = 10). In addition, ten healthy volunteers formed the comparison/control group. MiRNA expression was measured in duodenal biopsies from patients (CD-inactiveMU, n = 10) after in vitro exposure to PT gliadin and 33-mer peptide. MiRNAs expression was measured in plasma and in peripheral blood mononuclear cells (PBMCs) and monocytes, before and after in vitro exposure to native gliadin (gliadinN). Expression levels of miRNA-146a, miRNA-155, and miRNA-21 in PBMCs, miRNA-155 in monocytes and miRNA-155, miRNA-21, and miRNA-125b in plasma were elevated in both groups of celiac patients. After in vitro exposure with gliadinN, miRNA-146a and miRNA-155 expression markedly increased in PBMCs and monocytes, while miRNA-155 and miRNA-21 increased in the CD-active group. MiRNAs expression in intestinal mucosa did not change. MiRNA-146a and miRNA-155 expression showed high sensitivity and specificity for the presence of CD, irrespective of the current dietary treatment. Selected inflammation-related miRNAs expression is elevated in the peripheral blood of celiac. This suggests their participation in the immune processes underlying the pathology. Their similar response in active and inactive CD suggests that they should be further evaluated, as potential diagnostic biomarkers for CD.

Supplemental Digital Content is available in the text., Background. Primary graft dysfunction, infections, and acute rejection (AR) worsen lung transplantation (LTx) outcome and patient survival. Despite significant efforts, reliable biomarkers of acute lung allograft dysfunction are lacking. To address this issue, we profiled the bronchoalveolar lavage (BAL) miRNome in LTx patients. Methods. BAL-microRNAs (miRNAs) from 16 patients were collected 7 days (T0), 15 days (T1), and 3 months (T2) after bilateral LTx and profiled on low-density array. Unsupervised and supervised analyses were used to identify miRNAs associated with clinical features, pneumonia, or AR. Prognostic markers were identified using the Cox model. Targeted signaling pathways were predicted in silico. A second series of 11 patients were used to validate AR-associated miRNAs. Results. Variation in BAL-miRNAs was associated with acute lung allograft dysfunction. Increased levels of miR-23b-3p at T2 were detected in patients with pneumonia, whereas let-7f-5p, miR-146b-3p, miR-22-5p, miR-29c-5p, miR-362-5p, and miR-452-5p were upregulated at T2 in patients with AR. miR-148b-5p and miR-744-3p distinguished LTx patients with AR in both cohorts. Low miR-148b-5p and high miR-744-3p expression levels were significantly associated with a shorter time to AR either within the first year after LTx or during follow-up. Combination of the 2 miRNAs identified LTx patients with higher AR risk independently of clinical variables. Conclusions. Our data provide new insights into the roles of BAL-miRNAs in regulating the pulmonary environment after transplantation and suggest that these miRNAs could serve as biomarkers of early- or mid-stage events. If validated, these findings could pave the way to a personalized clinical approach in LTx patients.

Purpose In a previous study on miRNA profiling in bronchoalveolar lavage (BAL) fluid of lung transplanted (LTx) patients we obtained signatures of deregulated miRNAs associated to acute lung allograft dysfunction (ALAD). Now we extended this analysis to chronic dysfunction (CLAD) and to pathways potentially modulated by AR- or CLAD-miRNAs. Methods BALs were obtained from 16 patients who underwent bilateral LTx at our Institution. Mean follow-up time was 21 months (range 17-25). MiRNAs expression in BAL samples collected 7 days (T0), 15 days (T1) and 3 months (T2) after LTx was analyzed using TLDA. The association of miRNAs with pneumonia, acute rejection (AR) or CLAD was examined using ANOVA or Cox regression. The false discovery rate was applied for multiple comparison correction. miRNAs targets were search with miRTarget Link and imported in STRING or WebGestalt databases for functional annotation. Potential targets expression was analyzed by immunohistochemistry (IHC) on transbrochial biopsies. Results We identified one, 9 and 4 BAL-miRNAs whose upregulation is associated with pneumonia, AR or CLAD respectively (Fig. 1A-C). At T1, six BAL-miRNAs predicted CLAD by Cox model. Pathways prediction revealed that AR- or CLAD-miRNAs regulated cell dedifferentiation or WNT signaling, respectively, through the modulation of Sp1 or Bcl2 and cMYC factors (Fig. 1D,E). By IHC, Sp1 was more expressed in biopsies from patients with AR (Fig. 1F) whereas Bcl2 was predominantly detected in lungs from patients with CLAD (Fig. 1G). Conclusion Our results, although preliminary, uncover novel miRNAs-networks involved in AR and CLAD pathogenesis.

An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC). This analysis is crucial for the selection of patients with advanced-stage tumors eligible for treatment with pembrolizumab and potentially with other anti-PD-1/PD-L1 checkpoint inhibitors. This multicentric retrospective study evaluated the reproducibility of PD-L1 testing in the Italian scenario both for closed and open platforms. In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche’s Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. With regard to the performances by using open platforms, the combination of 22C3 with Dako Omnis or Benchmark obtained good results basically, while the 28,8 clone seemed to be associated with worse scores.

Background The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF).

Background Breast cancers that harbor mismatch-repair (MMR) deficiency and/or microsatellite instability (MSI) might be sensitive to immune checkpoint blockade, but there are currently no specific guidelines for assessing MMR status in breast cancer. Here, we sought to define the clinical value of MMR immunohistochemistry (IHC) and MSI analysis in breast cancers. Methods We subjected 444 breast cancers to MMR IHC and MSI analysis. Cases were classified as MMR-proficient (pMMR), MMR-deficient (dMMR), and MMR-heterogeneous (hMMR) based on the loss of immunoreactivity; MSI was defined by instability in the five indicators recommended by the National Cancer Institute for endometrial and colorectal cancers. Correlation of MMR status with patients’ survival was assessed using the Kaplan-Meier estimator. Statistical tests were two-sided. Results Loss of MMR proteins was homogeneous (dMMR) in 75 patients (17%) and heterogeneous (hMMR) in 55 (12%). Among luminal breast cancers, there were similar frequencies of dMMR and hMMR tumors. Overall, the rate of discrepancy between IHC and MSI analysis was high (91%). Women with Luminal B-like dMMR carcinomas (n = 44) showed shorter overall survival (median = 77 months, range = 0–115 months) than those with pMMR (n = 205) or hMMR (n = 35) tumors (median = 84 months, range = 0–127 months) (P = .008). On the contrary, patients with estrogen receptor-negative breast cancers treated with chemotherapy lived longer in cases of dMMR (n = 9) than pMMR (n = 33) or hMMR (n = 7) tumors, with 87 months of median survival (range = 73–123 months) for the former compared with 79 months (range = 8–113 months) for the latter two categories (P Conclusions Immunohistochemistry and MSI are not interchangeable tests in breast carcinomas. MMR protein loss is a more common event than MSI and shows intra-tumor heterogeneity. MMR IHC allows the identification of clinically relevant subclasses of breast cancer patients, provided that multiple areas of the tumor are analyzed.

Background Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients' cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0·03, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund This work was supported by Fondazione Cariplo (2014–1148 to VV), Fondazione IRCCS Ca' Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV).

Background Gliadins are involved in gluten-related disorders and are responsible for the alteration of the cellular redox balance. It is not clear if the gliadin-related oxidative stress can induce DNA damage in enterocytes. Aim To investigate any possible genotoxicity caused by gliadin and to assess its relationship with oxidative stress in vitro and ex vivo. Methods Caco-2 cells were exposed for 6–12–24 h to increasing concentrations (250 μg/mL–1000 μg/mL) of digested gliadin. We investigated: cytotoxicity, oxidative balance (reactive oxygen species, ROS), DNA damage (comet assay and γ-H2AX detection), transglutaminase type 2 (TG2) activity and annexin V expression. H2AX and 8-OHG immunohistochemistry has been evaluated on duodenal biopsies of celiac subjects and controls. Results Gliadin induced a significant increase (+50%) of ROS after 12 h of exposition starting with a 500 μg/mL dose of gliadin. Comet assay and γ-H2AX demonstrated DNA damage, evident at the gliadin concentration of 500 μg/mL after 24 h. TG2 activity increased in chromatin and cytoskeleton cellular compartments at different gliadin doses (250/500/1000 μg/mL). The γ-H2AX and 8-OHG immunohistochemistry was altered in the duodenal biopsies of celiac patients. Conclusions Gliadin induces cellular oxidative stress, DNA damage and pro-apoptotic stimulation in Caco-2 cells and in the duodenal mucosa of celiac patients.

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Funding Statement: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Primary culture were obtained from fresh glioma specimens using surgical surplus of neoplastic or non-neoplastic tissue. All patients received a gross total resection of the tumor at Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (Milan, Italy) and no patient received neoadjuvant therapy. The study was approved by an Institutional Review Board (IRB#275/2013) and signed informed consent was obtained from all enrolled subjects.

Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. We investigated deregulation of V-ATPase and associated signaling in aggressive gliomas. In vivo, we found that V-ATPase subunit expression correlates with glioma growth. Using patients' series, we observed that glioblastoma (GBM) and relapsed gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis. Molecularly, GBM-like V-ATPase expression correlated with switch in epigenetic enzymes and upregulation of homeobox genes. GBM neurospheres influenced the non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via large oncosomes (LO). LO reprogrammed recipient cells to grow as spheres and to migrate. Moreover, LO were particularly abundant in the circulation of GBM and of patients who died during follow-up. Finally, impairment of V-ATPase reduced LO activity. These data identify a global cell fate change that underlies glioma aggressiveness and suggest new entry points for glioma stratification, follow-up, and therapy. Funding Statement: This work was supported by Fondazione Cariplo (2014-1148 to VV), Fondazione IRCCS Ca’ Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV) and by the Ricerca Corrente program 2017 (to SF). AMS and MF were supported by a Fellowship from the Doctorate School in Molecular and Translational Medicine of Milan University. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Animal experiments were carried out in compliance with the institutional guidelines for the care and use of experimental animals (European Directive 2010/63/UE and the Italian law 26/2014), authorized by the Italian Ministry of Health and approved by the Animal Use and Care Committee of the University of Milan. The patient series of the study: The study was approved by an Institutional Review Board (IRB#275/2013) and signed informed consent was obtained from all patients.

Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers., Breast cancer tumors originating from mammary luminal epithelial cells are highly heterogeneous. Here, the authors show MYC-driven tumor initiation is reliant on cell reprogramming via an epigenetic program which leads to mammary luminal epithelial cells acquiring basal/stem cell-like properties.

Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes resulted difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Over-expression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.

Purpose We previously showed that microRNAs (miRNA) discriminate patients with primary graft dysfunction (PGD) from healthy subjects after bilateral lung transplantation . Now we investigated whether miRNAs measured at different time points could identify patients who undergo acute rejection (AR) in the first year after transplant. Methods Patients who underwent bilateral lung transplantation at our Institution between May and Dicember 2017 were included (n=16). Mean follow-up time was 9 months. MiRNAs expression in BAL samples collected 7 days (T0), 15 days (T1) and 3 months (T2) after surgery during routine bronchoscopy was analyzed using TLDA platform. The association of miRNAs with clinical variables, PGD (any grade) within the first 72 hours after transplantation, and acute rejection was examined using both unsupervised and supervised tests (R studio). Results Globally, miRNAs show a drop in expression at later time points respect to T0 (Fig.1A). By unsupervised principal component analysis (PCA), miRNA profiles obtained at T0 discriminate patients according to patients’ disease, the Oto score and PGD (Fig. 1B), whereas T1-miRNAs discriminate patients with PGD (Fig.1C). Interestingly T2-miRNAs discriminate patients according to AR (Fig. 1D). More importantly, we identified a signature of 14 miRNAs measured at T0 that distinguish patients who develop AR during follow-up (Fig.1E). Conclusion Our results show that miRNAs are novel non-invasive molecular markers associated with lung transplantation pathogenesis and outcome, with the potential to detect patients at higher risk of complications at both early (such as PGD) and late (such as AR) time points.

The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted ‘Division of Pathology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy’. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as ‘Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico “P.Giaccone”, Università di Palermo, Palermo, 90127, Italy’. The correct affiliation is ‘Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy’. These errors have now been corrected in both the PDF and HTML versions of the Article.

Aims: The role of tumour metabolic and proliferative indices in predicting non-small-cell lung cancer (NSCLC) patients' prognosis is unclear. We correlated fluorine 18 ((18) F)-fluorodeoxyglucose (FDG)-positron emission tomography (PET) value and Ki67 index to patients' survival, taking into account tumour heterogeneity, disease characteristics and genetic aberrations. Methods and results: A series of 383 NSCLCs was arranged into tissue microarrays and Ki67 staining was analysed by immunohistochemistry. The maximum standardized uptake (SUV(MAX) ) value detected by (18) F-FDG-PET analysis was calculated over a region of interest. Large-cell and squamous cell carcinomas had higher proliferative and metabolic activities than adenocarcinomas, and the two measures were correlated significantly. The hot-spot Ki67 value was correlated with patients' survival and the cut-off to discriminate patients in the survival risk groups was 20%. Ki67 hot-spot values were greater in anaplastic lymphoma kinase (ALK) rearranged tumours. Adenocarcinomas showed the highest intratumour heterogeneity in proliferative activity and the hot-spot Ki67 value predicted only the prognosis of patients in this group. Although tumour metabolic activity was not associated with patients' prognosis, a SUV(MAX) > 2 was related to nodal metastases, tumour size and grade. Conclusions: Our results highlight how tumour heterogeneity influences evaluation of prognostic biomarkers. Our data support Ki67 evaluation to estimate NSCLC patients' prognosis, particularly for adenocarcinoma.

The coexistence of mesothelioma and other primary malignancies has been previously reported in literature, but the finding of a pleural mesothelioma with a synchronous peritoneal mesothelioma has not been reported so far. We report a case of a 58-years-old woman that came to our attention for the incidental finding of an inguinal mass. Fine-needle biopsies of the mass and a thoracoscopy with pleural biopsies were performed, after imaging studies showed pleural thickenings suspicious for malignancy. Histological morphology and growth pattern were similar in both cases. Both tumors stained for calretinin, but only the pleural mesothelioma showed positivity for Wilms-Tumor 1 antibody. We tried to demonstrate with molecular biology techniques whether they were synchronous or one was the metastasis of the other, but our studies did not give informative results. The prognosis in this case is poor, and after 6 months the patient is still following a chemotherapy regimen, which is the only practicable approach given the extent of the disease.

CD (coeliac disease) is a frequent autoimmune disorder of the small bowel, which is characterized by an immunological reaction against gluten and transglutaminase in genetically predisposed subjects. However, the molecular determinants underpinning CD pathogenesis are yet to be fully elucidated and little data are available about the involvement of miRNAs (microRNAs) in CD. In the present study, the duodenal mucosa miRNA expression was profiled in adult untreated CD presenting with a classic phenotype or iron-deficiency anaemia, treated patients with or without duodenal normalization, and non-CD subjects as controls. Deregulation of seven miRNAs (miR-31-5p, miR-192-3p, miR-194-5p, miR-551a, miR-551b-5p, miR-638 and miR-1290) was determined in a larger series of CD patients with different clinical phenotypes compared with non-CD subjects. These seven microRNAs were then analysed in duodenal fibroblasts obtained from CD patients and incubated with gliadin peptides (13- and 33-mer). The miRNA cluster miR-192/194, involved in matrix remodelling, was deregulated in CD according to the different clinical presentations, and miR-192-3p levels were modulated by gliadin peptides in vitro. In conclusion, the analysis of miRNAs deserves further consideration for its potential use in the treatment and management of CD. © 2014 Biochemical Society.

Abstract Imaging appearance of cyst-like changes is most frequently described in primary neuroendocrine lesions, especially pancreatic NETs. The imaging finding of a pseudocystic lesion of the liver puts in differential diagnosis many pathologies such as infectious diseases, simple biliary cysts up to biliary cystadenomas and eventually to primary or metastatic malignancies. Primary or metastatic hepatic malignancies with pseudocystic aspects are rare, and a pseudocystic aspect is reported only after neo-adjuvant treatment. Liver metastasis of untreated neuroendocrine tumors are usually solid and, to our knowledge, only two cases of neuroendocrine cystic hepatic metastases of ileal atypical carcinoids have been reported so far. We present a case of a 67 years old man with synchronous finding of an untreated hepatic pseudocystic lesion and an ileal mass histologically diagnosed as a well differentiated (G1) neuroendocrine tumor. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1443883503102967.

Background Tumors with a massive inflammatory infiltration are described in several organs. There is agreement about considering the inflammatory infiltration as the host’s immune response to neoplastic cells; such neoplasms indeed have a better prognostic outcome than non-inflammatory counterparts. Only seventeen cases of pulmonary adenocarcinoma with massive lymphocytic infiltration (AMLI) have been reported in literature so far. Case presentation We present a case of pulmonary adenocarcinoma with massive lymphocytic infiltration occurring in a 71 years old male smoker. He came under our attention because of dyspnea, and underwent a left lower lobectomy. Histological examination showed a moderately differentiated (G2) acinar adenocarcinoma associated with a stromal desmoplastic reaction and a massive inflammatory infiltration, made up mostly of CD3+ lymphocytes. pTNM stage was pT2a, N0 (clinical stage: Ib). Molecular testing of EGFR gene showed no mutations and immunohistochemistry for ALK resulted negative. EBV infection was ruled out by EBV in situ hybridization. Conclusions Literature review showed seventeen similar cases, with a 16/1 male/female ratio and a mean age of 70,2 years. In eight out of seventeen cases EBV-infection was demonstrated with immunohistochemical or molecular biology techniques. Similarly to the cases previously reported in literature our patient is a male smoker, without lymph node metastasis and he is still alive after a follow-up period of six months without recurrent or residual disease. Because of histological, biological and clinical peculiarity, we propose to take into account pulmonary adenocarcinomas with massive inflammatory infiltration for a separate pathological classification.

Background: Penile cancer (PC) is a rare tumor, and therapeutic options are limited for this disease, with an overall 5-year overall survival around 65-70%. Adjuvant therapy is not recommended for patients with N0-1 disease, despite up to 60% of these patients will die within 5 years from diagnosis. Methods: Medical records of all patients who underwent radical surgery at University Federico II of Naples and at National Tumor Institute "Pascale" of Naples for early squamous cell carcinoma of the penis from January, 2000 to December, 2011 were retrieved. Paraffin wax embedded tissue specimens were retrieved from the pathology archives of the participating Institutions for all patients. Expression of p-EGFR, EGFR and positivity to HPV were evaluated along with other histological variables of interest. Demographic data of eligible patients were retrieved along with clinical characteristics such as type of surgical operation, time of follow up, time of recurrence, overall survival. A multivariable model was constructed using a forward stepwise selection procedure. Results: Thirty eligible patients were identified. All patients were positive for EGFR by immunohistochemistry, while 13 and 16 were respectively positive for nuclear and cytosolic p-EGFR. No EGFR amplification was detected by FISH. Eight patients were positive for high-risk HPV by ISH. On univariable analysis, corpora cavernosa infiltration (OR 7.8; 95% CI = 0,8 to 75,6; P = 0,039) and positivity for cytosolic p-EGFR (OR 7.6; 95% CI = 1.49 to 50; P = 0.009) were predictive for recurrence, while only positivity for cytosolic p-EGFR (HR = 9.0; 95% CI 1.0-100; P = 0,0116) was prognostic for poor survival. Conclusion: It is of primary importance to identify patients with N0-1 disease who are at increased risk of recurrence, as they do not normally receive any adjuvant therapy. Expression of p-EGFR was found in this series to be strongly related to increase risk of recurrence and shorter overall survival. This finding is consistent with the role of p-EGFR in other solid malignancies. Integration of p-EGFR with classic prognostic factors and other histology markers should be pursued to establish optimal adjuvant therapy for N0-1 PC patients.

Background. Extensive neoangiogenesis is a distinctive feature of both the spleen and the bone marrow (BM) of patients with myelofibrosis (MF). We and others have previously reported that mature endothelial cells obtained from microvessels of the spleen, either by laser microdissection or by cell sorting, harbor the V617FJAK2 mutation, whereas nothing is known about the existence of such mutated endothelial cells in the BM. The origin of the mutated endothelium has not been clarified up to now, although it has been proposed that it could derive from the transdifferentiation of a mutated CD34+ hematopoietic progenitor cells into an endothelial cell, an event similar to what already observed in solid tumors. Aim. To assess the presence of the V617FJAK2 mutation in mature endothelial cells from the BM of patients with MF and to evaluate the presence of transitional cells expressing both myeloid and endothelial cell markers in the BM of MF patients. Patients and Methods. Paraffin-embedded BM sections from 5 patients with MF, harboring a V617FJAK2 mutation in their granulocytes, were used both for isolating mature endothelial cells by laser microdissection and for immunostaining with antibodies directed against CD34, CD33, and CD144 (VE-cadherin) antigens. Sections from patients with lymphomas who underwent a BM biopsy for staging of the disease were used as controls (n=2). The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGE J software. DNA was extracted from 50 microdissected endothelial cells by a commercial kit and the presence of V617FJAK2 mutation assessed by an allele-specific real-time quantitative PCR. Results. The V617FJAK2 mutation was detected in at least 1 sample of endothelial cells of 5/5 patients, indicating that a mutated endothelium, in addition to the spleen, is also detectable in the BM of patients with MF. The allelic burden ranged from 51.4% to 84.1% of mutated alleles, and was similar to the allelic burden of hematopoietic cells in 1 case, whereas it was lower in 4. Staining of BM sections from 4 patients with antibodies specific for hematopoiesis (CD33) and endothelium (VE-cadherin), as well as common to the 2 lineages (CD34), while confirming the presence of increased neoangiogenetic processes in samples from MF patients, allowed the detection of cells, usually located close to neovessels, co-expressing both hematopoietic (CD33, CD34) and endothelial (VE-cadherin, CD34) markers. These "triple positive" cells were found in all the slides of all patients whereas they were never observed in the BM sections of controls. Conclusions. V617FJAK2 positive endothelial cells can be detected in the BM of patients with MF, similarly to what observed in the spleen. These mutated endothelial cells could derive from the transdifferentiation of hematopoietic mutated cells as suggested by the detection of cells co-expressing both myeloid and endothelial markers. These observations support the hypothesis that the endothelial lineage is involved in the process of malignant transformation of MF, providing new perspectives in our understanding of the phenotype, evolution, and therapy of the disease. Disclosures No relevant conflicts of interest to declare.

// Andrea Di Cristofori 1,2,* , Stefano Ferrero 3,4,* , Irene Bertolini 1,3 , Gabriella Gaudioso 1,3 , Maria Veronica Russo 1,3 , Valeria Berno 5 , Marco Vanini 6 , Marco Locatelli 2 , Mario Zavanone 1,2 , Paolo Rampini 2 , Thomas Vaccari 7 , Manuela Caroli 2 and Valentina Vaira 3,5 1 Department of Pathophysiology and Organ Transplantation, University of Milan, Milan, Italy 2 Division of Neurosurgery, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy 3 Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy 4 Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy 5 Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy 6 Surgical Pathology Unit, St. Anna Hospital, Como, Italy 7 IFOM - The FIRC Institute of Molecular Oncology, Milan, Italy * These authors have equally contributed to this work Correspondence to: Manuela Caroli, email: // Valentina Vaira, email: // Keywords : glioblastoma, vacuolar H+-ATPase, bafilomycin A1, cancer stem cells, organotypic tissue cultures Received : January 16, 2015 Accepted : May 02, 2015 Published : May 22, 2015 Abstract The vacuolar H + ATPase (V-ATPase) is a proton pump responsible for acidification of cellular microenvironments, an activity exploited by tumors to survive, proliferate and resist to therapy. Despite few observations, the role of V-ATPase in human tumorigenesis remains unclear. We investigated the expression of ATP6V0C, ATP6V0A2, encoding two subunits belonging to the V-ATPase V0 sector and ATP6V1C, ATP6V1G1, ATPT6V1G2, ATP6V1G3, which are part of the V1 sector, in series of adult gliomas and in cancer stem cell-enriched neurospheres isolated from glioblastoma (GBM) patients. ATP6V1G1 expression resulted significantly upregulated in tissues of patients with GBM and correlated with shorter patients’ overall survival independent of clinical variables. ATP6V1G1 knockdown in GBM neurospheres hampered sphere-forming ability, induced cell death, and decreased matrix invasion, a phenotype not observed in GBM monolayer cultures. Treating GBM organotypic cultures or neurospheres with the selective V-ATPase inhibitor bafilomycin A1 reproduced the effects of ATP6V1G1 siRNA and strongly suppressed expression of the stem cell markers Nestin, CD133 and transcription factors SALL2 and POU3F2 in neurospheres. These data point to ATP6V1G1 as a novel marker of poor prognosis in GBM patients and identify V-ATPase inhibition as an innovative therapeutic strategy for GBM.