Publications by authors named "Valentina Vaira"

80 Publications

Addition of 5% CO2 to Inspiratory Gas Prevents Lung Injury in an Experimental Model of Pulmonary Artery Ligation.

Am J Respir Crit Care Med 2021 Jul 12. Epub 2021 Jul 12.

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, 9339, Department of Anesthesia, Critical Care and Emergency, Milan, Italy.

Rationale: Unilateral ligation of the pulmonary artery may induce lung injury through multiple mechanisms, which might be dampened by inhaled CO2.

Objectives: To characterize bilateral lung injury due to unilateral ligation of pulmonary artery in healthy swine undergoing controlled mechanical ventilation and its prevention by 5% CO2 inhalation. To investigate relevant pathophysiological mechanisms.

Methods: Sixteen healthy pigs were allocated to surgical ligation of the left pulmonary artery (Ligation group), 7 to surgical ligation of the left pulmonary artery and inhalation of 5% CO2 (Ligation + FiCO2 5%), and 6 to no intervention (No Ligation). Then, all animals received mechanical ventilation with tidal volume (Vt) 10 ml/Kg, positive end-expiratory pressure 5 cmH2O, respiratory rate 25 bpm and FiO2 50% (±FiCO2 5%) for 48 hours or until development of severe lung injury.

Measurements And Main Results: Histological, physiological and quantitative CT-scan data were compared between groups to characterize lung injury. Electrical impedance tomography and immunohistochemistry analysis were performed in a subset of animals to explore mechanisms of injury. Animals from the Ligation group developed bilateral lung injury as assessed by significantly higher histological score, larger increase in lung weight, poorer oxygenation, and worse respiratory mechanics in comparison to the Ligation + FiCO2 5% group. In the Ligation group, the right lung received larger fraction of Vt, and inflammation was more represented, while CO2 dampened both processes.

Conclusions: Mechanical ventilation induces bilateral lung injury within 48 hours in healthy pigs undergoing left pulmonary artery ligation. Inhalation of 5% CO2 prevents injury, likely through decreased stress to the right lung and anti-inflammatory effects. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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http://dx.doi.org/10.1164/rccm.202101-0122OCDOI Listing
July 2021

An EBC/Plasma miRNA Signature Discriminates Lung Adenocarcinomas From Pleural Mesothelioma and Healthy Controls.

Front Oncol 2021 15;11:643280. Epub 2021 Jun 15.

Division of Thoracic Surgery and Lung Transplantation, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Background: Despite significant improvement in screening programs for cancers of the respiratory district, especially in at-risk subjects, early disease detection is still a major issue. In this scenario, new molecular and non-invasive biomarkers are needed to improve early disease diagnosis.

Methods: We profiled the miRNome in exhaled breath condensate (EBC) and plasma samples from fourteen patients affected by lung AdCa, nine healthy subjects. miRNA signatures were then analyzed in another neoplasia of the respiratory district, i.e. pleural mesothelioma (n = 23) and subjects previously exposed to asbestos were used as controls for this cohort (n = 19). Selected miRNAs were analyzed in purified pulmonary neoplastic or normal epithelial and stromal cell subpopulation from AdCa patients. Finally, the plasmatic miRNA signature was analyzed in a publicly available cohort of NSCLC patients for data validation and analysis was performed with predicted miRNA targets using the multiMiR tool and STRING database.

Results: miR-597-5p and miR-1260a are significantly over-expressed in EBC from lung AdCa and are associated with AdCa. Similarly, miR-1260a is also up-regulated in the plasma of AdCa patients together with miR-518f-3p and correlates with presence of lung cancer, whereas let-7f-5p is under-expressed. Analysis of these circulating miRNAs in pleural mesothelioma cases confirmed that up-regulation of miR-518f-3p, -597-5p and -1260a, is specific for lung AdCa. Lastly, quantification of the miRNAs in laser-assisted microdissected lung tissues revealed that miR-518f-3p, 597-5p and miR-1260a are predominantly expressed in tumor epithelial cells. Validation analysis confirmed miR-518f-3p as a possible circulating biomarker of NSCLC. analysis of the potentially modulated biological processes by these three miRNAs, shows that tumor bioenergetics are the most affected pathways.

Conclusions: Overall, our data suggest a 3-miRNAs signature as a non-invasive and accurate biomarker of lung AdCa. This approach could supplement the current screening approaches for early lung cancer diagnosis.
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http://dx.doi.org/10.3389/fonc.2021.643280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239300PMC
June 2021

The Core Stem Genes , , and Are Expressed in Human Parathyroid Tumors and Modulated by , , and β-catenin Pathways Activation.

Biomedicines 2021 Jun 2;9(6). Epub 2021 Jun 2.

Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy.

Tumors of the parathyroid glands are the second most common endocrine neoplasia. Epigenetic studies revealed an embryonic signature involved in parathyroid tumorigenesis. Here, we investigated the expression of the stem core genes and . Rare cells within normal parathyroid glands expressed and , while was undetectable. Nuclear expression was detectable in 18% of parathyroid adenomas (PAds, = 34) involving 5-30% of cells, while and were expressed at the nuclear level in a more consistent subset of PAds involving 15-40% of cells. Most parathyroid carcinomas expressed the core stem genes. -expressing cells co-expressed parathormone (PTH). In PAds-derived primary cultures, silencing of the tumor suppressor gene induced the expression of , likely through a axis, while calcium-sensing receptor activation increased mRNA levels through activation. In addition, inducing nuclear β-catenin accumulation in PAds-derived primary cultures by short-term incubation with lithium chloride (LiCl), and expression levels increased, while transcripts were reduced, and LiCl long-term incubation induced an opposite pattern of gene expression. In conclusion, detection of the core stem genes in parathyroid tumors supports their embryogenic signature, which is modulated by crucial genes involved in parathyroid tumorigenesis.
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http://dx.doi.org/10.3390/biomedicines9060637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227846PMC
June 2021

Imaging Metformin Efficacy as Add-On Therapy in Cells and Mouse Models of Human EGFR Glioblastoma.

Front Oncol 2021 3;11:664149. Epub 2021 May 3.

Department of Medicine and Surgery and Tecnomed Foundation, University of Milano - Bicocca, Monza, Italy.

Glioblastoma (GBM) is a highly aggressive tumor of the brain. Despite the efforts, response to current therapies is poor and 2-years survival rate ranging from 6-12%. Here, we evaluated the preclinical efficacy of Metformin (MET) as add-on therapy to Temozolomide (TMZ) and the ability of [F]FLT (activity of thymidine kinase 1 related to cell proliferation) and [F]VC701 (translocator protein, TSPO) Positron Emission Tomography (PET) radiotracers to predict tumor response to therapy. Indeed, TSPO is expressed on the outer mitochondrial membrane of activated microglia/macrophages, tumor cells, astrocytes and endothelial cells. TMZ-sensitive (Gli36ΔEGFR-1 and L0627) or -resistant (Gli36ΔEGFR-2) GBM cell lines representative of classical molecular subtype were tested and in orthotopic mouse models. Our results indicate that , MET increased the efficacy of TMZ on TMZ-sensitive and on TMZ-resistant cells by deregulating the balance between pro-survival () and pro-apoptotic () Bcl-family members and promoting early apoptosis in both Gli36ΔEGFR-1 and Gli36ΔEGFR-2 cells. , MET add-on significantly extended the median survival of tumor-bearing mice compared to TMZ-treated ones and reduced the rate of recurrence in the TMZ-sensitive models. PET studies with the cell proliferation radiopharmaceutical [F]FLT performed at early time during treatment were able to distinguish responder from non-responder to TMZ but not to predict the duration of the effect. On the contrary, [F]VC701 uptake was reduced only in mice treated with MET plus TMZ and levels of uptake negatively correlated with animals' survival. Overall, our data showed that MET addition improved TMZ efficacy in GBM preclinical models representative of classical molecular subtype increasing survival time and reducing tumor relapsing rate. Finally, results from PET imaging suggest that the reduction of cell proliferation represents a common mechanism of TMZ and combined treatment, whereas only the last was able to reduce TSPO. This reduction was associated with the duration of treatment response. TSPO-ligand may be used as a complementary molecular imaging marker to predict tumor microenvironment related treatment effects.
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http://dx.doi.org/10.3389/fonc.2021.664149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126706PMC
May 2021

V-ATPase controls tumor growth and autophagy in a Drosophila model of gliomagenesis.

Autophagy 2021 May 12:1-11. Epub 2021 May 12.

Department of Biosciences, Università Degli Studi Di Milano, Milan, Italy.

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors , we used an established model of glioma based on overexpression in larval glial cells of an active human and of the PI3K homolog . Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of , or of the Tor (Target of rapamycin) complex 1 (TORC1) component prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, our data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.
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http://dx.doi.org/10.1080/15548627.2021.1918915DOI Listing
May 2021

Emergency Lung Transplantation after COVID-19: Immunopathological Insights on Two Affected Patients.

Cells 2021 03 10;10(3). Epub 2021 Mar 10.

Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, 20122 Milan, Italy.

We herein characterize the immunopathological features of two Italian COVID-19 patients who underwent bilateral lung transplantation (bLTx). Removed lungs underwent histopathological evaluation. Gene expression profiling (GEP) for immune-related signatures was performed on lung specimens and SARS-CoV-2-stimulated peripheral blood mononuclear cells (PBMCs). Cytokine levels were measured on lungs, bronchoalveolar lavage fluids and in culture supernatants. Pathological assessment showed extensive lung damage with the pattern of proliferative to fibrotic phases, with diffuse alveolar damage mimicking usual interstitial pneumonia (UIP). Lungs' GEP revealed overexpression of pathogen recognition receptors, effector cytokines and chemokines, immune activation receptors and of the inflammasome components. Multiplex cytokine analysis confirmed a proinflammatory state, with high levels of monocyte/macrophage chemotactic and activating factors and of IL-6 and TNF-α. A similar profile was observed in SARS-CoV-2-stimulated PBMCs collected 7 days after transplant. The pattern of tissue damage observed in the lungs suggests that this may represent the output of protracted disease, resembling a diffuse UIP-like picture. The molecular immune profiling supports the paradigm of a persistent proinflammatory state and sustained humoral immunity, conditions that are maintained despite the iatrogenic immunosuppression.
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http://dx.doi.org/10.3390/cells10030611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999589PMC
March 2021

Triple negative aggressive phenotype controlled by miR-135b and miR-365: new theranostics candidates.

Sci Rep 2021 Mar 22;11(1):6553. Epub 2021 Mar 22.

Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F.Cervi 93, 20090, Segrate-Milan, Milan, Italy.

Triple negative breast cancer (TNBC) accounts for about a fifth of all breast cancers and includes a diverse group of cancers. The heterogeneity of TNBC and the lack of target receptors on the cell surface make it difficult to develop specific therapeutic treatments. These aspects cause the high negative prognosis of patients with this type of tumor. The analysis of the molecular profiles of TNBC samples has allowed a better characterization of this tumor, supporting the search for new reliable diagnostic markers. To this end, we have developed a bioinformatic approach to integrate networks of genes differentially expressed in basal breast cancer compared to healthy tissues, with miRNAs able to regulate their expression. We studied the role of these miRNAs in TNBC subtype cell lines. We therefore identified two miRNAs, namely miR-135b and miR-365, with a central role in regulating the altered functional pathways in basal breast cancer. These two miRNAs are differentially expressed in human TNBC immunohistochemistry-selected tissues, and their modulation has been shown to play a role in the proliferation of tumor control and its migratory and invasive capacity in TNBC subtype cell lines. From the perspective of personalized medicine, we managed to modulate the expression of the two miRNAs in organotypic cultures, suggesting their possible use as diagnostic and therapeutic molecules. miR-135b and miR-365 have a key role in TNBC, controlling proliferation and invasion. Their detection could be helpful in TNBC diagnosis, while their modulation could become a new therapeutic tool for TNBC.
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http://dx.doi.org/10.1038/s41598-021-85746-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985188PMC
March 2021

Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors.

Int J Mol Sci 2021 Feb 18;22(4). Epub 2021 Feb 18.

Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy.

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, silencing reduces the expression of the key parathyroid oncosuppressor , while silencing increases expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets , , and , and this effect was blunted by silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.
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http://dx.doi.org/10.3390/ijms22042016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922006PMC
February 2021

Usefulness of autofluorescence bronchoscopy in early diagnosis of airway complications after lung transplantation.

Sci Rep 2020 12 18;10(1):22316. Epub 2020 Dec 18.

Thoracic Surgery and Lung Transplant Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Via Francesco Sforza, 35, 20122, Milan, Italy.

Despite the promising results achieved so far in long-term survival after lung transplantation (LuTx), airway complications (ACs) still arise in the post-operative period. Early diagnosis and prompt treatment of ACs play a critical role in preventing their onset. Specifically, large bronchi ischemia has been recognized as a triggering factor for ACs. Autofluorescence bronchoscopy, which was first introduced for early cancer diagnosis, displays ischemic mucosae as red spots, while normal vascularized mucosae appear in green. The aim of this study is to investigate whether a significant correlation exists between ACs and the red/green (RG) ratio detected on scheduled autofluorescence bronchoscopy up to 1 year after LuTx. This prospective, observational, single-center cohort study initially considered patients who underwent LuTx between July 2014 and February 2016. All patients underwent concomitant white-light and autofluorescence bronchoscopy at baseline (immediately after LuTx), on POD7, POD14, POD21, POD28, POD45, 3 months, 6 months, and 1 year after LuTx. An autofluorescence image of the first bronchial carina distal to the anastomosis was captured and analyzed using histograms for red and green pixels; the R/G ratio was then recorded. Potential ACs were classified according according to the presence of a white-light following the MDS (macroscopic aspect, diameter and suture) criteria. The authors assessed the association between the R/G ratio and the ACs occurrence using a generalized estimating equations model. Thirty-one patients met the inclusion criteria and were therefore selected. Out of a total of 53 bronchial anastomoses, 8 developed complications (late bronchial stenosis), with an average onset time of 201 days after LuTx. ACs showed a similar baseline covariate value when compared to anastomoses that involved no complication. Generalized estimating equations regression indicated a clear association over time between the R/G ratio and the rise of complications (p = 0.023). The authors observed a significant correlation between post-anastomotic stenosis and the delayed decrease of the R/G ratio. Preliminary outcomes suggest that autofluorescence bronchoscopy may be an effective and manageable diagnostic tool, proving complementary to other instruments for early diagnosis of ACs after LuTx. Further research is needed to confirm and detail preliminary findings.
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http://dx.doi.org/10.1038/s41598-020-79442-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749104PMC
December 2020

How to manage celiac disease and gluten-free diet during the COVID-19 era: proposals from a tertiary referral center in a high-incidence scenario.

BMC Gastroenterol 2020 Nov 19;20(1):387. Epub 2020 Nov 19.

Center for Prevention and Diagnosis of Celiac Disease, Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.

The outbreak of COVID-19 and SARS-CoV-2 infection is spreading worldwide as the first coronavirus pandemic. The clinical picture is variable but flu-like symptoms are common with bilateral interstitial pneumonia being the most frightening presentation. No specific therapies nor vaccine have been developed to date and the only way to limit the virus diffusion is by modifying one's lifestyle limiting social life and following strict hygienic precautions. No data is available on the risk of COVID-19 and its outcomes in celiac disease (CeD). The restrictions applied to counter COVID-19 can impact on CeD treatment and gluten-free dieting, the only available therapy for CeD. With the present manuscript, we aim to support gastroenterologists and nutritionists in the management of CeD patients in the new pandemic scenario, being conscious that availability and local situations are extremely various.
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http://dx.doi.org/10.1186/s12876-020-01524-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675390PMC
November 2020

miR-126-3p contributes to parathyroid tumor angiogenesis.

Endocr Relat Cancer 2021 Jan;28(1):53-63

Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy.

Tumors of the parathyroid glands are highly vascularized and display a microRNA (miRNA) profile divergent from normal parathyroid glands (PaNs). Angiogenic miRNAs, namely miR-126-3p, miR-126-5p, and miR-296-5p, have been found downregulated in parathyroid tumors. Here, we show that miR-126-3p expression levels are reduced in parathyroid adenomas (PAds; n = 12) compared with PaNs (n = 4). In situ hybridization (ISH) of miR-126-3p and miR-296-5p in 10 PAds show that miR-126-3p is expressed by endothelial cells lining the walls of great vessels and by cells within the thin stroma surrounding acinar structures. At variance, miR-296-5p was detectable in most PAd epithelial cells. Combining ISH for miR-126-3p with immunohistochemistry for the endothelial and mesenchymal markers CD34, CD31 and α-smooth muscle actin (αSMA), we could identify that miR-126-3p is localized in the αSMA-positive thin stroma. Further, miR-126-3p-expressing cells are enriched in the CD34-positive stromal cells surrounding epithelial cell acinar structures, a cellular pattern consistent with tumor-associated myofibroblasts (TAMs). In line with this, CD34-positive cells, sorted by FACS from PAds tissues, express miR-126-3p at higher levels than CD34-negative cells, suggesting that miR-126-3p downregulation promotes the endothelial-to-αSMA+ mesenchymal transition. In human mesenchymal stem cells derived from bone marrow (hBM-MSCs), a model of TAMs, the co-culture with PAds-derived cells for 5 days decreases miR-126-3p, while it increases VEGFA expression. At variance, adrenomedullin (ADM) expression is unaffected. Finally, overexpression of the miR-126-3p mimic in both hBM-MSCs and PAds-derived explants downregulates VEGFA expression levels. In conclusion, miR-126-3p is expressed by both endothelial cells and TAMs in PAds, and its downregulation promotes neoangiogenesis, possibly through VEGFA overexpression.
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http://dx.doi.org/10.1530/ERC-20-0313DOI Listing
January 2021

Characterization of the immune microenvironment in malignant pleural mesothelioma reveals prognostic subgroups of patients.

Lung Cancer 2020 12 2;150:53-61. Epub 2020 Oct 2.

Division of Pathology, Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milan, Italy; Department of Biomedical, Surgical, and Dental Sciences, University of Milan, Italy.

Malignant pleural mesothelioma (MPM) is a rare tumor with an extremely poor prognosis. Its pathogenesis is related to an immune response against asbestos fibers. The T-lymphocytes, including CD8 and CD4 cells, are an important part of the MPM immune microenvironment, and likely contribute to the therapy resistance observed in these tumors. Here, we sought to characterize the MPM-specific lymphocytes subpopulations within the tumor immune microenvironment to identify novel clinically relevant immunologic subtypes of tumors. Representative formalin-fixed, paraffin-embedded (FFPE) tissue blocks of 88 MPMs were included in tissue microarrays and subjected to tumor-infiltrating lymphocytes (TILs) quantification and subtyping by immunohistochemistry (IHC) with antibodies specific for CD4, CD8, and CD19. Further, PD-L1 (clone 22C3) expression was assessed by IHC as a combined positive score (CPS). Our data show that PD-L1 expression by tumor cells or the presence of a sarcomatoid component is related to increased stromal TILs presence in MPM. Survival analyses showed that low CD4 and high CD8 stromal TILs are associated with poor patients' survival. In MPMs with PD-L1 CPS > 1, stromal CD8 was a poor prognostic factor, akin stromal CD4 peritumoral TILs correlated with a worse prognosis. Furthermore, we demonstrated that a high CD4/CD8 ratio in the tumor immune microenvironment is an independent prognostic factor for survival. Finally, we provided evidence that the characterization of the stromal immune landscape of MPM predicts responses to chemotherapy in subgroups of MPM. The results of this study provide novel insights into the clinical scenario of immune-related biomarkers in MPM.
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http://dx.doi.org/10.1016/j.lungcan.2020.09.026DOI Listing
December 2020

Laser capture microdissection on formalin-fixed and paraffin-embedded renal transplanted biopsies: Technical perspectives for clinical practice application.

Exp Mol Pathol 2020 10 25;116:104516. Epub 2020 Aug 25.

Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Unit of Nephrology, Dialysis and Renal Transplant, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. Electronic address:

Renal biopsy (RBx) is an essential tool in the diagnostic and therapeutic process of most native kidney diseases and in the renal transplanted graft. Laser capture microdissection (LCM), combined with molecular biology, might improve the diagnostic power of RBx. However, the limited amount of available renal tissue is often an obstacle for achieving a satisfactory qualitative and quantitative analysis. In our work we present a method which allows us to obtain good quality and quantity of RNA from formalin-fixed and paraffin-embedded (FFPE) renal tissue derived from RBx performed in transplanted patients. Histology, immunohistochemistry, LCM, pre-amplify system and qRT-PCR of biomarkers related to tubular damage, inflammation and fibrosis on FFPE RBx were performed. Glomeruli, tubules and interstitium of three RBx (RB-A: no alteration; RB-B and -C: the progressive rise of creatinine) were compared. The method proposed, could well be useful in future clinical practice. It is quick, easy to perform and allows the analyses of many biomarkers. In addition, it could be extended to all types of RBx without any limitation on the sample amount. Nevertheless, the need for a higher number of well-trained technicians might represent some limitation, counterbalanced by the opportunity to elaborate more accurate diagnosis and, consequently, more targeted therapies.
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http://dx.doi.org/10.1016/j.yexmp.2020.104516DOI Listing
October 2020

Small Extracellular Vesicle Regulation of Mitochondrial Dynamics Reprograms a Hypoxic Tumor Microenvironment.

Dev Cell 2020 10 10;55(2):163-177.e6. Epub 2020 Aug 10.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, PA 19104, USA; Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium.
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http://dx.doi.org/10.1016/j.devcel.2020.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606608PMC
October 2020

The Oncosuppressors MEN1 and CDC73 Are Involved in lncRNA Deregulation in Human Parathyroid Tumors.

J Bone Miner Res 2020 12 23;35(12):2423-2431. Epub 2020 Sep 23.

Division of Pathology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

A role for long non-coding RNAs (lncRNAs) in endocrine cancer pathogenesis is emerging. However, knowledge regarding their expression pattern, correlation with known genetic defects, and clinical implications in parathyroid tumors is still unclear. Here, we profiled 90 known lncRNAs in a first series of normal (PaN = 2), adenomatous (PAd = 12), and carcinomatous (PCa = 4) parathyroid glands and we confirmed deregulation of 11 lncRNAs using an independent cohort of patients (PaN = 4; PAd = 26; PCa = 9). Expression of lncRNAs was correlated with cytogenetic aberrations, status of genes multiple endocrine neoplasia 1 (MEN1) and cell division cycle 73 (CDC73), or clinical features. Globally, lncRNAs discriminate according to tissue histology. BC200 consistently identifies parathyroid cancers from adenomas and atypical adenomas. Loss-of-heterozygosity (LOH) at chromosomes 1, 11, 15, 21, and 22 significantly impacts expression of lncRNAs in PAds. Silencing of the key parathyroid gene MEN1 modulates the expression of six lncRNAs in primary PAds-derived cultures. Analogous levels of lncRNAs are measured in PAds with the mutation in the MEN1 gene compared with PAds with wild-type MEN1. Similarly, carcinomas with mutated CDC73 differ from PCas with wild-type protein in terms of expression of lncRNAs. PCas harboring CDC73 mutations overexpress BC200 compared to wild-type carcinomas. Overall, these findings shed light on deregulation of lncRNAs in human parathyroid tumors and propose that circuits between lncRNAs and the oncosuppressors MEN1 or CDC73 may have a role in parathyroid tumorigenesis as epigenetic modulators. © 2020 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4154DOI Listing
December 2020

Interplay Between V-ATPase G1 and Small EV-miRNAs Modulates ERK1/2 Activation in GBM Stem Cells and Nonneoplastic Milieu.

Mol Cancer Res 2020 11 4;18(11):1744-1754. Epub 2020 Aug 4.

Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EV). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures. To this end, small EVs were isolated from patients-derived GSCs grown as neurospheres (NS) with high (V1G1-NS) or low (V1G1-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA contents, and cellular responses in recipient cultures. Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The miRNA profile of V1G1-NS-derived small EVs differed significantly from that of V1G1-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the prooncogenic effects of V1G1 small EV. These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration. IMPLICATIONS: Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase-dependent reprogramming of the brain microenvironment through the release of specialized EVs.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642188PMC
November 2020

Comprehensive Genomic Analysis Reveals the Prognostic Role of Copy-Number Variations in Human Malignancies.

Genes (Basel) 2020 07 24;11(8). Epub 2020 Jul 24.

Neurology Unit, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, 20122 Milan, Italy.

Genetic alterations of leucine-rich repeat kinase 2 (), one of the most important contributors to familial Parkinson's disease (PD), have been hypothesized to play a role in cancer development due to demographical and preclinical data. Here, we sought to define the prevalence and prognostic significance of somatic mutations across all types of human malignancies by querying the publicly available online genomic database cBioPortal. Ninety-six different studies with 14,041 cases were included in the analysis, and 761/14,041 (5.4%) showed genetic alterations in . Among these, 585 (76.9%) were point mutations, indels or fusions, 168 (22.1%) were copy number variations (CNVs), and 8 (1.0%) showed both types of alterations. One case showed the somatic mutation R1441C. A significant difference in terms of overall survival (OS) was noted between cases harboring somatic whole deletions, amplifications, and CNV-unaltered cases (median OS: 20.09, 57.40, and 106.57 months, respectively; = 0.0008). These results suggest that both amplifications and whole gene deletions could play a role in cancer development, paving the way for future research in terms of potential treatment with LRRK2 small molecule inhibitors for -amplified cases.
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http://dx.doi.org/10.3390/genes11080846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465025PMC
July 2020

Quantitative Multivolume Proton-Magnetic Resonance Imaging in Lung Transplant Recipients: Comparison With Computed Tomography and Spirometry.

Acad Radiol 2020 Jul 8. Epub 2020 Jul 8.

Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano, Italy.

Rationale And Objectives: Acute and chronic graft rejection remains the major problem in clinical surveillance of lung-transplanted patients and early detection of complications is of capital importance to allow the optimal therapeutic option. The aim of this study was to investigate the role of quantitative non contrast-enhanced magnetic resonance imaging (MRI) as a non-ionizing imaging modality to assess ventilation impairment in patients who have undergone lung transplantation, in comparison with quantitative computed tomography (CT) and spirometry.

Materials And Methods: Ten lung-transplanted patients (39 ±12 years, forced-expiratory volume in 1 second (FEV1) = 81 ± 27%, forced vital capacity (FVC) = 87 ± 27%) were acquired in breath-hold at full-expiration and full-inspiration with 1.5T MRI and CT. Maps of expiratory-inspiratory difference in MR signal-intensity and CT-density were computed to estimate regional ventilation. Based on expiratory, inspiratory, and expiratory-inspiratory difference values, each pixel was classified as healthy (H), low ventilation (LV), air trapping (AT), and consolidation (C) and the percent extent of each class was quantified.

Results: Overall, expiratory-inspiratory difference in MR signal-intensity correlated to CT-density (r = 0.64, p < 0.0001) and to FEV1 (ρ = 0.71, p = 0.02). The linear correlation between MRI and CT functional maps considering all the four classes is r = 0.93 (p < 0.0001). MRI percent volumes of H, AT, and C correlated to FEV1 %pred, with the highest correlation reported for AT (ρ = -0.82).

Conclusion: Results demonstrated a good agreement between MRI and CT ventilation imaging and between the corresponding percent volumes of lung damage. Quantitative MRI may represent an accurate non-ionizing imaging technique for longitudinal monitoring of lung transplant recipients.
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http://dx.doi.org/10.1016/j.acra.2020.05.026DOI Listing
July 2020

Bronchoalveolar Lavage-microRNAs Are Potential Novel Biomarkers of Outcome After Lung Transplantation.

Transplant Direct 2020 May 9;6(5):e547. Epub 2020 Apr 9.

Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

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.
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http://dx.doi.org/10.1097/TXD.0000000000000994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213607PMC
May 2020

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

Clin Sci (Lond) 2020 05;134(10):1151-1166

Center for the Prevention and Diagnosis of Celiac Disease, Division of Gastroenterology and Endoscopy, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

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.
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http://dx.doi.org/10.1042/CS20200032DOI Listing
May 2020

Transbronchial Cryobiopsies in Lung Allograft Recipients for Surveillance Purposes: Initial Results.

Transplant Proc 2020 Jun 26;52(5):1601-1604. Epub 2020 Mar 26.

Thoracic Surgery and Lung Transplant Unit Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milan, Italy.

Transbronchial biopsy (TBB) using standard forceps is the main procedure to establish the presence of lung allograft rejection (AR) after lung transplantation. Few studies report the use of the transbronchial cryobiopsy (TCB) as a scheduled procedure for surveillance purposes in lung allograft, despite this the technique yields larger biopsies. We aimed to analyze the diagnostic yield and potential complications of TCB compared with conventional forceps biopsy for acute rejection surveillance in lung transplantation. In our center, TCBs are performed to monitor lung allografts at 3, 6, and 12 months after transplantation. From March 2018 to September 2019 TCBs were performed in 54 lung transplanted patients for surveillance purposes. Clinical and functional data, complications, and histologic results were collected. We analyzed through a retrospective study our first 75 cases of cryobiopsies for surveillance purposes in lung allograft recipients. The diagnostic rate of AR using TCB was 100% compared with 83% using conventional TBB. Also, diagnostic rate of airway inflammation and chronic rejection was 17% and 21% higher, respectively, for TCB compared with TBB. The overall major complication rate was 9%: 1 pneumothorax case required chest tube drainage and 6 moderate bleedings. Bleeding rate in the scheduled TCB group (8%) seems to be higher if compared with scheduled TBB group (1%). TCB seems to be safe and effective for diagnosis of lung AR compared with transbronchial conventional forceps biopsy.
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http://dx.doi.org/10.1016/j.transproceed.2020.02.052DOI Listing
June 2020

Parathyroid Tumor Microenvironment.

Adv Exp Med Biol 2020 ;1226:37-50

Department of Biomedical, Surgical and Odontoiatric Sciences, University of Milan, Milan, Italy.

Parathyroid tumors are the second most common endocrine neoplasia, and it is almost always associated with hypersecretion of the parathormone (PTH), involved in calcium homeostasis, causing primary hyperparathyroidism (PHPT). Parathyroid neoplasia has a stromal component particularly represented in atypical adenomatous and carcinomatous lesions. Recently, data about the features and the function of the parathyroid tumor microenvironment (TME) have been accumulated. Parathyroid TME includes heterogeneous cells: endothelial cells, myofibroblasts, lymphocytes and macrophages, and mesenchymal stem cells have been identified, each of them presenting a phenotype consistent with tumor-associated cells. Parathyroid tumors overexpress proangiogenic molecules including vascular endothelial growth factor (VEGF-A), fibroblast growth factor-2 (FGF-2), and angiopoietins that promote both recruitment and proliferation of endothelial cell precursors, thus resulting in a microvessel density higher than that detected in normal parathyroid glands. Moreover, parathyroid tumor endocrine cells operate multifaceted interactions with stromal cells, partly mediated by the CXCL12/CXCR4 pathway, while, at present, the immune landscape of parathyroid tumors has just begun to be investigated. Studies about TME in parathyroid adenomas provide an example of the role of TME in benign tumors, whose molecular mechanisms and functions comprehension are limited.
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http://dx.doi.org/10.1007/978-3-030-36214-0_3DOI Listing
February 2020

A miRNA-Based Blood and Mucosal Approach for Detecting and Monitoring Celiac Disease.

Dig Dis Sci 2020 07 28;65(7):1982-1991. Epub 2019 Nov 28.

Laboratory of Immunogenetics, Institute of Nutrition and Food Technology, INTA, University of Chile, Av. El Líbano, 5524, Macul, Santiago, Chile.

Background: The role of microRNAs (miRNAs) in celiac disease (CD) is unclear.

Aims: We evaluated inflammation-related miRNA-146a, miRNA-155, miRNA-21, and miRNA-125b expression in peripheral blood and intestinal mucosa of CD adults.

Methods: 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-inactive, n = 10). In addition, ten healthy volunteers formed the comparison/control group. MiRNA expression was measured in duodenal biopsies from patients (CD-inactive, 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 (gliadin).

Results: 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 gliadin, 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.

Conclusions: 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.
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http://dx.doi.org/10.1007/s10620-019-05966-zDOI Listing
July 2020

MFF Regulation of Mitochondrial Cell Death Is a Therapeutic Target in Cancer.

Cancer Res 2019 Dec 3;79(24):6215-6226. Epub 2019 Oct 3.

Prostate Cancer Discovery and Development Program.

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..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911621PMC
December 2019

Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer.

EBioMedicine 2019 Oct 18;48:353-363. Epub 2019 Sep 18.

Prostate Cancer Discovery and Development Program, USA; Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

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, Ca 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, Ca 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.
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http://dx.doi.org/10.1016/j.ebiom.2019.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838406PMC
October 2019

A miRNome analysis of drug-free manic psychotic bipolar patients versus healthy controls.

Eur Arch Psychiatry Clin Neurosci 2020 Oct 17;270(7):893-900. Epub 2019 Aug 17.

Department of Psychiatry, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico Milano, Università degli Studi di Milano, Via F. Sforza 35, 20122, Milan, Italy.

The lifetime presence of psychotic symptoms is associated with more clinical severity, poorer outcome and biological changes in patients affected by bipolar disorder (BD). Epigenetic mechanisms have been evoked to explain the onset of psychotic symptoms in BD as well as the associated biological changes. The main objective of the present study was to evaluate the expression profiles of circulating microRNAs (miRNAs) in drug-free manic psychotic bipolar patients versus healthy controls (HC), to identify possible non-invasive molecular markers of the disorder. 15 drug-free manic psychotic bipolar patients and 9 HC were enrolled and 800 miRNAs expression profile was measured by Nanostring nCounter technology on plasma samples and validated through qPCR. Overall, twelve miRNAs showed a significantly altered expression between the two groups (p < 0.05). Functional annotation of predicted miRNAs targets by MultiMIR R tool showed repression in bipolar patients of genes with a role in neurodevelopment and neurogenesis, and upregulation of genes involved in metabolism regulation. We identified a signature of circulating miRNA characteristic of manic psychotic bipolar patients, suggesting a possible role in neurodevelopment and metabolic processes regulation.
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http://dx.doi.org/10.1007/s00406-019-01057-2DOI Listing
October 2020

Mismatch Repair Protein Loss as a Prognostic and Predictive Biomarker in Breast Cancers Regardless of Microsatellite Instability.

JNCI Cancer Spectr 2018 Oct 13;2(4):pky056. Epub 2018 Dec 13.

Division of Pathology, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.

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) ( = .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 ( < .001).

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.
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http://dx.doi.org/10.1093/jncics/pky056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6649738PMC
October 2018

p65BTK is a novel potential actionable target in KRAS-mutated/EGFR-wild type lung adenocarcinoma.

J Exp Clin Cancer Res 2019 Jun 14;38(1):260. Epub 2019 Jun 14.

Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.

Background: Lung cancer is still the main cause of cancer death worldwide despite the availability of targeted therapies and immune-checkpoint inhibitors combined with chemotherapy. Cancer cell heterogeneity and primary or acquired resistance mechanisms cause the elusive behaviour of this cancer and new biomarkers and active drugs are urgently needed to overcome these limitations. p65BTK, a novel isoform of the Bruton Tyrosine Kinase may represent a new actionable target in non-small cell lung cancer (NSCLC).

Methods: p65BTK expression was evaluated by immunohistochemistry in 382 NSCLC patients with complete clinico-pathological records including smoking habit, ALK and EGFR status, and in metastatic lymph nodes of 30 NSCLC patients. NSCLC cell lines mutated for p53 and/or a component of the RAS/MAPK pathway and primary lung cancer-derived cells from Kras/Trp53 null mice were used as a preclinical model. The effects of p65BTK inhibition by BTK Tyrosine Kinase Inhibitors (TKIs) (Ibrutinib, AVL-292, RN486) and first-generation EGFR-TKIs (Gefitinib, Erlotinib) on cell viability were evaluated by MTT. The effects of BTK-TKIs on cell growth and clonogenicity were assessed by crystal violet and colony assays, respectively. Cell toxicity assays were performed to study the effect of the combination of non-toxic concentrations of BTK-TKIs with EGFR-TKIs and standard-of-care (SOC) chemotherapy (Cisplatin, Gemcitabine, Pemetrexed).

Results: p65BTK was significantly over-expressed in EGFR-wild type (wt) adenocarcinomas (AdC) from non-smoker patients and its expression was also preserved at the metastatic site. p65BTK was also over-expressed in cell lines mutated for KRAS or for a component of the RAS/MAPK pathway and in tumors from Kras/Trp53 null mice. BTK-TKIs were more effective than EGFR-TKIs in decreasing cancer cell viability and significantly impaired cell proliferation and clonogenicity. Moreover, non-toxic doses of BTK-TKIs re-sensitized drug-resistant NSCLC cell lines to both target- and SOC therapy, independently from EGFR/KRAS status.

Conclusions: p65BTK results as an emerging actionable target in non-smoking EGFR-wt AdC, also at advanced stages of disease. Notably, these patients are not eligible for EGFR-TKIs-based therapy due to a lack of EGFR mutation. The combination of BTK-TKIs with EGFR-TKIs is cytotoxic for EGFR-wt/KRAS-mutant/p53-null tumors and BTK-TKIs re-sensitizes drug-resistant NSCLC to SOC chemotherapy. Therefore, our data suggest that adding BTK-TKIs to SOC chemotherapy and EGFR-targeted therapy may open new avenues for clinical trials in currently untreatable NSCLC.
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http://dx.doi.org/10.1186/s13046-019-1199-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570906PMC
June 2019

Demystifying autoimmune small bowel enteropathy.

Curr Opin Gastroenterol 2019 05;35(3):243-249

Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

Purpose Of Review: We reviewed the current 'state of the art' on autoimmune enteropathy and small-bowel mucosal atrophy, with the aim of supporting clinicians in a frequently challenging diagnosis through different therapeutic options and prognosis.

Recent Findings: The diagnosis of small-bowel diseases has radically changed over the last 10 years. The possibility to 'easily' obtain bioptic samples from the jejunum and ileum by means of the enteroscopic techniques (particularly, device-assisted enteroscopy) and the novel cross-sectional imaging studies have opened the window to new insights on intestinal disorders. Consequentially, the detection of small-bowel mucosal atrophy has become a frequent finding in patients undergoing endoscopic investigation and its differential diagnosis can be challenging at times. Among the 'typical' causes of mucosal atrophy, autoimmune enteropathy has become more frequent than previously thought. However, the final diagnosis of autoimmune enteropathy is a 'puzzle' composed by serological, endoscopic, histological and molecular markers, which should be correctly dealt with in order to reach a certain diagnosis.

Summary: In conclusion, there is an emerging body of literature about autoimmune enteropathy and small-bowel atrophy. The herein presented practical review on autoimmune enteropathy can be of help to clinicians in their daily practice.
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http://dx.doi.org/10.1097/MOG.0000000000000515DOI Listing
May 2019

Specific V-ATPase expression sub-classifies IDHwt lower-grade gliomas and impacts glioma growth in vivo.

EBioMedicine 2019 Mar 5;41:214-224. Epub 2019 Feb 5.

Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; Fondazione Istituto Nazionale Genetica Molecolare 'Romeo ed Enrica Invernizzi', Milan, Italy. Electronic address:

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).
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http://dx.doi.org/10.1016/j.ebiom.2019.01.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441867PMC
March 2019