128 results on '"Lavitrano M"'
Search Results
2. Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach.
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Lado S, Thannesberger J, Spettel K, Arpović J, Ferreira BI, Lavitrano M, and Steininger C
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- Humans, Mutation, Genome, Viral, Genetic Variation, Evolution, Molecular, SARS-CoV-2 genetics, COVID-19 virology, COVID-19 diagnosis, High-Throughput Nucleotide Sequencing methods, Spike Glycoprotein, Coronavirus genetics
- Abstract
Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus-host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus's genetic landscape.
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- 2024
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3. Correction: A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.
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Grassilli E, Pisano F, Cialdella A, Bonomo S, Missaglia C, Cerrito MG, Masiero L, Ianzano L, Giordano F, Cicirelli V, Narloch R, D'Amato F, Noli B, Ferri GL, Leone BE, Stanta G, Bonin S, Helin K, Giovannoni R, and Lavitrano M
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- 2024
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4. Olfactory receptor genes and chromosome 11 structural aberrations: Players or spectators?
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Redaelli S, Grati FR, Tritto V, Giannuzzi G, Recalcati MP, Sala E, Villa N, Crosti F, Roversi G, Malvestiti F, Zanatta V, Repetti E, Rodeschini O, Valtorta C, Catusi I, Romitti L, Martinoli E, Conconi D, Dalprà L, Lavitrano M, Riva P, and Bentivegna A
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- Humans, Comparative Genomic Hybridization, In Situ Hybridization, Fluorescence, Chromosome Aberrations, Translocation, Genetic genetics, Chromosomes, Human, Pair 11, Receptors, Odorant genetics
- Abstract
The largest multi-gene family in metazoans is the family of olfactory receptor (OR) genes. Human ORs are organized in clusters over most chromosomes and seem to include >0.1% the human genome. Because 369 out of 856 OR genes are mapped on chromosome 11 (HSA11), we sought to determine whether they mediate structural rearrangements involving this chromosome. To this aim, we analyzed 220 specimens collected during diagnostic procedures involving structural rearrangements of chromosome 11. A total of 222 chromosomal abnormalities were included, consisting of inversions, deletions, translocations, duplications, and one insertion, detected by conventional chromosome analysis and/or fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array-CGH). We verified by bioinformatics and statistical approaches the occurrence of breakpoints in cytobands with or without OR genes. We found that OR genes are not involved in chromosome 11 reciprocal translocations, suggesting that different DNA motifs and mechanisms based on homology or non-homology recombination can cause chromosome 11 structural alterations. We also considered the proximity between the chromosomal territories of chromosome 11 and its partner chromosomes involved in the translocations by using the deposited Hi-C data concerning the possible occurrence of chromosome interactions. Interestingly, most of the breakpoints are located in regions highly involved in chromosome interactions. Further studies should be carried out to confirm the potential role of chromosome territories' proximity in promoting genome structural variation, so fundamental in our understanding of the molecular basis of medical genetics and evolutionary genetics., Competing Interests: Declaration of interests At the time of data collection, F.R.G. was, together with F.M., V.Z., and E.R., a full-time employee of TOMA Advanced Biomedical Assays S.p.A. (Impact Lab) without ownership shares. F.R.G. is currently a full-time employee of Menarini Silicon Biosystems, Reproductive Precision Medicine Unit., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2024
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5. Vacuolar Proton-Translocating ATPase May Take Part in the Drug Resistance Phenotype of Glioma Stem Cells.
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Giambra M, Di Cristofori A, Raimondo F, Rigolio R, Conconi D, Chiarello G, Tabano SM, Antolini L, Nicolini G, Bua M, Ferlito D, Carrabba G, Giussani CG, Lavitrano M, and Bentivegna A
- Subjects
- Humans, Drug Resistance, Phenotype, Neoplastic Stem Cells metabolism, Vacuolar Proton-Translocating ATPases metabolism, Glioma pathology, Glioblastoma pathology, Macrolides
- Abstract
The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.
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- 2024
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6. Differential Expression of NOTCH-1 and Its Molecular Targets in Response to Metronomic Followed by Conventional Therapy in a Patient with Advanced Triple-Negative Breast Cancer.
- Author
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Ilari A, Cogliati V, Sherif N, Grassilli E, Ramazzotti D, Cordani N, Cazzaniga G, Di Bella C, Lavitrano M, Cazzaniga ME, and Cerrito MG
- Abstract
A group of 27 patients diagnosed with metastatic triple-negative breast cancer (mTNBC) was randomly distributed into two groups and underwent different lines of metronomic treatment (mCHT). The former group (N 14) received first-line mCHT and showed a higher overall survival rate than the second group (N 13), which underwent second-line mCHT. Analysis of one patient still alive from the first group, diagnosed with mTNBC in 2019, showed a complete metabolic response (CMR) after a composite approach implicating first-line mCHT followed by second-line epirubicin and third-line nab-paclitaxel, and was chosen for subsequent molecular characterization. We found altered expression in the cancer stemness-associated gene NOTCH-1 and its corresponding protein. Additionally, we found changes in the expression of oncogenes, such as MYC and AKT , along with their respective proteins. Overall, our data suggest that a first-line treatment with mCHT followed by MTD might be effective by negatively regulating stemness traits usually associated with the emergence of drug resistance.
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- 2024
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7. High-sensitivity analysis of clonal hematopoiesis reveals increased clonal complexity of potential-driver mutations in severe COVID-19 patients.
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Ronchini C, Caprioli C, Tunzi G, D'Amico FF, Colombo E, Giani M, Foti G, Conconi D, Lavitrano M, Passerini R, Pase L, Capizzi S, Mastrilli F, Alcalay M, Orecchia R, Natoli G, and Pelicci PG
- Subjects
- Humans, RNA, Viral, SARS-CoV-2 genetics, Mutation, Clonal Hematopoiesis genetics, COVID-19 genetics
- Abstract
Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ronchini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2024
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8. TWIST1 Upregulation Is a Potential Target for Reversing Resistance to the CDK4/6 Inhibitor in Metastatic Luminal Breast Cancer Cells.
- Author
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Cordani N, Mologni L, Piazza R, Tettamanti P, Cogliati V, Mauri M, Villa M, Malighetti F, Di Bella C, Jaconi M, Cerrito MG, Cavaletti G, Lavitrano M, and Cazzaniga ME
- Subjects
- Humans, Female, Up-Regulation, Cyclin-Dependent Kinase 4, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Progression-Free Survival, Cyclin-Dependent Kinase 6, Antineoplastic Combined Chemotherapy Protocols pharmacology, Nuclear Proteins genetics, Nuclear Proteins metabolism, Twist-Related Protein 1 genetics, Twist-Related Protein 1 metabolism, Breast Neoplasms drug therapy, Breast Neoplasms genetics, Breast Neoplasms metabolism
- Abstract
Cyclin-dependent kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival in hormone-receptor-positive (HR+), human-epidermal-growth-factor-receptor-type-2-negative (HER2-) metastatic luminal breast cancer (mLBC). Several studies have shown that in patients with endocrine-sensitive or endocrine-resistant LBC, the addition of CDK4/6 inhibitors to endocrine therapy significantly prolongs progression-free survival. However, the percentage of patients who are unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers have been validated to select a priori responders or refractory patients. The selection of mutant clones in the target oncoprotein is the main cause of resistance. Other mechanisms such as oncogene amplification/overexpression or mutations in other pathways have been described in several models. In this study, we focused on palbociclib, a selective CDK4/6 inhibitor. We generated a human MCF-7 luminal breast cancer cell line that was able to survive and proliferate at different concentrations of palbociclib and also showed cross-resistance to abemaciclib. The resistant cell line was characterized via RNA sequencing and was found to strongly activate the epithelial-to-mesenchymal transition. Among the top deregulated genes, we found a dramatic downregulation of the CDK4 inhibitor CDKN2B and an upregulation of the TWIST1 transcription factor. TWIST1 was further validated as a target for the reversal of palbociclib resistance. This study provides new relevant information about the mechanisms of resistance to CDK4/6 inhibitors and suggests potential new markers for patients' follow-up care during treatment.
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- 2023
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9. Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.
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Mierzejewska P, Di Marzo N, Zabielska-Kaczorowska MA, Walczak I, Slominska EM, Lavitrano M, Giovannoni R, Kutryb-Zajac B, and Smolenski RT
- Abstract
The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.
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- 2023
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10. Progression after First-Line Cyclin-Dependent Kinase 4/6 Inhibitor Treatment: Analysis of Molecular Mechanisms and Clinical Data.
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Villa F, Crippa A, Pelizzoni D, Ardizzoia A, Scartabellati G, Corbetta C, Cipriani E, Lavitrano M, and Ardizzoia A
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- Humans, Female, Cyclin-Dependent Kinase 4, Cell Cycle, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Cyclin-Dependent Kinase 6, Breast Neoplasms drug therapy, Breast Neoplasms pathology
- Abstract
Cyclin-dependent kinase 4/6 inhibitors (CDK4/6iss) are widely used in first-line metastatic breast cancer. For patients with progression under CDK4/6is, there is currently no standard treatment recommended at the category 1 level in international guidelines. The purpose of this article is to review the cellular mechanisms underlying the resistance to CDK4/6is, as well as treatment strategies and the clinical data about the efficacy of subsequent treatments after CDK4/6is-based therapy. In the first part, this review mainly discusses cell-cycle-specific and cell-cycle-non-specific resistance to CDK4/6is, with a focus on early and late progression. In the second part, this review analyzes potential therapeutic approaches and the available clinical data on them: switching to other CDK4/6is, to another single hormonal therapy, to other target therapies (PI3K, mTOR and AKT) and to chemotherapy.
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- 2023
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11. Evaluating [ 18 F]FDG and [ 18 F]FLT Radiotracers as Biomarkers of Response for Combined Therapy Outcome in Triple-Negative and Estrogen-Receptor-Positive Breast Cancer Models.
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Rainone P, Valtorta S, Villa C, Todde S, Cadamuro M, Bertoli G, Conconi D, Lavitrano M, and Moresco RM
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- Humans, Female, Animals, Mice, Fluorodeoxyglucose F18, Positron Emission Tomography Computed Tomography, Biomarkers, Estrogens, Triple Negative Breast Neoplasms diagnostic imaging, Triple Negative Breast Neoplasms drug therapy, Metformin
- Abstract
Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [
18 F]FDG and [18 F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial-mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18 F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.- Published
- 2023
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12. Genomic Complexity and Complex Chromosomal Rearrangements in Genetic Diagnosis: Two Illustrative Cases on Chromosome 7.
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Villa N, Redaelli S, Farina S, Conconi D, Sala EM, Crosti F, Mariani S, Colombo CM, Dalprà L, Lavitrano M, Bentivegna A, and Roversi G
- Abstract
Complex chromosomal rearrangements are rare events compatible with survival, consisting of an imbalance and/or position effect of one or more genes, that contribute to a range of clinical presentations. The investigation and diagnosis of these cases are often difficult. The interpretation of the pattern of pairing and segregation of these chromosomes during meiosis is important for the assessment of the risk and the type of imbalance in the offspring. Here, we investigated two unrelated pediatric carriers of complex rearrangements of chromosome 7. The first case was a 2-year-old girl with a severe phenotype. Conventional cytogenetics evidenced a duplication of part of the short arm of chromosome 7. By array-CGH analysis, we found a complex rearrangement with three discontinuous trisomy regions (7p22.1p21.3, 7p21.3, and 7p21.3p15.3). The second case was a newborn investigated for hypodevelopment and dimorphisms. The karyotype analysis promptly revealed a structurally altered chromosome 7. The array-CGH analysis identified an even more complex rearrangement consisting of a trisomic region at 7q11.23q22 and a tetrasomic region of 4.5 Mb spanning 7q21.3 to q22.1. The mother's karyotype examination revealed a complex rearrangement of chromosome 7: the 7q11.23q22 region was inserted in the short arm at 7p15.3. Finally, array-CGH analysis showed a trisomic region that corresponds to the tetrasomic region of the son. Our work proved that the integration of several technical solutions is often required to appropriately analyze complex chromosomal rearrangements in order to understand their implications and offer appropriate genetic counseling.
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- 2023
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13. Influence of Bruton's Tyrosine Kinase (BTK) on Epithelial-Mesenchymal Transition (EMT) Processes and Cancer Stem Cell (CSC) Enrichment in Head and Neck Squamous Cell Carcinoma (HNSCC).
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Leichtle F, Betzler AC, Eizenberger C, Lesakova K, Ezić J, Drees R, Greve J, Schuler PJ, Laban S, Hoffmann TK, Cordes N, Lavitrano M, Grassilli E, and Brunner C
- Subjects
- Humans, Carcinogenesis, Cytokines, Neoplastic Stem Cells, NF-kappa B, Squamous Cell Carcinoma of Head and Neck, Agammaglobulinaemia Tyrosine Kinase, Epithelial-Mesenchymal Transition, Head and Neck Neoplasms genetics
- Abstract
Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1
+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.- Published
- 2023
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14. AhRR and PPP1R3C: Potential Prognostic Biomarkers for Serous Ovarian Cancer.
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Ardizzoia A, Jemma A, Redaelli S, Silva M, Bentivegna A, Lavitrano M, and Conconi D
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- Female, Humans, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Carcinoma, Ovarian Epithelial pathology, DNA Copy Number Variations genetics, Intracellular Signaling Peptides and Proteins metabolism, Neoplastic Stem Cells metabolism, Prognosis, Cystadenocarcinoma, Serous diagnosis, Cystadenocarcinoma, Serous genetics, Cystadenocarcinoma, Serous metabolism, Ovarian Neoplasms diagnosis, Ovarian Neoplasms genetics, Ovarian Neoplasms metabolism
- Abstract
The lack of effective screening and successful treatment contributes to high ovarian cancer mortality, making it the second most common cause of gynecologic cancer death. Development of chemoresistance in up to 75% of patients is the cause of a poor treatment response and reduced survival. Therefore, identifying potential and effective biomarkers for its diagnosis and prognosis is a strong critical need. Copy number alterations are frequent in cancer, and relevant for molecular tumor stratification and patients' prognoses. In this study, array-CGH analysis was performed in three cell lines and derived cancer stem cells (CSCs) to identify genes potentially predictive for ovarian cancer patients' prognoses. Bioinformatic analyses of genes involved in copy number gains revealed that AhRR and PPP1R3C expression negatively correlated with ovarian cancer patients' overall and progression-free survival. These results, together with a significant association between AhRR and PPP1R3C expression and ovarian cancer stemness markers, suggested their potential role in CSCs. Furthermore, AhRR and PPP1R3C's increased expression was maintained in some CSC subpopulations, reinforcing their potential role in ovarian cancer. In conclusion, we reported for the first time, to the best of our knowledge, a prognostic role of AhRR and PPP1R3C expression in serous ovarian cancer.
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- 2023
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15. Pathologist Validation of a Machine Learning-Derived Feature for Colon Cancer Risk Stratification.
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L'Imperio V, Wulczyn E, Plass M, Müller H, Tamini N, Gianotti L, Zucchini N, Reihs R, Corrado GS, Webster DR, Peng LH, Chen PC, Lavitrano M, Liu Y, Steiner DF, Zatloukal K, and Pagni F
- Subjects
- Male, Humans, Aged, Pathologists, Artificial Intelligence, Machine Learning, Risk Assessment, Colonic Neoplasms diagnosis, Adenocarcinoma
- Abstract
Importance: Identifying new prognostic features in colon cancer has the potential to refine histopathologic review and inform patient care. Although prognostic artificial intelligence systems have recently demonstrated significant risk stratification for several cancer types, studies have not yet shown that the machine learning-derived features associated with these prognostic artificial intelligence systems are both interpretable and usable by pathologists., Objective: To evaluate whether pathologist scoring of a histopathologic feature previously identified by machine learning is associated with survival among patients with colon cancer., Design, Setting, and Participants: This prognostic study used deidentified, archived colorectal cancer cases from January 2013 to December 2015 from the University of Milano-Bicocca. All available histologic slides from 258 consecutive colon adenocarcinoma cases were reviewed from December 2021 to February 2022 by 2 pathologists, who conducted semiquantitative scoring for tumor adipose feature (TAF), which was previously identified via a prognostic deep learning model developed with an independent colorectal cancer cohort., Main Outcomes and Measures: Prognostic value of TAF for overall survival and disease-specific survival as measured by univariable and multivariable regression analyses. Interpathologist agreement in TAF scoring was also evaluated., Results: A total of 258 colon adenocarcinoma histopathologic cases from 258 patients (138 men [53%]; median age, 67 years [IQR, 65-81 years]) with stage II (n = 119) or stage III (n = 139) cancer were included. Tumor adipose feature was identified in 120 cases (widespread in 63 cases, multifocal in 31, and unifocal in 26). For overall survival analysis after adjustment for tumor stage, TAF was independently prognostic in 2 ways: TAF as a binary feature (presence vs absence: hazard ratio [HR] for presence of TAF, 1.55 [95% CI, 1.07-2.25]; P = .02) and TAF as a semiquantitative categorical feature (HR for widespread TAF, 1.87 [95% CI, 1.23-2.85]; P = .004). Interpathologist agreement for widespread TAF vs lower categories (absent, unifocal, or multifocal) was 90%, corresponding to a κ metric at this threshold of 0.69 (95% CI, 0.58-0.80)., Conclusions and Relevance: In this prognostic study, pathologists were able to learn and reproducibly score for TAF, providing significant risk stratification on this independent data set. Although additional work is warranted to understand the biological significance of this feature and to establish broadly reproducible TAF scoring, this work represents the first validation to date of human expert learning from machine learning in pathology. Specifically, this validation demonstrates that a computationally identified histologic feature can represent a human-identifiable, prognostic feature with the potential for integration into pathology practice.
- Published
- 2023
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16. Insights into the Peritumoural Brain Zone of Glioblastoma: CDK4 and EXT2 May Be Potential Drivers of Malignancy.
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Giambra M, Di Cristofori A, Conconi D, Marzorati M, Redaelli S, Zambuto M, Rocca A, Roumy L, Carrabba G, Lavitrano M, Roversi G, Giussani C, and Bentivegna A
- Subjects
- Humans, Brain metabolism, Gene Expression Profiling, Cyclin-Dependent Kinase 4 genetics, Cyclin-Dependent Kinase 4 metabolism, Glioblastoma metabolism, Brain Neoplasms metabolism, Glioma metabolism
- Abstract
Despite the efforts made in recent decades, glioblastoma is still the deadliest primary brain cancer without cure. The potential role in tumour maintenance and progression of the peritumoural brain zone (PBZ), the apparently normal area surrounding the tumour, has emerged. Little is known about this area due to a lack of common definition and due to difficult sampling related to the functional role of peritumoural healthy brain. The aim of this work was to better characterize the PBZ and to identify genes that may have role in its malignant transformation. Starting from our previous study on the comparison of the genomic profiles of matched tumour core and PBZ biopsies, we selected CDK4 and EXT2 as putative malignant drivers of PBZ. The gene expression analysis confirmed their over-expression in PBZ, similarly to what happens in low-grade glioma and glioblastoma, and CDK4 high levels seem to negatively influence patient overall survival. The prognostic role of CDK4 and EXT2 was further confirmed by analysing the TCGA cohort and bioinformatics prediction on their gene networks and protein-protein interactions. These preliminary data constitute a good premise for future investigations on the possible role of CDK4 and EXT2 in the malignant transformation of PBZ.
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- 2023
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17. BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.
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Betzler AC, Strobel H, Abou Kors T, Ezić J, Lesakova K, Pscheid R, Azoitei N, Sporleder J, Staufenberg AR, Drees R, Weissinger SE, Greve J, Doescher J, Theodoraki MN, Schuler PJ, Laban S, Kibe T, Kishida M, Kishida S, Idel C, Hoffmann TK, Lavitrano M, Grassilli E, and Brunner C
- Abstract
Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.
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- 2023
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18. Co-targeting triple-negative breast cancer cells and endothelial cells by metronomic chemotherapy inhibits cell regrowth and migration via downregulation of the FAK/VEGFR2/VEGF axis and autophagy/apoptosis activation.
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Scagliotti A, Capizzi L, Cazzaniga ME, Ilari A, De Giorgi M, Cordani N, Gallazzi M, Bruno A, Pelosi G, Albini A, Lavitrano M, Grassilli E, and Cerrito MG
- Abstract
High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs' migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo . In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Scagliotti, Capizzi, Cazzaniga, Ilari, De Giorgi, Cordani, Gallazzi, Bruno, Pelosi, Albini, Lavitrano, Grassilli and Cerrito.)
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- 2022
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19. Tumor Microenvironment and Immune Escape in the Time Course of Glioblastoma.
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Virtuoso A, De Luca C, Cirillo G, Riva M, Romano G, Bentivegna A, Lavitrano M, Papa M, and Giovannoni R
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- Animals, Cell Line, Tumor, Humans, Mice, Mice, Inbred C57BL, Microglia metabolism, Tenascin metabolism, Brain Neoplasms immunology, Brain Neoplasms pathology, Glioblastoma immunology, Glioblastoma pathology, Glioma immunology, Glioma pathology, Tumor Escape, Tumor Microenvironment
- Abstract
Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a malignant prognosis. GBM is characterized by high cellular heterogeneity and its progression relies on the interaction with the central nervous system, which ensures the immune-escape and tumor promotion. This interplay induces metabolic, (epi)-genetic and molecular rewiring in both domains. In the present study, we aim to characterize the time-related changes in the GBM landscape, using a syngeneic mouse model of primary GBM. GL261 glioma cells were injected in the right striatum of immuno-competent C57Bl/6 mice and animals were sacrificed after 7, 14, and 21 days (7D, 14D, 21D). The tumor development was assessed through 3D tomographic imaging and brains were processed for immunohistochemistry, immunofluorescence, and western blotting. A human transcriptomic database was inquired to support the translational value of the experimental data. Our results showed the dynamic of the tumor progression, being established as a bulk at 14D and surrounded by a dense scar of reactive astrocytes. The GBM growth was paralleled by the impairment in the microglial/macrophagic recruitment and antigen-presenting functions, while the invasive phase was characterized by changes in the extracellular matrix, as shown by the analysis of tenascin C and metalloproteinase-9. The present study emphasizes the role of the molecular changes in the microenvironment during the GBM progression, fostering the development of novel multi-targeted, time-dependent therapies in an experimental model similar to the human disease., (© 2022. The Author(s).)
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- 2022
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20. Exome Sequencing in an ADSHE Family: VUS Identification and Limits.
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Villa C, Arrigoni F, Rivellini E, Lavitrano M, De Gioia L, Ferini-Strambi L, and Combi R
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- Humans, Exome Sequencing methods, Mutation, Pedigree, Epilepsy, Exome
- Abstract
Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is the familial form of a focal epilepsy characterized by hyperkinetic focal seizures, mainly arising during non-rapid eye movements (NREM) sleep. Mutations associated with ADSHE account for a small proportion of the genetically determined cases, suggesting the existence of other disease-causing genes. Here, we reported the results obtained by performing trio-based whole-exome sequencing (WES) in an Italian family showing ADSHE and investigated the structural impact of putative variants by in silico modeling analysis. We identified a p.(Trp276Gly) variant in MOXD1 gene encoding the monooxigenase DBH like 1 protein, cosegregating with the disease and annotated as VUS under the ACMG recommendations. Structural bioinformatic analysis predicted a high destabilizing effect of this variant, due to the loss of important hydrophilic bonds and an expansion of cavity volume in the protein hydrophobic core. Although our data support a functional effect of the p.(Trp276Gly) variant, we highlight the need to identify additional families carrying MOXD1 mutations or functional analyses in suitable models to clarify its role in ADSHE pathogenesis. Moreover, we discuss the importance of VUS reporting due to the low rate of pathogenic variant identification by NGS in epilepsy and for future reinterpretation studies.
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- 2022
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21. Paediatric biobanking for health: The ethical, legal, and societal landscape.
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Casati S, Ellul B, Mayrhofer MT, Lavitrano M, Caboux E, and Kozlakidis Z
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- Adult, Child, Europe, Humans, Parents, Translational Research, Biomedical, Biological Specimen Banks, Research Personnel
- Abstract
Biobanks play a central role in pediatric translational research, which deals primarily with genetic data from sample-based research. However, participation of children in biobanking has received only limited attention in the literature, even though research in general and in clinical trials in particular have a long history in involving minors. So, we resolved to explore specific challenging ethical, legal, and societal issues (ELSI) in the current pediatric biobanking landscape to propose a way forward for biobanking with children as partners in research. Methodologically, we first established the accessibility and utilization of pediatric biobanks, mainly in Europe. This was supported by a literature review related to children's participation, taking into account not only academic papers but also relevant guidelines and best-practices. Our findings are discussed under five themes: general vulnerability; ethical issues-balancing risks and benefits, right to an open future, return of results including secondary findings; legal issues-capacity and legal majority; societal issues-public awareness and empowerment; and responsible research with children. Ultimately, we observed an on-going shift from the parents'/guardians' consent being a sine-qua-non condition to the positive minor's agreement: confirming that the minor is the participant, not the parent(s)/guardian(s). This ethical rethinking is paving the way toward age-appropriate, dynamic and participatory models of involving minors in decision-making. However, we identified a requirement for dynamic tools to assess maturity, a lack of co-produced engagement tools and paucity of shared best practices. We highlight the need to provide empowerment and capability settings to support researchers and biobankers, and back this with practical examples. In conclusion, equipping children and adults with appropriate tools, and ensuring children's participation is at the forefront of responsible pediatric biobanking, is an ethical obligation, and a cornerstone for research integrity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Casati, Ellul, Mayrhofer, Lavitrano, Caboux and Kozlakidis.)
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- 2022
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22. Analysis of copy number alterations in bladder cancer stem cells revealed a prognostic role of LRP1B.
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Conconi D, Jemma A, Giambra M, Redaelli S, Croci GA, Dalprà L, Lavitrano M, and Bentivegna A
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- Humans, Neoplasm Recurrence, Local, Neoplastic Stem Cells, Prognosis, Receptors, LDL genetics, Urinary Bladder pathology, DNA Copy Number Variations genetics, Urinary Bladder Neoplasms genetics, Urinary Bladder Neoplasms pathology
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Purpose: Bladder cancer is the most common malignancy of the urinary tract and one of the most prevalent cancers worldwide. It represents a spectrum of diseases, from recurrent non-invasive tumors (NMIBCs) managed chronically, to muscle infiltrating and advanced-stage disease (MIBC) that requires multimodal and invasive treatment. Multiple studies have underlined the complexity of bladder tumors genome, highlighting many specific genetic lesions and genome-wide occurrences of copy-number alterations (CNAs). In this study, we analyzed CNAs of selected genes in our cohorts of cancer stem cells (CSCs) and in The Cancer Genome Atlas (TCGA-BLCA) cohort with the aim to correlate their frequency with patients' prognosis., Methods: CNAs have been verified on our array-CGH data previously reported on 19 bladder cancer biopsies (10 NMIBCs and 9 MIBCs) and 16 matched isolated CSC cultures. In addition, CNAs data have been consulted on the TCGA database, to search correlations with patients' follow-up. Finally, mRNA expression levels of LRP1B in TGCA cohort were obtained from The Human Protein Atlas., Results: We firstly identified CNAs differentially represented between TGCA data and CSCs derived from NMIBCs and MIBCs, and we correlated the presence of these CNAs with patients' follow-up. LRP1B loss was significantly increased in CSCs and linked to short-term poor prognosis, both at genomic and transcriptomic level, confirming its pivotal role in bladder cancer tumorigenesis., Conclusion: Our study allowed us to identify potential "predictive" prognostic CNAs for bladder cancer, implementing knowledge for the ultimate goal of personalized medicine., (© 2022. The Author(s).)
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- 2022
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23. BTK, the new kid on the (oncology) block?
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Grassilli E, Cerrito MG, and Lavitrano M
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In the last decade data piled up indicating that BTK - for twenty years considered as a "private matter" of bone marrow-derived cells - it is expressed and plays important and different roles also outside of the hematopoietic compartment and, most notably, in tumor cells. Initial evidence that BTK plays a critical role in B cell-derived malignancies prompted the chase for specific inhibitors, the forefather of which entered the clinic in a record time and paved the way for an ever increasing number of new molecules to be trialed. The growing interests in BTK also led to the discovery that, in solid tumors, two novel isoforms are mainly expressed and actionable liabilities for target therapy. Remarkably, the different isoforms appear to be involved in different signaling pathways which will have to be attentively specified in order to define the area of therapeutic intervention. In this perspective we briefly summarize the progress made in the last decade in studying BTK and its isoforms in cancer cells and define the open questions to be addressed in order to get the most benefits from its targeting for therapeutic purposes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grassilli, Cerrito and Lavitrano.)
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- 2022
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24. A Systematic Review of the Current Status and Quality of Radiomics for Glioma Differential Diagnosis.
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Brancato V, Cerrone M, Lavitrano M, Salvatore M, and Cavaliere C
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Radiomics is a promising tool that may increase the value of imaging in differential diagnosis (DDx) of glioma. However, implementation in clinical practice is still distant and concerns have been raised regarding the methodological quality of radiomic studies. Therefore, we aimed to systematically review the current status of radiomic studies concerning glioma DDx, also using the radiomics quality score (RQS) to assess the quality of the methodology used in each study. A systematic literature search was performed to identify original articles focused on the use of radiomics for glioma DDx from 2015. Methodological quality was assessed using the RQS tool. Spearman's correlation (ρ) analysis was performed to explore whether RQS was correlated with journal metrics and the characteristics of the studies. Finally, 42 articles were selected for the systematic qualitative analysis. Selected articles were grouped and summarized in terms of those on DDx between glioma and primary central nervous system lymphoma, those aiming at differentiating glioma from brain metastases, and those based on DDx of glioma and other brain diseases. Median RQS was 8.71 out 36, with a mean RQS of all studies of 24.21%. Our study revealed that, despite promising and encouraging results, current studies on radiomics for glioma DDx still lack the quality required to allow its introduction into clinical practice. This work could provide new insights and help to reach a consensus on the use of the radiomic approach for glioma DDx.
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- 2022
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25. Matrix metalloproteinases, purinergic signaling, and epigenetics: hubs in the spinal neuroglial network following peripheral nerve injury.
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De Luca C, Virtuoso A, Cerasuolo M, Gargano F, Colangelo AM, Lavitrano M, Cirillo G, and Papa M
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- Animals, Gliosis metabolism, Matrix Metalloproteinases metabolism, Rats, Rats, Sprague-Dawley, Sciatic Nerve injuries, Sciatic Nerve metabolism, Spinal Cord metabolism, Peripheral Nerve Injuries metabolism
- Abstract
Activation of glial cells (reactive gliosis) and the purinergic pathway, together with metalloproteinase (MMP)-induced remodeling of the neural extracellular matrix (nECM), drive maladaptive changes in the spinal cord following peripheral nerve injury (PNI). We evaluated the effects on spinal maladaptive plasticity through administration of oxidized ATP (oxATP), an antagonist of P2X receptors (P2XR), and/or GM6001, an inhibitor of MMPs, in rats following spared nerve injury (SNI) of the sciatic nerve. With morpho-molecular techniques, we demonstrated a reduction in spinal reactive gliosis and changes in the neuro-glial-nECM crosstalk via expression remodeling of P2XR, nerve growth factor (NGF) receptors (TrkA and p75), and histone deacetylase 2 (HDAC2) after treatments with oxATP/GM6001. Altogether, our data suggest that MMPs and purinergic inhibition have a modulatory impact on key proteins in the neuro-glial-nECM network, acting at different levels from intracellular signaling to epigenetic modifications., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)
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- 2022
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26. Characterization of Chromosomal Breakpoints in 12 Cases with 8p Rearrangements Defines a Continuum of Fragility of the Region.
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Redaelli S, Conconi D, Sala E, Villa N, Crosti F, Roversi G, Catusi I, Valtorta C, Recalcati MP, Dalprà L, Lavitrano M, and Bentivegna A
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- Comparative Genomic Hybridization, Gene Rearrangement, Humans, In Situ Hybridization, Fluorescence, Chromosome Aberrations, Genome
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Improvements in microarray-based comparative genomic hybridization technology have allowed for high-resolution detection of genome wide copy number alterations, leading to a better definition of rearrangements and supporting the study of pathogenesis mechanisms. In this study, we focused our attention on chromosome 8p. We report 12 cases of 8p rearrangements, analyzed by molecular karyotype, evidencing a continuum of fragility that involves the entire short arm. The breakpoints seem more concentrated in three intervals: one at the telomeric end, the others at 8p23.1, close to the beta-defensin gene cluster and olfactory receptor low-copy repeats. Hypothetical mechanisms for all cases are described. Our data extend the cohort of published patients with 8p aberrations and highlight the need to pay special attention to these sequences due to the risk of formation of new chromosomal aberrations with pathological effects.
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- 2022
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27. Can SARS-CoV-2 Infection Exacerbate Alzheimer's Disease? An Overview of Shared Risk Factors and Pathogenetic Mechanisms.
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Villa C, Rivellini E, Lavitrano M, and Combi R
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The current coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus (SARS-CoV)-2, is affecting every aspect of global society, including public healthcare systems, medical care access, and the economy. Although the respiratory tract is primarily affected by SARS-CoV-2, emerging evidence suggests that the virus may also reach the central nervous system (CNS), leading to several neurological issues. In particular, people with a diagnosis of Alzheimer's disease (AD) are a vulnerable group at high risk of contracting COVID-19, and develop more severe forms and worse outcomes, including death. Therefore, understanding shared links between COVID-19 and AD could aid the development of therapeutic strategies against both. Herein, we reviewed common risk factors and potential pathogenetic mechanisms that might contribute to the acceleration of neurodegenerative processes in AD patients infected by SARS-CoV-2.
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- 2022
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28. Inhibition of plasminogen/plasmin system retrieves endogenous nerve growth factor and adaptive spinal synaptic plasticity following peripheral nerve injury.
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Virtuoso A, Colangelo AM, Korai SA, Izzo S, Todisco A, Giovannoni R, Lavitrano M, Papa M, and Cirillo G
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- Animals, Behavior, Animal, Gliosis, Injections, Spinal, Male, Neuralgia psychology, Neuropeptides administration & dosage, Neuropeptides therapeutic use, Peripheral Nerve Injuries psychology, Rats, Rats, Sprague-Dawley, Recovery of Function, Sciatic Nerve injuries, Serpins administration & dosage, Serpins therapeutic use, Neuroserpin, Fibrinolysin antagonists & inhibitors, Nerve Growth Factors metabolism, Neuronal Plasticity, Peripheral Nerve Injuries metabolism, Peripheral Nerve Injuries physiopathology, Plasminogen antagonists & inhibitors, Spinal Cord physiopathology
- Abstract
Dysfunctions of the neuronal-glial crosstalk and/or impaired signaling of neurotrophic factors represent key features of the maladaptive changes in the central nervous system (CNS) in neuroinflammatory as neurodegenerative disorders. Tissue plasminogen activator (tPA)/plasminogen (PA)/plasmin system has been involved in either process of maturation and degradation of nerve growth factor (NGF), highlighting multiple potential targets for new therapeutic strategies. We here investigated the role of intrathecal (i.t.) delivery of neuroserpin (NS), an endogenous inhibitor of plasminogen activators, on neuropathic behavior and maladaptive synaptic plasticity in the rat spinal cord following spared nerve injury (SNI) of the sciatic nerve. We demonstrated that SNI reduced spinal NGF expression, induced spinal reactive gliosis, altering the expression of glial and neuronal glutamate and GABA transporters, reduced glutathione (GSH) levels and is associated to neuropathic behavior. Beside the increase of NGF expression, i.t. NS administration reduced reactive gliosis, restored synaptic homeostasis, GSH levels and reduced neuropathic behavior. Our results hereby highlight the essential role of tPA/PA system in the synaptic homeostasis and mechanisms of maladaptive plasticity, sustaining the beneficial effects of NGF-based approach in neurological disorders., (Copyright © 2021. Published by Elsevier Ltd.)
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- 2021
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29. The CORBEL matrix on informed consent in clinical studies: a multidisciplinary approach of Research Infrastructures Building Enduring Life-science Services.
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Colombo C, Mayrhofer MT, Kubiak C, Battaglia S, Matei M, Lavitrano M, Casati S, Chico V, Schluender I, Carapina T, and Mosconi P
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- Humans, Information Dissemination, Research Design, Research Personnel, Clinical Studies as Topic, Informed Consent
- Abstract
Background: Informed consent forms for clinical research are several and variable at international, national and local levels. According to the literature, they are often unclear and poorly understood by participants. Within the H2020 project CORBEL-Coordinated Research Infrastructures Building Enduring Life-science Services-clinical researchers, researchers in ethical, social, and legal issues, experts in planning and management of clinical studies, clinicians, researchers in citizen involvement and public engagement worked together to provide a minimum set of requirements for informed consent in clinical studies., Methods: The template was based on a literature review including systematic reviews and guidelines searched on PubMed, Embase, Cochrane Library, NICE, SIGN, GIN, and Clearinghouse databases, and on comparison of templates gathered through an extensive search on the websites of research institutes, national and international agencies, and international initiatives. We discussed the draft versions step-by-step and then we referred to it as the "matrix" to underline its modular character and indicate that it allows adaptation to the context in which it will be used. The matrix was revised by representatives of two international patient groups., Results: The matrix covers the process of ensuring that the appropriate information, context and setting are provided so that the participant can give truly informed consent. It addresses the key topics and proposes wording on how to clarify the meaning of placebo and of non-inferiority studies, the importance of individual participants' data sharing, and the impossibility of knowing in advance how the data might be used in future studies. Finally, it presents general suggestions on wording, format, and length of the information sheet., Conclusions: The matrix underlines the importance of improving the process of communication, its proper conditions (space, time, setting), and addresses the participants' lack of knowledge on how clinical research is conducted. It can be easily applied to a specific setting and could be a useful tool to identify the appropriate informed consent format for any study. The matrix is mainly intended to support multicentre interventional randomized clinical studies, but several suggestions also apply to non-interventional research., (© 2021. The Author(s).)
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- 2021
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30. p65BTK Is a Novel Biomarker and Therapeutic Target in Solid Tumors.
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Grassilli E, Cerrito MG, Bonomo S, Giovannoni R, Conconi D, and Lavitrano M
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Bruton's tyrosine kinase (BTK) is a non-receptor intracellular kinase playing a key role in the proliferation and survival of normal and malignant B-lymphocytes. Its targeting by Ibrutinib, the first specific inhibitor, represented a turning point for the therapy of certain types of B-cell leukemias/lymphomas and several more BTK inhibitors are today in the clinic or advanced clinical trials. BTK expression was successively found to occur also outside of the hematopoietic compartment. In fact, we identified p65BTK, a novel 65 kDa isoform lacking an N-term stretch of 86 amino acids (compared to the 77 kDa protein expressed in B cells) as highly expressed in colon cancer patients. We demonstrated that p65BTK is a powerful oncogene acting downstream of the RAS/MAPK pathway and necessary for RAS-mediated transformation. Notably, the kinase domain is conserved and therefore inhibited by the available BTK-targeting drugs (Ibrutinib, Spebrutinib, etc.) which we used to demonstrate that p65BTK is an actionable target in drug-resistant colorectal carcinomas. We found p65BTK expressed also in >50% non-small cell lung cancers (NSCLC) and demonstrated that it is an actionable target in KRAS -mutated/ EGFR -wild type drug-resistant NSCLC models (for which no targeted therapy is available). We also reported a significant correlation between p65BTK expression and low-grade tumors and overall survival of patients with grade III gliomas and showed that its targeting induced a significant decrease in the viability of in glioma stem cells. Finally, in ovarian cancer patients, p65BTK expression levels correlate with early relapse and shorter progression-free survival, both indicators of resistance to therapy. Remarkably, Ibrutinib is more effective than standard of care (SOC) therapeutics in in vitro and ex vivo settings. On the whole, our preclinical data indicate that, depending on the tumor type, BTK inhibitors used alone can induce cytotoxicity (gliomas), be more effective than SOC chemotherapy (ovarian cancer) or can kill drug-resistant tumor cells when used in combination with SOC chemotherapy (colon cancer and NSCLC) or targeted therapy (NSCLC and ovarian cancer), thus suggesting that p65BTK may be an actionable target in different solid tumors. In addition, our data also give the proof-of-concept for starting clinical trials using BTK inhibitors, alone or in combination, to improve the therapeutic options for solid tumors treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grassilli, Cerrito, Bonomo, Giovannoni, Conconi and Lavitrano.)
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- 2021
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31. Human Chromosome 18 and Acrocentrics: A Dangerous Liaison.
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Villa N, Redaelli S, Sala E, Conconi D, Romitti L, Manfredini E, Crosti F, Roversi G, Lavitrano M, Rodeschini O, Recalcati MP, Piazza R, Dalprà L, Riva P, and Bentivegna A
- Subjects
- Adult, Cell Line, Tumor, Female, Humans, Infant, Male, Pregnancy, Chromosomes, Human, Pair 18 genetics, Translocation, Genetic
- Abstract
The presence of thousands of repetitive sequences makes the centromere a fragile region subject to breakage. In this study we collected 31 cases of rearrangements of chromosome 18, of which 16 involved an acrocentric chromosome, during genetic screening done in three centers. We noticed a significant enrichment of reciprocal translocations between the centromere of chromosome 18 and the centromeric or pericentromeric regions of the acrocentrics. We describe five cases with translocation between chromosome 18 and an acrocentric chromosome, and one case involving the common telomere regions of chromosomes 18p and 22p. In addition, we bring evidence to support the hypothesis that chromosome 18 preferentially recombines with acrocentrics: (i) the presence on 18p11.21 of segmental duplications highly homologous to acrocentrics, that can justify a NAHR mechanism; (ii) the observation by 2D-FISH of the behavior of the centromeric regions of 18 respect to the centromeric regions of acrocentrics in the nuclei of normal subjects; (iii) the contact analysis among these regions on published Hi-C data from the human lymphoblastoid cell line (GM12878).
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- 2021
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32. The Glioblastoma Microenvironment: Morphology, Metabolism, and Molecular Signature of Glial Dynamics to Discover Metabolic Rewiring Sequence.
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Virtuoso A, Giovannoni R, De Luca C, Gargano F, Cerasuolo M, Maggio N, Lavitrano M, and Papa M
- Subjects
- Animals, Brain Neoplasms pathology, Glioblastoma pathology, Humans, Tumor Hypoxia, Warburg Effect, Oncologic, Brain Neoplasms metabolism, Glioblastoma metabolism, Tumor Microenvironment
- Abstract
Different functional states determine glioblastoma (GBM) heterogeneity. Brain cancer cells coexist with the glial cells in a functional syncytium based on a continuous metabolic rewiring. However, standard glioma therapies do not account for the effects of the glial cells within the tumor microenvironment. This may be a possible reason for the lack of improvements in patients with high-grade gliomas therapies. Cell metabolism and bioenergetic fitness depend on the availability of nutrients and interactions in the microenvironment. It is strictly related to the cell location in the tumor mass, proximity to blood vessels, biochemical gradients, and tumor evolution, underlying the influence of the context and the timeline in anti-tumor therapeutic approaches. Besides the cancer metabolic strategies, here we review the modifications found in the GBM-associated glia, focusing on morphological, molecular, and metabolic features. We propose to analyze the GBM metabolic rewiring processes from a systems biology perspective. We aim at defining the crosstalk between GBM and the glial cells as modules. The complex networking may be expressed by metabolic modules corresponding to the GBM growth and spreading phases. Variation in the oxidative phosphorylation (OXPHOS) rate and regulation appears to be the most important part of the metabolic and functional heterogeneity, correlating with glycolysis and response to hypoxia. Integrated metabolic modules along with molecular and morphological features could allow the identification of key factors for controlling the GBM-stroma metabolism in multi-targeted, time-dependent therapies.
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- 2021
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33. Patient-Derived Induced Pluripotent Stem Cells (iPSCs) and Cerebral Organoids for Drug Screening and Development in Autism Spectrum Disorder: Opportunities and Challenges.
- Author
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Villa C, Combi R, Conconi D, and Lavitrano M
- Abstract
Autism spectrum disorder (ASD) represents a group of neurodevelopmental diseases characterized by persistent deficits in social communication, interaction, and repetitive patterns of behaviors, interests, and activities. The etiopathogenesis is multifactorial with complex interactions between genetic and environmental factors. The clinical heterogeneity and complex etiology of this pediatric disorder have limited the development of pharmacological therapies. The major limit to ASD research remains a lack of relevant human disease models which can faithfully recapitulate key features of the human pathology and represent its genetic heterogeneity. Recent advances in induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of patients into all types of patient-specific neural cells, have provided a promising cellular tool for disease modeling and development of novel drug treatments. The iPSCs technology allowed not only a better investigation of the disease etiopathogenesis but also opened up the potential for personalized therapies and offered new opportunities for drug discovery, pharmacological screening, and toxicity assessment. Moreover, iPSCs can be differentiated and organized into three-dimensional (3D) organoids, providing a model which mimics the complexity of the brain's architecture and more accurately recapitulates tissue- and organ-level disease pathophysiology. The aims of this review were to describe the current state of the art of the use of human patient-derived iPSCs and brain organoids in modeling ASD and developing novel therapeutic strategies and to discuss the opportunities and major challenges in this rapidly moving field.
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- 2021
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34. Genomic and Epigenomic Profile of Uterine Smooth Muscle Tumors of Uncertain Malignant Potential (STUMPs) Revealed Similarities and Differences with Leiomyomas and Leiomyosarcomas.
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Conconi D, Redaelli S, Lissoni AA, Cilibrasi C, Perego P, Gautiero E, Sala E, Paderno M, Dalprà L, Landoni F, Lavitrano M, Roversi G, and Bentivegna A
- Subjects
- Adult, Aged, Case-Control Studies, DNA Methylation, Female, Follow-Up Studies, Genomics, Humans, Leiomyoma genetics, Leiomyosarcoma genetics, Middle Aged, Prognosis, Smooth Muscle Tumor genetics, Uterine Neoplasms genetics, Biomarkers, Tumor genetics, Epigenomics, Gene Expression Regulation, Neoplastic, Leiomyoma pathology, Leiomyosarcoma pathology, Smooth Muscle Tumor pathology, Uterine Neoplasms pathology
- Abstract
Uterine smooth muscle tumors of uncertain malignant potential (STUMPs) represent a heterogeneous group of tumors that cannot be histologically diagnosed as unequivocally benign or malignant. For this reason, many authors are working to obtain a better definition of diagnostic and prognostic criteria. In this work, we analyzed the genomic and epigenomic profile of uterine smooth muscle tumors (USMTs) in order to find similarities and differences between STUMPs, leiomyosarcomas (LMSs) and leiomyomas (LMs), and possibly identify prognostic factors in this group of tumors. Array-CGH data on 23 USMTs demonstrated the presence of a more similar genomic profile between STUMPs and LMSs. Some genes, such as PRKDC and PUM2 , with a potential prognostic value, were never previously associated with STUMP. The methylation data appears to be very promising, especially with regards to the divergent profile found in the sample that relapsed, characterized by an overall CGI hypomethylation. Finally, the Gene Ontology analysis highlighted some cancer genes that could play a pivotal role in the unexpected aggressive behavior that can be found in some of these tumors. These genes could prove to be prognostic markers in the future.
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- 2021
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35. Persistence of Anti-SARS-CoV-2 Antibodies in Non-Hospitalized COVID-19 Convalescent Health Care Workers.
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Bruni M, Cecatiello V, Diaz-Basabe A, Lattanzi G, Mileti E, Monzani S, Pirovano L, Rizzelli F, Visintin C, Bonizzi G, Giani M, Lavitrano M, Faravelli S, Forneris F, Caprioli F, Pelicci PG, Natoli G, Pasqualato S, Mapelli M, and Facciotti F
- Abstract
Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length Nucleocapsid protein (N). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in using serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.
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- 2020
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36. Molecular and Imaging Biomarkers in Alzheimer's Disease: A Focus on Recent Insights.
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Villa C, Lavitrano M, Salvatore E, and Combi R
- Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly, affecting millions of people worldwide and clinically characterized by a progressive and irreversible cognitive decline. The rapid increase in the incidence of AD highlights the need for an easy, efficient and accurate diagnosis of the disease in its initial stages in order to halt or delay the progression. The currently used diagnostic methods rely on measures of amyloid-β (Aβ), phosphorylated (p-tau) and total tau (t-tau) protein levels in the cerebrospinal fluid (CSF) aided by advanced neuroimaging techniques like positron emission tomography (PET) and magnetic resonance imaging (MRI). However, the invasiveness of these procedures and the high cost restrict their utilization. Hence, biomarkers from biological fluids obtained using non-invasive methods and novel neuroimaging approaches provide an attractive alternative for the early diagnosis of AD. Such biomarkers may also be helpful for better understanding of the molecular mechanisms underlying the disease, allowing differential diagnosis or at least prolonging the pre-symptomatic stage in patients suffering from AD. Herein, we discuss the advantages and limits of the conventional biomarkers as well as recent promising candidates from alternative body fluids and new imaging techniques.
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- 2020
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37. Instability of Short Arm of Acrocentric Chromosomes: Lesson from Non-Acrocentric Satellited Chromosomes. Report of 24 Unrelated Cases.
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Redaelli S, Conconi D, Villa N, Sala E, Crosti F, Corti C, Catusi I, Garzo M, Romitti L, Martinoli E, Patrizi A, Malgara R, Recalcati MP, Dalprà L, Lavitrano M, Riva P, Roversi G, and Bentivegna A
- Subjects
- Cytogenetic Analysis, Humans, In Situ Hybridization, Fluorescence, Karyotyping, Chromosome Aberrations, Chromosomes genetics, DNA, Satellite genetics, Translocation, Genetic
- Abstract
Satellited non-acrocentric autosomal chromosomes (ps-qs-chromosomes) are the result of an interchange between sub- or telomeric regions of autosomes and the p arm of acrocentrics. The sequence homology at the rearrangement breakpoints appears to be, among others, the most frequent mechanism generating these variant chromosomes. The unbalanced carriers of this type of translocation may or may not display phenotypic abnormalities. With the aim to understand the causative mechanism, we revised all the ps-qs-chromosomes identified in five medical genetics laboratories, which used the same procedures for karyotype analysis, reporting 24 unrelated cases involving eight chromosomes. In conclusion, we observed three different scenarios: true translocation, benign variant and complex rearrangement. The detection of translocation partners is essential to evaluate possible euchromatic unbalances and to infer their effect on phenotype. Moreover, we emphasize the importance to perform both, molecular and conventional cytogenetics methods, to better understand the behavior of our genome., Competing Interests: The authors declare no conflict of interest.
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- 2020
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38. BTK inhibitors synergise with 5-FU to treat drug-resistant TP53-null colon cancers.
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Lavitrano M, Ianzano L, Bonomo S, Cialdella A, Cerrito MG, Pisano F, Missaglia C, Giovannoni R, Romano G, McLean CM, Voest EE, D'Amato F, Noli B, Ferri GL, Agostini M, Pucciarelli S, Helin K, Leone BE, Canzonieri V, and Grassilli E
- Subjects
- Agammaglobulinaemia Tyrosine Kinase antagonists & inhibitors, Agammaglobulinaemia Tyrosine Kinase metabolism, Animals, Antineoplastic Combined Chemotherapy Protocols pharmacology, Apoptosis drug effects, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Disease Progression, Drug Resistance, Neoplasm drug effects, Drug Synergism, E2F Transcription Factors metabolism, Fluorouracil administration & dosage, Fluorouracil pharmacology, Genes, p53, Humans, Mice, Nude, Molecular Targeted Therapy methods, Neoplasm Staging, Organoids drug effects, Protein Isoforms antagonists & inhibitors, Protein Isoforms metabolism, Protein Kinase Inhibitors pharmacology, Transforming Growth Factor beta1 metabolism, Tumor Cells, Cultured, Xenograft Model Antitumor Assays methods, Antineoplastic Combined Chemotherapy Protocols therapeutic use, Colonic Neoplasms drug therapy, Protein Kinase Inhibitors administration & dosage
- Abstract
Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK - a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2020
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39. Sperm-Mediated Genetic Modifications.
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Lavitrano M, Farina L, Cerrito MG, and Giovannoni R
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- Animals, DNA genetics, Fertilization in Vitro methods, Male, Mice, Spermatozoa metabolism, Animals, Genetically Modified genetics, Gene Editing methods, Gene Transfer Techniques, Spermatozoa growth & development
- Abstract
The ability to introduce controlled modifications of the genome of animals represents an important tool for biomedical and veterinary research. Among transgenic techniques, we describe here the sperm-mediated gene transfer method that is based on the spontaneous ability of sperm cells to bind and internalize exogenous DNA and to carry it to the oocyte during fertilization, producing genetically modified animals.
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- 2020
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40. Correction: Data in question: A survey of European biobank professionals on ethical, legal and societal challenges of biobank research.
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Goisauf M, Martin G, Bentzen HB, Budin-Ljøsne I, Ursin L, Durnová A, Leitsalu L, Smith K, Casati S, Lavitrano M, Mascalzoni D, Boeckhout M, and Mayrhofer MT
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0221496.].
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- 2019
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41. Long Non-Coding RNAs and Related Molecular Pathways in the Pathogenesis of Epilepsy.
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Villa C, Lavitrano M, and Combi R
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- Animals, Apoptosis genetics, Biomarkers, Cell Differentiation genetics, Epigenesis, Genetic, Gene Expression Profiling, Humans, Neuronal Plasticity genetics, Neurons cytology, Neurons metabolism, RNA Interference, Epilepsy etiology, Epilepsy metabolism, Gene Expression Regulation, RNA, Long Noncoding genetics, Signal Transduction
- Abstract
Epilepsy represents one of the most common neurological disorders characterized by abnormal electrical activity in the central nervous system (CNS). Recurrent seizures are the cardinal clinical manifestation. Although it has been reported that the underlying pathological processes include inflammation, changes in synaptic strength, apoptosis, and ion channels dysfunction, currently the pathogenesis of epilepsy is not yet completely understood. Long non-coding RNAs (lncRNAs), a class of long transcripts without protein-coding capacity, have emerged as regulatory molecules that are involved in a wide variety of biological processes. A growing number of studies reported that lncRNAs participate in the regulation of pathological processes of epilepsy and they are dysregulated during epileptogenesis. Moreover, an aberrant expression of lncRNAs linked to epilepsy has been observed both in patients and in animal models. In this review, we summarize latest advances concerning the mechanisms of action and the involvement of the most dysregulated lncRNAs in epilepsy. However, the functional roles of lncRNAs in the disease pathogenesis are still to be explored and we are only at the beginning. Additional studies are needed for the complete understanding of the underlying mechanisms and they would result in the use of lncRNAs as diagnostic biomarkers and novel therapeutic targets., Competing Interests: The authors declare no conflicts of interest.
- Published
- 2019
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42. Data in question: A survey of European biobank professionals on ethical, legal and societal challenges of biobank research.
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Goisauf M, Martin G, Bentzen HB, Budin-Ljøsne I, Ursin L, Durnová A, Leitsalu L, Smith K, Casati S, Lavitrano M, Mascalzoni D, Boeckhout M, and Mayrhofer MT
- Subjects
- Europe, Humans, Informed Consent ethics, Internet, Surveys and Questionnaires, Biological Specimen Banks, Biomedical Research ethics, Biomedical Research legislation & jurisprudence
- Abstract
Biobanks have evolved, and their governance procedures have undergone important transformations. Our paper examines this issue by focusing on the perspective of the professionals working in management or scientific roles in research-based biobanks, who have an important impact on shaping these transformations. In particular, it highlights that recent advances in molecular medicine and genomic research have raised a range of ethical, legal and societal implications (ELSI) related to biobank-based research, impacting directly on regulations and local practices of informed consent (IC), private-public partnerships (PPPs), and engagement of participants. In our study, we investigate the ways that these concerns influence biobanking practices and assess the level of satisfaction of the cross-national biobanking research communities with the ELSI related procedures that are currently in place. We conducted an online survey among biobankers and researchers to investigate secondary use of data, informing and/or re-contacting participants, sharing of data with third parties from industry, participant engagement, and collaboration with industrial partners. Findings highlight the need for a more inclusive and transparent biobanking practice where biobanks are seen in a more active role in providing information and communicating with participants; the need to improve the current IC procedures and the role of biobanks in sharing of samples and data with industry partners and different countries, and the need for practical, tangible and hands-on ethical and legal guidance., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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43. Role of Bruton's Tyrosine Kinase in Stage III Colorectal Cancer.
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Basile D, Gerratana L, Buonadonna A, Garattini SK, Perin T, Grassilli E, Miolo G, Cerrito MG, Belluco C, Bertola G, De Paoli A, Cannizzaro R, Lavitrano M, Puglisi F, and Canzonieri V
- Abstract
Background: Bruton's tyrosine kinase (BTK) is involved in the immune response and its deficiency impairs B cell maturation. We evaluated the expression of a novel BTK isoform, p65BTK, in colorectal cancer (CRC), to identify its impact on survival., Materials and Methods: This retrospective study evaluated 87 consecutive stage III CRC patients treated at the National Cancer Institute of Aviano (1999-2017). Multiple specimens were collected and analyzed for staining intensity and percentage of tumor cells positive for p65BTK. Prognostic impact was tested by univariate Cox regression analysis., Results: After a median follow-up of 82.59 months, median disease-free survival (DFS) and overall survival (OS) were 11.67 months and 31.33 months, respectively. Interestingly, 10% of patients did not express p65BTK. For the immunohistochemistry IHC intensity 1, the best cutoff point was 1% of p65BTK positivity; for IHC intensity 2, it was 50%; and for IHC intensity 3, it was 80%. Through univariate analysis, patients with highly expressed p65BTK (IHC intensity 3 and ≥80%) were shown to have the worst prognosis in terms of DFS (HR: 6.23; p = 0.005; 95% C.I. 1.75-22.79) and OS (HR: 2.54; p = 0.025; 95% C.I. 1.12-5.76)., Conclusions: p65BTK is frequently expressed in CRC and, if highly expressed, is an unfavourable prognostic factor. However, further confirmation is needed and its potential targeting needs to be studied.
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- 2019
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44. p65BTK is a novel potential actionable target in KRAS-mutated/EGFR-wild type lung adenocarcinoma.
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Giordano F, Vaira V, Cortinovis D, Bonomo S, Goedmakers J, Brena F, Cialdella A, Ianzano L, Forno I, Cerrito MG, Giovannoni R, Ferri GL, Tasciotti E, Vicent S, Damarco F, Bosari S, Lavitrano M, and Grassilli E
- Subjects
- Adenocarcinoma of Lung drug therapy, Adenocarcinoma of Lung pathology, Agammaglobulinaemia Tyrosine Kinase antagonists & inhibitors, Agammaglobulinaemia Tyrosine Kinase genetics, Animals, Antineoplastic Agents pharmacology, Cell Line, Tumor, Cell Proliferation, Cell Survival genetics, Drug Synergism, ErbB Receptors genetics, Gene Expression, Gene Expression Regulation, Neoplastic, Humans, Immunohistochemistry, Mice, Neoplasm Staging, Protein Isoforms, Protein Kinase Inhibitors pharmacology, Signal Transduction, Adenocarcinoma of Lung genetics, Adenocarcinoma of Lung metabolism, Agammaglobulinaemia Tyrosine Kinase metabolism, Biomarkers, Tumor, Mutation, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
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.
- Published
- 2019
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45. Specific Expression of a New Bruton Tyrosine Kinase Isoform (p65BTK) in the Glioblastoma Gemistocytic Histotype.
- Author
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Sala L, Cirillo G, Riva G, Romano G, Giussani C, Cialdella A, Todisco A, Virtuoso A, Cerrito MG, Bentivegna A, Grassilli E, Ardizzoia A, Bonoldi E, Giovannoni R, Papa M, and Lavitrano M
- Abstract
Bruton's tyrosine-kinase (BTK) is a non-receptor tyrosine kinase recently associated with glioma tumorigenesis and a novel prognostic marker for poor survival in patients with glioma. The p65BTK is a novel BTK isoform involved in different pathways of drug resistance of solid tumors, thus we aimed to investigate the expression and the putative role of p65BTK in tumors of the central nervous system (CNS). We selected a large cohort of patients with glial tumors ( n = 71) and analyzed the expression of p65BTK in different histotypes and correlation with clinical parameters. Sections were stained with glial fibrillary acidic protein (GFAP), p53, epidermal growth factor receptor (EGFR), S100, vimentin, and epithelial membrane antigen (EMA) antibodies. Glioma stem cell (GSC) lines, isolated from glioblastoma multiforme (GBM), were treated with different concentrations of ibrutinib, a specific inhibitor of BTK, in order to evaluate their metabolic activity, mitotic index and mortality. Moreover, an orthotopic xenotransplant of GSC from human GBM was used to evaluate the expression of p65BTK in the brain of immunodeficient mice. p65BTK was expressed in GSC and in gemistocytes in human gliomas at different histological grade. We found a significant correlation between BTK expression and low-grade (LG) tumors ( p ≤ 0.05) and overall survival (OS) of patients with grade III gliomas ( p ≤ 0.05), suggestive of worst prognosis. Interestingly, the expression of p65BTK remained restricted exclusively to gemistocytic cells in the xenograft mouse model. Ibrutinib administration significantly reduced metabolic activity and mitotic index and increased mortality in GSC, highlighting the specific role of p65BTK in cell proliferation and survival. In conclusion, our data demonstrated that p65BTK is expressed in glioma tumors, restricted to gemistocytic cells, has a key role in GSC and has a bad prognostic value, thus highlighting the importance of future research for targeted therapy of human gliomas.
- Published
- 2019
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46. APOA-1Milano muteins, orally delivered via genetically modified rice, show anti-atherogenic and anti-inflammatory properties in vitro and in Apoe -/- atherosclerotic mice.
- Author
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Romano G, Reggi S, Kutryb-Zajac B, Facoetti A, Chisci E, Pettinato M, Giuffrè MR, Vecchio F, Leoni S, De Giorgi M, Avezza F, Cadamuro M, Crippa L, Leone BE, Lavitrano M, Rivolta I, Barisani D, Smolenski RT, and Giovannoni R
- Subjects
- Administration, Oral, Animals, Atherosclerosis metabolism, Atherosclerosis pathology, Dose-Response Relationship, Drug, Food, Genetically Modified, Male, Mice, Mice, Knockout, Plaque, Atherosclerotic metabolism, Plaque, Atherosclerotic pathology, Anti-Inflammatory Agents administration & dosage, Apolipoprotein A-I administration & dosage, Apolipoproteins E deficiency, Atherosclerosis drug therapy, Oryza genetics, Plaque, Atherosclerotic drug therapy
- Abstract
Background: Atherosclerosis is a slowly progressing, chronic multifactorial disease characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue that drives to the formation of asymmetric focal thickenings in the tunica intima of large and mid-sized arteries. Despite the high therapeutic potential of ApoA-1 proteins, the purification and delivery into the disordered organisms of these drugs is still limited by low efficiency in these processes., Methods and Results: We report here a novel production and delivery system of anti-atherogenic APOA-1Milano muteins (APOA-1M) by means of genetically modified rice plants. APOA-1M, delivered as protein extracts from transgenic rice seeds, significantly reduced macrophage activation and foam cell formation in vitro in oxLDL-loaded THP-1 model. The APOA-1M delivery method and therapeutic efficacy was tested in healthy mice and in Apoe
-/- mice fed with high cholesterol diet (Western Diet, WD). APOA-1M rice milk significantly reduced atherosclerotic plaque size and lipids composition in aortic sinus and aortic arch of WD-fed Apoe-/- mice as compared to wild type rice milk-treated, WD-fed Apoe-/- mice. APOA-1M rice milk also significantly reduced macrophage number in liver of WD-fed Apoe-/- mice as compared to WT rice milk treated mice., Translational Impact: The delivery of therapeutic APOA-1M full length proteins via oral administration of rice seeds protein extracts (the 'rice milk') to the disordered organism, without any need of purification, might overcome the main APOA1-based therapies' limitations and improve the use of this molecules as therapeutic agents for cardiovascular patients., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2018
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47. Valproic Acid Inhibits Proliferation and Reduces Invasiveness in Glioma Stem Cells Through Wnt/β Catenin Signalling Activation.
- Author
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Riva G, Cilibrasi C, Bazzoni R, Cadamuro M, Negroni C, Butta V, Strazzabosco M, Dalprà L, Lavitrano M, and Bentivegna A
- Abstract
Glioblastoma is the most common malignant brain tumour in adults. The failure of current therapies can be ascribed to glioma stem cells (GSCs), which can rapidly repopulate the tumour following the initial treatment. The study of histone deacetylase inhibitors, such as valproic acid (VPA), is becoming an attractive field in cancer research. However, the exact mechanisms underlying its anti-cancer effect remain to be elucidated due to its pleiotropic effects on several cell-signalling pathways. Ingenuity Pathway Analysis (IPA) bioinformatics analysis was performed on genome-wide data regarding GSCs methylome to identify the signalling pathways mainly affected by methylation changes induced by VPA. Real time PCR and luciferase reporter assay were used to better investigate VPA effects on Wnt/β-catenin signalling pathway. VPA effect on GSC proliferation was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Trypan blue assays. Finally, VPA impact on GSC motility was demonstrated by Boyden chamber assay and further confirmed evaluating the expression levels or localisation, through western blot or immunofluorescence, of Twist1, Snail1, E-Cadherin and N-Cadherin. The bioinformatics analyses performed on GSCs methylome highlighted that Wnt/β-catenin signalling was affected by the methylation changes induced by VPA, which could influence its activation status. In particular, we pointed out a general activation of this pathway after VPA exposure, which was accompanied by an inhibitory potential on GSCs proliferation. Finally, we also proved VPA's ability to inhibit GSCs invasion through Snail1 and Twist1 downregulation and E-Cadherin relocalisation. VPA treatment may represent a new, interesting therapeutic approach to affect GSC proliferation and motility, but further investigations are certainly needed.
- Published
- 2018
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48. Familiar unbalanced complex rearrangements involving 13 p-arm: description of two cases.
- Author
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Conconi D, Villa N, Redaelli S, Sala E, Crosti F, Maitz S, Rigoldi M, Parini R, Dalprà L, Lavitrano M, and Roversi G
- Abstract
Background: Copy number variations (CNVs) are largely known today, but their position is rarely established by fluorescence in situ hybridization (FISH) or karyotype analysis., Case Presentation: We described two families with copy number gain in which FISH analysis with the specific subtelomeric probe of chromosome 4q and 7q evidenced a third signal at band 13p11.2. Genomic study by array comparative genomic hybridization defined the triple dose segment. In the first case, the duplicate tract is free of known genes, in the second one it contained three expressed genes., Conclusions: The CNV localization on the short arm of an acrocentric chromosome could explain the lack of phenotypic effect, being known the regulatory role of heterochromatin in the position-effect silencing. Furthermore, we would like to underline the importance of using complementary techniques such as FISH and array-CGH to obtain a better definition of genomic rearrangements., Competing Interests: Not applicable.Written informed consent was obtained from the patient’s parents for publication of this case report and any accompanying images.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- Published
- 2018
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49. Metronomic combination of Vinorelbine and 5Fluorouracil is able to inhibit triple-negative breast cancer cells. Results from the proof-of-concept VICTOR-0 study.
- Author
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Cerrito MG, De Giorgi M, Pelizzoni D, Bonomo SM, Digiacomo N, Scagliotti A, Bugarin C, Gaipa G, Grassilli E, Lavitrano M, Giovannoni R, Bidoli P, and Cazzaniga ME
- Abstract
Triple Negative Breast Cancer (TNBC) is an aggressive neoplasia with median Overall Survival (OS) less than two years. Despite the availability of new drugs, the chance of survival of these patients did not increase. The combination of low doses of drugs in a metronomic schedule showed efficacy in clinical trials, exhibiting an anti-proliferative and anti-tumour activity. In Victor-2 study we recently evaluated a new metronomic combination (mCHT) of Capecitabine (CAPE) and Vinorelbine (VNR) in breast cancer patients showing a disease control rate with a median Progression-Free Survival (PFS) of 4.7 months in 28 TNBC patients. Here in Victor-0 study, we examined the effect of mCHT vs standard (STD) schedule of administration of different combinations of 5-Fluorouracil (5FU), the active metabolite of CAPE, and VNR in TNBC cell lines MDA-MB-231 and BT-549. A significant anti-proliferative activity was observed in cells treated with metronomic vs STD administration of 5FU or VNR alone. Combination of the two drugs showed an additive inhibitor effect on cell growth in both cell lines. Moreover, after exposure of cells to 5FU and VNR under mCHT or conventional schedule of administration we also observed a downregulation of chemoresistance factor Bcl-2, changes in pro-apoptotic protein Bax and in cleaved effector caspase-3 and increased expression of LC3A/B autophagy protein. Our results therefore suggest that molecular mechanisms implicated in apoptosis and autophagy as well as the cross-talk between these two forms of cell death in MDA-MB-231 and BT-549 cells treated with 5FU and VNR is dose- and schedule-dependent and provide some insights about the roles of autophagy and senescence in 5FU/VNR-induced cell death., Competing Interests: CONFLICTS OF INTEREST None.
- Published
- 2018
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50. Enhancing Reuse of Data and Biological Material in Medical Research: From FAIR to FAIR-Health.
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Holub P, Kohlmayer F, Prasser F, Mayrhofer MT, Schlünder I, Martin GM, Casati S, Koumakis L, Wutte A, Kozera Ł, Strapagiel D, Anton G, Zanetti G, Sezerman OU, Mendy M, Valík D, Lavitrano M, Dagher G, Zatloukal K, van Ommen GB, and Litton JE
- Subjects
- Guidelines as Topic, Humans, Biological Specimen Banks organization & administration, Biological Specimen Banks standards, Confidentiality standards, Databases, Factual standards, Information Dissemination methods
- Abstract
The known challenge of underutilization of data and biological material from biorepositories as potential resources for medical research has been the focus of discussion for over a decade. Recently developed guidelines for improved data availability and reusability-entitled FAIR Principles (Findability, Accessibility, Interoperability, and Reusability)-are likely to address only parts of the problem. In this article, we argue that biological material and data should be viewed as a unified resource. This approach would facilitate access to complete provenance information, which is a prerequisite for reproducibility and meaningful integration of the data. A unified view also allows for optimization of long-term storage strategies, as demonstrated in the case of biobanks. We propose an extension of the FAIR Principles to include the following additional components: (1) quality aspects related to research reproducibility and meaningful reuse of the data, (2) incentives to stimulate effective enrichment of data sets and biological material collections and its reuse on all levels, and (3) privacy-respecting approaches for working with the human material and data. These FAIR-Health principles should then be applied to both the biological material and data. We also propose the development of common guidelines for cloud architectures, due to the unprecedented growth of volume and breadth of medical data generation, as well as the associated need to process the data efficiently.
- Published
- 2018
- Full Text
- View/download PDF
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