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Callari M.,Fondazione IRCCS Instituto Nazionale dei Tumori | Lembo A.,University of Turin | Musella V.,Fondazione IRCCS Instituto Nazionale dei Tumori | Cappelletti V.,Fondazione IRCCS Instituto Nazionale dei Tumori | And 3 more authors.
PLoS ONE | Year: 2014

Formalin fixed paraffin-embedded (FFPE) tumor specimens are the conventionally archived material in clinical practice, representing an invaluable tissue source for biomarkers development, validation and routine implementation. For many prospective clinical trials, this material has been collected allowing for a prospective-retrospective study design which represents a successful strategy to define clinical utility for candidate markers. Gene expression data can be obtained even from FFPE specimens with the broadly used Affymetrix HG-U133 Plus 2.0 microarray platform. Nevertheless, important major discrepancies remain in expression data obtained from FFPE compared to fresh-frozen samples, prompting the need for appropriate data processing which could help to obtain more consistent results in downstream analyses. In a publicly available dataset of matched frozen and FFPE expression data, the performances of different normalization methods and specifically designed Chip Description Files (CDFs) were compared. The use of an alternative CDFs together with fRMA normalization significantly improved frozen-FFPE sample correlations, frozen-FFPE probeset correlations and agreement of differential analysis between different tumor subtypes. The relevance of our optimized data processing was assessed and validated using two independent datasets. In this study we demonstrated that an appropriate data processing can significantly improve the reliability of gene expression data derived from FFPE tissues using the standard Affymetrix platform. Tools for the implementation of our data processing algorithm are made publicly available at http://www.biocut.unito.it/cdf-ffpe/. © 2014 Callari et al.


Callari M.,Fondazione IRCCS Instituto Nazionale Dei Tumori | Cappelletti V.,Fondazione IRCCS Instituto Nazionale Dei Tumori | D'Aiuto F.,Fondazione IRCCS Instituto Nazionale Dei Tumori | Musella V.,Fondazione IRCCS Instituto Nazionale Dei Tumori | And 9 more authors.
Clinical Cancer Research | Year: 2016

Purpose: In spite of improvements of average benefit from adjuvant/neoadjuvant treatments, there are still individual patients with early breast cancer at high risk of relapse. We explored the association with outcome of robust gene cluster based metagenes linked to proliferation, ER-related genes, and immune response to identify those high-risk patients. Experimental Design: A total of 3,847 publicly available geneexpression profiles were analyzed (untreated, N = 826; tamoxifen- treated, N = 685; chemotherapy-treated, N = 1,150). Genes poorly performing in formalin-fixed samples were removed. Outcomes of interest were pathologic-complete response (PCR) and distant metastasis-free survival (DMFS). In ERHER2, the proliferation and ER-related metagenes were combined to define three risk groups. In HER2 and ER HER2 risk groups were defined by tertiles of an immune-related metagene. Results: The high-proliferation/low-ER group of ERHER2 breast cancer had significantly higher PCR rate [OR, 5.01 (1.76 17.99), P = 0.005], but poorer outcome [HR = 3.73 (1.638.51), P = 0.0018] than the low-proliferation/high-ER. A similar association with outcome applied to patients with residual disease (RD) after neoadjuvant chemotherapy (P = 0.01). In ER HER2 and HER2 breast cancer, immune metagene in the high tertile was linked to higher PCR [33.7% vs. 11.6% in high and low tertile, respectively; OR, 3.87 (1.798.95); P = 0.0009]. In ER HER2, after adjuvant/neoadjuvant chemotherapy, 5-year DMFS was 85.4% for high-tertile immune metagene, and 43.9% for low tertile. The outcome association was similar in patients with RD (P = 0.0055). In HER2 breast cancer treated with chemotherapy the association with risk of relapse was not significant. Conclusions: Wedeveloped metagene-based predictors able to define low and high risk of relapse after adjuvant/neoadjuvant therapy. High-risk patients so defined should be preferably considered for trials with investigational agents. © 2015 American Association for Cancer Research.


Piemonti L.,San Raffaele Scientific Institute | Everly M.J.,Terasaki Foundation Laboratory | Maffi P.,San Raffaele Scientific Institute | Scavini M.,San Raffaele Scientific Institute | And 15 more authors.
Diabetes | Year: 2013

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes. © 2013 by the American Diabetes Association.


Carrera P.,Unit of Genomics for Diagnosis of Human Pathologies | Carrera P.,Laboratory of Clinical Molecular Biology | Di Resta C.,Unit of Genomics for Diagnosis of Human Pathologies | Volonteri C.,Vita-Salute San Raffaele University | And 38 more authors.
Clinica Chimica Acta | Year: 2015

Background: Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infancy, affecting preterm children with low birth weight. The disease has a multifactorial aetiology with a significant genetic component; until now published association studies have identified several candidate genes but only few of these data has been replicated. In this pilot study, we approached exome sequencing aimed at identifying non-common variants, which are expected to have a stronger phenotypic effect. Materials and methods: We performed this study on 26 Italian severely affected BPD preterm unrelated newborns, homogeneously selected from a large prospective cohort. We used an Illumina HiSeq 2000 for sequencing. Data analysis was focussed on genes previously associated to BPD susceptibility and to new candidates in related pathways, highlighted by a prioritization analysis performed using ToppGene Suite. Results: By exome sequencing, we identified 3369 novel variants, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (. TLR) family. All of them have been confirmed with Sanger sequencing. Conclusions: Potential candidate genes have been discovered in this preliminary study; the pathogenic role of identified variants will need to be confirmed with functional and segregation studies and possibly with further methods, able to evaluate the collective influence of rare variants.Moreover, additional candidates will be tested and genetic analysis will be extended to all affected children. © 2015 Elsevier B.V.


PubMed | University of Toronto, Vita-Salute San Raffaele University, Center for Translational Genomics and Bioinformatics and San Raffaele Telethon Institute for Gene Therapy
Type: Journal Article | Journal: Cancer cell | Year: 2016

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.


PubMed | Laboratory of Clinical Molecular Biology, Unit of Genomics for Diagnosis of Human Pathologies, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics
Type: Journal Article | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2015

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infancy, affecting preterm children with low birth weight. The disease has a multifactorial aetiology with a significant genetic component; until now published association studies have identified several candidate genes but only few of these data has been replicated. In this pilot study, we approached exome sequencing aimed at identifying non-common variants, which are expected to have a stronger phenotypic effect.We performed this study on 26 Italian severely affected BPD preterm unrelated newborns, homogeneously selected from a large prospective cohort. We used an Illumina HiSeq 2000 for sequencing. Data analysis was focussed on genes previously associated to BPD susceptibility and to new candidates in related pathways, highlighted by a prioritization analysis performed using ToppGene Suite.By exome sequencing, we identified 3369 novel variants, with a median of 400 variations per sample. The top candidate genes highlighted were NOS2, MMP1, CRP, LBP and the toll-like receptor (TLR) family. All of them have been confirmed with Sanger sequencing.Potential candidate genes have been discovered in this preliminary study; the pathogenic role of identified variants will need to be confirmed with functional and segregation studies and possibly with further methods, able to evaluate the collective influence of rare variants. Moreover, additional candidates will be tested and genetic analysis will be extended to all affected children.


Nucera S.,San Raffaele Telethon Institute for Gene Therapy | Giustacchini A.,San Raffaele Telethon Institute for Gene Therapy | Giustacchini A.,Vita-Salute San Raffaele University | Boccalatte F.,San Raffaele Telethon Institute for Gene Therapy | And 20 more authors.
Cancer Cell | Year: 2016

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden. © 2016 Elsevier Inc.

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