Publications by authors named "Cristiana Carniti"

33 Publications

T-cell immune response after mRNA SARS-CoV-2 vaccines is frequently detected also in the absence of seroconversion in patients with lymphoid malignancies.

Br J Haematol 2021 Oct 14. Epub 2021 Oct 14.

School of Medicine, University of Milano, Italy.

Patients affected by lymphoid malignancies (LM) are frequently immune-compromised, suffering increased mortality from COVID-19. This prospective study evaluated serological and T-cell responses after complete mRNA vaccination in 263 patients affected by chronic lymphocytic leukaemia, B- and T-cell lymphomas and multiple myeloma. Results were compared with those of 167 healthy subjects matched for age and sex. Overall, patient seroconversion rate was 64·6%: serological response was lower in those receiving anti-cancer treatments in the 12 months before vaccination: 55% vs 81·9% (P < 0·001). Anti-CD20 antibody plus chemotherapy treatment was associated with the lowest seroconversion rate: 17·6% vs. 71·2% (P < 0·001). In the multivariate analysis conducted in the subgroup of patients on active treatment, independent predictors for seroconversion were: anti-CD20 treatment (P < 0·001), aggressive B-cell lymphoma diagnosis (P = 0·002), and immunoglobulin M levels <40 mg/dl (P = 0·030). The T-cell response was evaluated in 99 patients and detected in 85 of them (86%). Of note, 74% of seronegative patients had a T-cell response, but both cellular and humoral responses were absent in 13·1% of cases. Our findings raise some concerns about the protection that patients with LM, particularly those receiving anti-CD20 antibodies, may gain from vaccination. These patients should strictly maintain all the protective measures.
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http://dx.doi.org/10.1111/bjh.17877DOI Listing
October 2021

Dose-Adjusted Epoch and Rituximab for the treatment of double expressor and double hit diffuse large B-cell lymphoma: impact of TP53 mutations on clinical outcome.

Haematologica 2021 Jul 22. Epub 2021 Jul 22.

Department of Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano; Chair of Hematology University of Milano.

Diffuse Large B-Cell Lymphoma (DLBCL) is a heterogeneous disease, including one-third of cases overexpressing MYC and BCL2 proteins (Double Expressor Lymphoma, DEL) and 5-10% of patients with chromosomal rearrangements of MYC, BCL2 and/or BCL-6 (Double/Triple-Hit Lymphomas, DH/TH). TP53 mutations are detected in 20-25% of DEL. We report the efficacy of dose-adjusted EPOCH and rituximab (DA-EPOCH-R) in a series of 122 consecutive patients, including DEL (n=81, 66%), DEL-MYC (n=9, 7%), DEL-BCL2 (n=13, 11%), or High-Grade Lymphomas (DH/TH) (n=19, 16%). Central nervous system (CNS) prophylaxis included intravenous methotrexate (n=66), intrathecal chemotherapy (IT) (n=40) or no prophylaxis (n=16). Sixty-seven pts (55%) had high-intermediate or high International Prognostic Index (IPI) and 30 (25%) had high CNS-IPI. The 2-year progressionfree survival (PFS) and overall survival (OS) for the entire study population were 74% and 84%, respectively. There was a trend for inferior OS for DH/TH (2-year OS: 66%, p=0.058) as compared to all the others. The outcome was significantly better for the IPI 0-2 versus IPI 3-5 (OS: 98% vs. 72%, p=0.002). DA-EPOCH-R did not overcome the negative prognostic value of TP53 mutations: 2-year OS of 62% versus 88% (p=0.036) were observed for mutated as compared to wild-type cases, respectively. Systemic CNS prophylaxis conferred a better 2-year OS (94%) as compared to IT or no prophylaxis (76% and 65%, respectively; p= 0.008). DA-EPOCH-R treatment resulted in a favorable outcome in patients with DEL and DEL with single rearrangement, whereas those with multiple genetic alterations such as DEL-DH/TH and TP53 mutated cases still have an inferior outcome.
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http://dx.doi.org/10.3324/haematol.2021.278638DOI Listing
July 2021

A rare biclonal Hairy Cell Leukemia disclosed by an integrated diagnostic approach: A case report.

Cytometry B Clin Cytom 2021 Nov 18;100(6):692-694. Epub 2020 Dec 18.

Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

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http://dx.doi.org/10.1002/cyto.b.21980DOI Listing
November 2021

CDKN2A deletion is a frequent event associated with poor outcome in patients with peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

Haematologica 2021 11 1;106(11):2918-2926. Epub 2021 Nov 1.

Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy; Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan.

Nodal peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) remains a diagnosis encompassing a heterogenous group of PTCL cases not fitting criteria for more homogeneous subtypes. They are characterized by a poor clinical outcome when treated with anthracycline-containing regimens. A better understanding of their biology could improve prognostic stratification and foster the development of novel therapeutic approaches. Recent targeted and whole exome sequencing studies have shown recurrent copy number abnormalities (CNAs) with prognostic significance. Here, investigating 5 formalin-fixed, paraffin embedded cases of PTCL-NOS by whole genome sequencing (WGS), we found a high prevalence of structural variants and complex events, such as chromothripsis likely responsible for the observed CNAs. Among them, CDKN2A and PTEN deletions emerged as the most frequent aberration, as confirmed in a final cohort of 143 patients with nodal PTCL. The incidence of CDKN2A and PTEN deletions among PTCL-NOS was 46% and 26%, respectively. Furthermore, we found that co-occurrence of CDKN2A and PTEN deletions is an event associated with PTCL-NOS with absolute specificity. In contrast, these deletions were rare and never co-occurred in angioimmunoblastic and anaplastic lymphomas. CDKN2A deletion was associated with shorter overall survival in multivariate analysis corrected by age, IPI, transplant eligibility and GATA3 expression (adjusted HR =2.53; 95% CI 1.006-6.3; p=0.048). These data suggest that CDKN2A deletions may be relevant for refining the prognosis of PTCL-NOS and their significance should be evaluated in prospective trials.
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http://dx.doi.org/10.3324/haematol.2020.262659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8561277PMC
November 2021

Timing the initiation of multiple myeloma.

Nat Commun 2020 04 21;11(1):1917. Epub 2020 Apr 21.

Myeloma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2-3 decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.
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http://dx.doi.org/10.1038/s41467-020-15740-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174344PMC
April 2020

Integrative analysis of the genomic and transcriptomic landscape of double-refractory multiple myeloma.

Blood Adv 2020 03;4(5):830-844

Department of Clinical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient.
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http://dx.doi.org/10.1182/bloodadvances.2019000779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065476PMC
March 2020

Tyrosine kinase inhibition to improve anthracycline-based chemotherapy efficacy in T-cell lymphoma.

Br J Cancer 2019 10 2;121(7):567-577. Epub 2019 Sep 2.

Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Background: Anthracycline-containing regimens, namely cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) or CHOEP (CHOP + etoposide), represent the current standard of care for patients with newly diagnosed peripheral T-cell lymphomas (PTCLs), although responses are unsatisfactory. In this study, we investigated the pathways able to mitigate the sensitivity to CHOP-based regimens in preclinical models of T-cell lymphoma (TCL) to select agents for the development of CHOP-based drug combinations.

Methods: We performed gene expression profiling of malignant T-cell lines exposed to CHOEP; flow cytometry was employed to study the effects of drug combinations on cell viability, cell cycle distribution, apoptosis and mitochondrial membrane depolarisation. Western blot analyses were performed to study cell signalling downstream of the T-cell receptor and apoptosis. The in vivo effect of the drug combination was tested in xenograft models.

Results: We highlighted a modulation of tyrosine kinases belonging to the T-cell receptor pathway upon chemotherapy that provided the rationale for combining the tyrosine kinase inhibitor dasatinib with CHOEP. Dasatinib improves CHOEP activity and reduces viability in vitro. Furthermore, combination treatment results in tumour growth inhibition in in vivo xenograft mouse models.

Conclusions: Our data provide the rationale for clinical testing of the dasatinib-CHOEP combination in patients with T-cell lymphoma.
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http://dx.doi.org/10.1038/s41416-019-0557-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889385PMC
October 2019

Integration of transcriptional and mutational data simplifies the stratification of peripheral T-cell lymphoma.

Am J Hematol 2019 06 19;94(6):628-634. Epub 2019 Mar 19.

Department of Oncology and Hemato-Oncology, University of Milan, Milan, Italy.

The histological diagnosis of peripheral T-cell lymphoma (PTCL) can represent a challenge, particularly in the case of closely related entities such as angioimmunoblastic T-lymphoma (AITL), PTCL-not otherwise specified (PTCL-NOS), and ALK-negative anaplastic large-cell lymphoma (ALCL). Although gene expression profiling and next generations sequencing have been proven to define specific features recurrently associated with distinct entities, genomic-based stratifications have not yet led to definitive diagnostic criteria and/or entered into the routine clinical practice. Herein, to improve the current molecular classification between AITL and PTCL-NOS, we analyzed the transcriptional profiles from 503 PTCLs stratified according to their molecular configuration and integrated them with genomic data of recurrently mutated genes (RHOA , TET2, IDH2 , and DNMT3A) in 53 cases (39 AITLs and 14 PTCL-NOSs) included in the series. Our analysis unraveled that the mutational status of RHOA , TET2, and DNMT3A poorly correlated, individually, with peculiar transcriptional fingerprints. Conversely, in IDH2 samples a strong transcriptional signature was identified that could act as a surrogate for mutational status. The integrated analysis of clinical, mutational, and molecular data led to a simplified 19-gene signature that retains high accuracy in differentiating the main nodal PTCL entities. The expression levels of those genes were confirmed in an independent cohort profiled by RNA-sequencing.
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http://dx.doi.org/10.1002/ajh.25450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684242PMC
June 2019

Noninvasive Molecular Monitoring in Multiple Myeloma Patients Using Cell-Free Tumor DNA: A Pilot Study.

J Mol Diagn 2018 11 28;20(6):859-870. Epub 2018 Aug 28.

Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy; Department of Hematology, Università degli Studi di Milano, Milano, Italy.

Novel treatments for multiple myeloma (MM) have increased rates of complete response, raising interest in more accurate methods to evaluate residual disease. Cell-free tumor DNA (cfDNA) analysis may represent a minimally invasive approach complementary to multiparameter flow cytometry (MFC) and molecular methods on bone marrow aspirates. A sequencing approach using the Ion Torrent Personal Genome Machine was applied to identify clonal IGH gene rearrangements in tumor plasma cells (PCs) and in serial plasma samples of 25 patients with MM receiving second-line therapy. The same clonal IGH rearrangement identified in tumor PCs was detected in paired plasma samples, and levels of IGH cfDNA correlated with outcome and mirrored tumor dynamics evaluated using conventional laboratory parameters. In addition, IGH cfDNA levels reflected the number of PCs enumerated by MFC immunophenotyping even in the complete response context. Patients determined by MFC to be free of minimal residual disease were characterized by low frequencies of tumor clonotypes in cfDNA and longer survival. This pilot study supports the clinical applicability of the noninvasive monitoring of tumor levels in plasma samples of patients with MM by IGH sequencing.
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http://dx.doi.org/10.1016/j.jmoldx.2018.07.006DOI Listing
November 2018

Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and disease subgroups.

Leukemia 2018 12 22;32(12):2604-2616. Epub 2018 May 22.

Harvard Medical School, LeBow Institute for Myeloma Therapeutics and Jerome Lipper Center for Multiple Myeloma Research, Dana-Farber Cancer Institute, Boston, MA, USA.

In multiple myeloma, next-generation sequencing (NGS) has expanded our knowledge of genomic lesions, and highlighted a dynamic and heterogeneous composition of the tumor. Here we used NGS to characterize the genomic landscape of 418 multiple myeloma cases at diagnosis and correlate this with prognosis and classification. Translocations and copy number abnormalities (CNAs) had a preponderant contribution over gene mutations in defining the genotype and prognosis of each case. Known and novel independent prognostic markers were identified in our cohort of proteasome inhibitor and immunomodulatory drug-treated patients with long follow-up, including events with context-specific prognostic value, such as deletions of the PRDM1 gene. Taking advantage of the comprehensive genomic annotation of each case, we used innovative statistical approaches to identify potential novel myeloma subgroups. We observed clusters of patients stratified based on the overall number of mutations and number/type of CNAs, with distinct effects on survival, suggesting that extended genotype of multiple myeloma at diagnosis may lead to improved disease classification and prognostication.
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http://dx.doi.org/10.1038/s41375-018-0037-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092251PMC
December 2018

Next-generation sequencing of a family with a high penetrance of monoclonal gammopathies for the identification of candidate risk alleles.

Cancer 2017 Oct 23;123(19):3701-3708. Epub 2017 May 23.

Department of Hematology and Pediatric Onco-Hematology, IRCCS Foundation National Cancer Institute, Milan, Italy.

Background: The authors describe a family with a high penetrance of plasma cell dyscrasias, suggesting inheritance of an autosomal dominant risk allele.

Methods: The authors performed whole-exome sequencing and reported on a combined approach aimed at the identification of causative variants and risk loci, using the wealth of data provided by this approach.

Results: The authors identified gene mutations and single-nucleotide polymorphisms of potential significance, and pinpointed a known risk locus for myeloma as a potential area of transmissible risk in the family.

Conclusions: To the authors' knowledge, the current study is the first to provide a whole-exome sequencing approach to such cases, and a framework analysis that could be applied to further understanding of the inherited risk of developing plasma cell dyscrasias. Cancer 2017;123:3701-3708. © 2017 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.30777DOI Listing
October 2017

Circulating miRNA panel for prediction of acute graft-versus-host disease in lymphoma patients undergoing matched unrelated hematopoietic stem cell transplantation.

Exp Hematol 2016 07 21;44(7):624-634.e1. Epub 2016 Mar 21.

Department of Hematology and Pediatric Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Acute graft-versus-host disease (aGVHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Noninvasive diagnostic and prognostic tests for aGVHD are currently lacking, but would be beneficial in predicting aGVHD and improving the safety of allo-HSCT. Circulating microRNAs exhibit marked stability and may serve as biomarkers in several clinical settings. Here, we evaluated the use of circulating microRNAs as predictive biomarkers of aGVHD in lymphoma patients after allo-HSCT from matched unrelated donors (MUDs). After receiving informed consent, we prospectively collected plasma samples from 24 lymphoma patients before and after unmanipulated MUD allo-HSCT; microRNAs were then isolated. Fourteen patients developed aGVHD symptoms at a median of 48 days (range: 32-90) post-transplantation. Two patients developed intestinal GVHD, eight cutaneous GVHD, and four multiorgan GVHD. The microRNA expression profile was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MicroRNAs 194 and 518f were significantly upregulated in aGVHD samples compared with samples taken from non-aGVHD patients. Remarkably, these upregulated microRNAs could be detected before the onset of aGVHD. Pathway prediction analysis indicated that these microRNAs may regulate critical pathways involved in aGVHD pathogenesis. Considering the noninvasive characteristics of plasma sampling and the feasibility of detecting miRNAs after allo-HSCT using real-time polymerase chain reaction, our results indicate that circulating microRNAs have the potential to enable an earlier aGVHD diagnosis and might assist in individualizing therapeutic strategies after MUD allo-HSCT. Nevertheless, standardization of blood sampling and analysis protocols is mandatory for the introduction of miRNA profiling into routine clinical use.
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http://dx.doi.org/10.1016/j.exphem.2016.03.005DOI Listing
July 2016

Pharmacologic Inhibition of JAK1/JAK2 Signaling Reduces Experimental Murine Acute GVHD While Preserving GVT Effects.

Clin Cancer Res 2015 Aug 14;21(16):3740-9. Epub 2015 May 14.

Department of Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Purpose: Immune-mediated graft-versus-tumor (GVT) effects can occur after allogeneic hematopoietic stem cell transplantation (HSCT), but GVT is tightly linked to its main complication, graft-versus-host disease (GVHD). Strategies aimed at modulating GVHD, while maintaining the GVT effect, are needed to improve the cure rate of transplant. Given the emerging role of Janus-activated kinase (JAK) signaling in lymphoproliferative and myeloproliferative diseases and its established function at dictating T-cell differentiation, we postulated that JAKs might be potential therapeutic targets through a pharmacologic approach.

Experimental Design: We examined the effect of JAK1/JAK2 modulation by ruxolitinib in a mouse model of fully MHC mismatched bone marrow transplant comprising in vivo tumor inoculation.

Results: JAK1/JAK2 inhibition by ruxolitinib improved both overall survival (P = 0.03) and acute GVHD pathologic score at target organs (P ≤ 0.001) of treated mice. In addition, treatment with ruxolitinib was associated with a preserved GVT effect, as evidenced by reduction of tumor burden (P = 0.001) and increase of survival time (P = 0.01). JAK1/JAK2 inhibition did not impair the in vivo acquisition of donor T-cell alloreactivity; this observation may account, at least in part, to the preserved GVT effect. Rather, JAK1/JAK2 inhibition of GVHD was associated with the modulation of chemokine receptor expression, which may have been one factor in the reduced infiltration of donor T cells in GVHD target organs.

Conclusions: These data provide further evidence that JAK inhibition represents a new and potentially clinically relevant approach to GVHD prevention.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-2758DOI Listing
August 2015

Role of naive-derived T memory stem cells in T-cell reconstitution following allogeneic transplantation.

Blood 2015 Apr 5;125(18):2855-64. Epub 2015 Mar 5.

Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy;

Early T-cell reconstitution following allogeneic transplantation depends on the persistence and function of T cells that are adoptively transferred with the graft. Posttransplant cyclophosphamide (pt-Cy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains undetermined. Here, we show that T memory stem cells (TSCM), which demonstrated superior reconstitution capacity in preclinical models, are the most abundant circulating T-cell population in the early days following haploidentical transplantation combined with pt-Cy and precede the expansion of effector cells. Transferred naive, but not TSCM or conventional memory cells preferentially survive cyclophosphamide, thus suggesting that posttransplant TSCM originate from naive precursors. Moreover, donor naive T cells specific for exogenous and self/tumor antigens persist in the host and contribute to peripheral reconstitution by differentiating into effectors. Similarly, pathogen-specific memory T cells generate detectable recall responses, but only in the presence of the cognate antigen. We thus define the cellular basis of T-cell reconstitution following pt-Cy at the antigen-specific level and propose to explore naive-derived TSCM in the clinical setting to overcome immunodeficiency. These trials were registered at www.clinicaltrials.gov as #NCT02049424 and #NCT02049580.
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http://dx.doi.org/10.1182/blood-2014-11-608406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424633PMC
April 2015

Graft monocytic myeloid-derived suppressor cell content predicts the risk of acute graft-versus-host disease after allogeneic transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.

Biol Blood Marrow Transplant 2014 Dec 20;20(12):2049-55. Epub 2014 Sep 20.

Hematology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. Electronic address:

Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Lin(low/neg)HLA-DR(-)CD11b(+)CD33(+)CD14(+)) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD.
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http://dx.doi.org/10.1016/j.bbmt.2014.09.011DOI Listing
December 2014

Impact of cytomegalovirus replication and cytomegalovirus serostatus on the outcome of patients with B cell lymphoma after allogeneic stem cell transplantation.

Biol Blood Marrow Transplant 2014 Jun 28;20(6):885-90. Epub 2014 Feb 28.

Department of Hematology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; Department of Hematology, Università degli Studi di Milano, Milano, Italy.

Cytomegalovirus (CMV) replication after allogeneic hematopoietic stem cell transplantation (HSCT) was historically associated with increased nonrelapse mortality (NRM). More recently, different groups have reported an association between CMV replication and reduced risk of acute myeloid leukemia (AML) relapse. Given the conflicting results, we evaluated the impact of CMV replication and other covariates on the outcome of a retrospective cohort of 265 adults with B cell lymphoma receiving allogeneic HSCT from HLA-identical siblings or alternative donors. In time-dependent multivariate analysis, CMV replication, evaluated by pp65 antigenemia, had no independent effect on the risk of relapse (hazard ratio [HR], 1.0; 95% confidence interval [CI], .6 to 1.6; P = .9), although it was associated with a reduced overall survival (HR, 2.0; 95% CI, 1.3 to 3.2; P = .001) and an increased NRM (HR, 2.5; 95% CI, 1.1 to 5.3; P = .01). Consistently, donor and/or recipient CMV seropositivity were not associated with a different outcome relative to CMV double-negative serostatus. In multivariate models, a diagnosis of follicular lymphoma (P < .0001) and pretransplantation complete remission status (P < .0001) were the main independent predictors for improved relapse-free survival. In summary, contrary to what is observed in patients with AML, this report identifies no independent role for CMV replication or serostatus on the relapse of patients with B cell lymphomas undergoing allogeneic HSCT.
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http://dx.doi.org/10.1016/j.bbmt.2014.02.015DOI Listing
June 2014

Molecular methods for detection of minimal residual disease following transplantation in lymphoid and plasma cell disorders.

Methods Mol Biol 2014 ;1109:209-37

Division of Hematology, Fondazione IRCCS Istituto Nazionale Tumori Milano, University of Milano, Milan, Italy.

Relapse represents the main cause of treatment failure after stem cell transplantation (SCT). Thus, monitoring of minimal residual disease (MRD) in allografted patients allows an early detection of recurrence and a subsequent intervention prior to clinically detectable relapse. MRD assessment by polymerase chain reaction-based methods is currently part of the routine clinical management of patients with chronic myeloid leukemia after allo-SCT. It is also recognized that it is a useful prognostic tool in several mature lymphoid and plasma cell disorders such as chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, and multiple myeloma. In some of these entities, clinical trials employing MRD as a decision-making tool are currently ongoing and will define whether sensitive MRD detection allows for earlier therapeutic intervention to improve the outcome of SCT. We here discuss the methods of MRD evaluation in lymphoid and plasma cell disorders following transplantation with the ultimate aim of providing critical information for the setup of molecular approaches to detect MRD.
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http://dx.doi.org/10.1007/978-1-4614-9437-9_12DOI Listing
September 2014

Long-term patterns of humoral and cellular response after vaccination against influenza A (H1N1) in patients with hematologic malignancies.

Eur J Haematol 2012 Aug 31;89(2):111-9. Epub 2012 May 31.

Hematology and Bone Marrow Transplant Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Objectives: The efficacy of a novel vaccine against influenza virus A (H1N1) in patients with hematologic malignancies is largely unknown.

Methods: We prospectively evaluated the humoral and cellular immune responses after one injection of monovalent adjuvanted 2009 H1N1 vaccine in 47 adults with hematologic malignancies and 77 controls by hemagglutination-inhibition assay and flow-cytometry analysis on day 0, 28, 50, and 90.

Results: On day 28 postvaccination, patients had lower seroprotection (95.2% vs. 75.2%, P < 0.01) and seroconversion (88.7% vs. 51.1%, P < 0.01) rates, as well as geometric mean titer (GMT; 256 vs. 134, P < 0.05), relative to controls. Response to vaccination varied according to the evaluated time point and the patient status: Patients not receiving chemotherapy had seroprotection and GMTs similar to controls in all time points, while patients receiving chemotherapy or allogeneic hematopoietic stem cell transplant (HSCT) had lower seroprotection and seroconversion levels than controls on day 28 and 50. EMEA cutoffs for efficacy were reached from day 28 by patients in follow-up or under treatment and only from day 90 by those with HSCT, especially if still under immunosuppressants. Patients treated with immunomodulatory drugs had higher antibody responses in terms of seroprotection and GMTs. T- and NK cell-mediated responses mounted from day 50 and did not differ between patients and controls.

Conclusions: According to EMEA recommendation, H1N1 vaccination strategy was effective at protecting most of the hematologic patients, but needed to be improved in those more immunocompromised.
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http://dx.doi.org/10.1111/j.1600-0609.2012.01793.xDOI Listing
August 2012

Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways.

J Proteome Res 2011 Sep 29;10(9):4196-207. Epub 2011 Jul 29.

Proteomics Laboratory, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and βIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.
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http://dx.doi.org/10.1021/pr200344nDOI Listing
September 2011

Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects.

Toxicology 2011 Apr 4;283(1):18-23. Epub 2011 Feb 4.

UO Ematologia 1-Centro Trapianti di Midollo, Milan, Italy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34+ cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34+ cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.
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http://dx.doi.org/10.1016/j.tox.2011.01.025DOI Listing
April 2011

Radioimmunotherapy and secondary leukemia: a case report.

Leuk Res 2010 Jan 15;34(1):e1-4. Epub 2009 May 15.

This study describes a patient with a relapsed, diffuse, large B cell lymphoma (DLBCL) treated with radioimmunotherapy (RIT) with yttrium-90 ((90)Y)-ibritumomab tiuxetan (Zevalin) who 5 months later developed acute myeloid leukemia (AML) with inversion of chromosome 16. Our data indicate that molecular biological techniques should be used in selected cases to integrate data obtained with standard cytogenetics: using RT-PCR we showed that inversion of chromosome 16 was already present before RIT, in striking contrast to the normal karyotype found with conventional cytogenetics. This approach will allow investigators to avoid misleading data and provide support for conclusions regarding the side effects of treatment.
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http://dx.doi.org/10.1016/j.leukres.2009.04.028DOI Listing
January 2010

Qualitative and quantitative polymerase chain reaction monitoring of minimal residual disease in relapsed chronic lymphocytic leukemia: early assessment can predict long-term outcome after reduced intensity allogeneic transplantation.

Haematologica 2009 May 18;94(5):654-62. Epub 2009 Apr 18.

Division of Hematology-Bone Marrow Transplantation, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, Milan, Italy.

Background: The graft-versus-leukemia effect is able to induce clinical responses in patients with chronic lymphocytic leukemia treated with a reduced intensity conditioning regimen, followed by allogeneic stem cell transplantation. We investigated whether molecular remissions could be attained after reduced intensity conditioning and allogeneic stem cell transplantation in patients with relapsed chronic lymphocytic leukemia and whether the assessment of minimal residual disease might be used to predict the clinical outcome.

Design And Methods: Minimal residual disease was monitored by polymerase chain reaction using the immunoglobulin heavy-chain gene rearrangement as a molecular marker in 29 relapsed patients who achieved complete remission following reduced intensity conditioning and allogeneic stem cell transplantation. A nested-polymerase chain reaction with patient-specific primers derived from complementarity determining regions (CDR2 and CDR3) was carried out in all the patients. Real-time polymerase chain reaction was performed in patients whose nested reaction gave positive or mixed results.

Results: Three patterns of minimal residual disease were observed: negative (31%), mixed (24%), and always positive (45%). The cumulative incidence of relapse according to the minimal residual disease status at 6 and 12 months after transplantation was significantly different between polymerase chain reaction-negative and -positive patients (p=0.031 and p=0.04, respectively). Two-year disease-free survival was 93% and 46% for polymerase chain reaction-negative and -positive patients at 6 months after transplantation, respectively (p=0.012). Similarly, 2-year disease-free survival was 100% and 57% for polymerase chain reaction-negative and -positive patients at 12 months, respectively (p=0.037). No clinical or biological factors were predictive of the achievement of polymerase chain reaction negativity after allogeneic stem cell transplantation. Graft-versus-host disease was more frequent in patients who did not relapse (p=0.04). Quantitative monitoring of minimal residual disease was able to identify polymerase chain reaction-positive patients with a higher risk of relapse.

Conclusions: These findings demonstrate that relapsed patients can achieve molecular remission after reduced intensity conditioning and allogeneic stem cell transplantation and suggest a minimal residual disease-driven intervention that might be useful to prevent overt hematologic relapse.
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http://dx.doi.org/10.3324/haematol.2008.000273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675677PMC
May 2009

Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery.

Blood 2009 May 11;113(19):4771-9. Epub 2009 Feb 11.

Division of Hematology and Bone Marrow Transplantation, Istituto Nazionale dei Tumori, Milan, Italy.

Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34(+) cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.
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http://dx.doi.org/10.1182/blood-2008-10-183723DOI Listing
May 2009

NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

Blood 2007 Oct 30;110(7):2600-9. Epub 2007 May 30.

Department of Clinical Medicine, University of Milano-Bicocca, Monza, Italy.

The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.
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http://dx.doi.org/10.1182/blood-2006-01-028647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1988934PMC
October 2007

RET is constitutively activated by novel tandem mutations that alter the active site resulting in multiple endocrine neoplasia type 2B.

Cancer Res 2006 Oct;66(20):10179-87

Cancer Research UK Department of Oncology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.

Constitutive activation of the RET receptor tyrosine kinase underlies the genesis and progression of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer predisposition. Importantly, although kinase activation represents a common theme in neoplasias, not all activating mutations are functionally equivalent. Consistent with this, we ascertained a patient with classical features of MEN 2B, but lacking either of the classical mutations in RET (M918T or A883F). Instead, the patient harbors a novel pair of germ line missense mutations in cis at codons 804 and 805. We evaluated the potential physiochemical effects of these substitutions in silico, predicting both to be moderately deleterious in isolation, but severely deleterious in combination. Consistent with this postulate, we show that the identified tandem mutations (V804M/E805K) are biologically active, transforming cells in culture and that their transforming capacity in combination is distinctly synergistic. Furthermore, the V804M/E805K tandem lesion confers resistance to the small molecule receptor tyrosine kinase inhibitor, PP1, suggesting a mode of action distinct from that known for classical MEN 2B mutations. To address this question, we used homology molecular modeling in silico to model the active site of RET. We predict that RET804 constitutes a critical gatekeeper residue that, when mutated in combination with RET805, induces a conformational change in the hinge region that locks the active site in a position permissive for ATP hydrolysis. Our findings have implications both in the clinic and in the successful development of novel kinase-targeted anticancer drugs.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-0884DOI Listing
October 2006

The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease.

Am J Pathol 2006 Apr;168(4):1262-75

Department of Experimental Oncology and Laboratories, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via G. Venezian 1, 20133 Milan, Italy.

In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung's disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1606559PMC
http://dx.doi.org/10.2353/ajpath.2006.050607DOI Listing
April 2006

A novel activating mutation in the RET tyrosine kinase domain mediates neoplastic transformation.

Mol Endocrinol 2006 Jul 9;20(7):1633-43. Epub 2006 Feb 9.

Cambridge Institute for Medical Research, Cancer Research UK Department of Oncology, University of Cambridge, Hills Road, Cambridge CB2 2XY, UK.

We report the finding of a novel missense mutation at codon 833 in the tyrosine kinase of the RET proto-oncogene in a patient with a carcinoma of the thyroid. In vitro experiments demonstrate that the R833C mutation induces transformed foci only when present in the long 3' splice isoform and, in keeping with a model in which the receptor has to dimerize to be completely activated, glial cell line-derived neurotrophic factor stimulation leads the RET(R833C) receptor to a higher level of activation. Tyrosine kinase assays show that the RET(R833C) long isoform has weak intrinsic kinase activity and phosphorylation of an exogenous substrate is not elevated even in the presence of glial cell line-derived neurotrophic factor. Furthermore, the R833C mutation is capable of sustaining the transformed phenotype in vivo but does not confer upon the transformed cells the ability to degrade the basement membrane in a manner analogous to metastasis. Our functional characterization of the R833C substitution suggests that, like the V804M and S891A mutations, this tyrosine kinase mutation confers a weak activating potential upon RET. This is the first report demonstrating that the introduction of an intracellular cysteine can activate RET. However, this does not occur via dimerization in a manner analogous to the extracellular cysteine mutants.
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http://dx.doi.org/10.1210/me.2004-0447DOI Listing
July 2006

RETMEN2A and RETMEN2B oncoproteins are targets of PP1 inhibitor.

Tumori 2003 Sep-Oct;89(5):550-2

Department of Experimental Oncology, National Cancer Institute, Milan, Italy.

Medullary thyroid carcinoma (MTC) responds very poorly to chemotherapy. Mutations in the RET gene are critical for MTC pathogenesis. RET therefore represents a rational target for the development of novel MTC therapies. The accumulation of evidence from laboratory studies strongly suggests that PP1 inhibitor is a cytostatic agent for cells expressing RET oncoproteins. PP1 functions as a potent and selective inhibitor of RET oncoprotein phosphorylation, promoting its proteasomal degradation.
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February 2004
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