Publications by authors named "Donatella Raspadori"

50 Publications

First reported case of secondary mixed phenotype acute leukemia after multiple myeloma.

Am J Blood Res 2021 20;11(1):123-131. Epub 2021 Feb 20.

Hematology Unit, University of Siena, Azienda Ospedaliera Universitaria Senese Siena, Italy.

In recent years the outcome of patients with multiple myeloma (MM) has significantly improved, due to new drugs. However, some agents, i.e. the alkylating drug melphalan, can be associated with an increased incidence of secondary malignancies. Myelodysplastic syndromes and acute myeloid leukemia are reported in the literature, and rarely acute lymphoblastic leukemia. Here we describe a unique case of a 56-years old female patient affected by MM since 2015 in complete remission after autologous stem cell transplant and in lenalidomide maintenance, who developed 2 years later mixed phenotype acute leukemia (MPAL). The patient, refractory to both lymphoblastic and myeloid acute leukemia regimens, achieved complete remission with bi-specific anti-CD19/anti-CD3 monoclonal antibody blinatumomab and with hypomethylating agent azacytidine plus the BCL-2 inhibitor venetoclax. She then underwent hematopoietic stem cell transplantation from HLA-identical sibling donor and she is still in complete remission after 9 months. To the best of our knowledge, there are no cases in the literature describing MPAL after autologous transplant for MM. Our patient was treated with blinatumomab and venetoclax and achieved complete remission 9 months from allogeneic transplant. The mechanism underlying the development of MPAL is not completely understood and therapies are still lacking. In this context the combination of blinatumomab, azacytidine and venetoclax successfully used in this patient may provide food for thought for further studies in this rare setting of patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010609PMC
February 2021

Long-Term Safety of Rapid Daratumumab Infusions in Multiple Myeloma Patients.

Front Oncol 2020 21;10:570187. Epub 2020 Dec 21.

Hematology Unit, University of Siena, Azienda Ospedaliero Universitaria Senese, Siena, Italy.

Multiple myeloma survival has significantly improved in recent years, due to novel agents that are available for treatment. The anti-CD38 monoclonal antibody Daratumumab is particularly efficient for patients with relapse/refractory disease, and many studies have shown its unprecedented efficacy also as a first treatment. However, to avoid the incidence of infusion reactions, long infusion schedules of 8 h at first dose and 4 h in the following doses are required, which can reduce the compliance of patients and health care professionals. A reduced infusion time of 90 min has been reported previously, but data are missing on the prolonged safety of this over time as well as the efficacy of this approach. In this work, we investigate the safety of 484 rapid Daratumumab infusions given early after the second dose over a 22 months period in 39 myeloma patients.
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http://dx.doi.org/10.3389/fonc.2020.570187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7783633PMC
December 2020

Venetoclax in association with decitabine as effective bridge to transplant in a case of relapsed early T-cell lymphoblastic leukemia.

Clin Case Rep 2020 Oct 7;8(10):2000-2002. Epub 2020 Jul 7.

Hematology Unit Azienda Ospedaliera Universitaria Senese University of Siena Siena Italy.

A case of an early-relapsed high-risk T-ALL with high BCL-2 expression on leukemic blasts was successfully treated with decitabine and venetoclax, achieving a CR. We suggest decitabine and venetoclax should be synergistic in BCL2-positive ALL.
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http://dx.doi.org/10.1002/ccr3.3041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562845PMC
October 2020

Minimal Residual Disease in Multiple Myeloma: State of the Art and Applications in Clinical Practice.

J Pers Med 2020 Sep 10;10(3). Epub 2020 Sep 10.

Division of Hematology, University of Siena, 53100 Siena, Italy.

Novel drugs have revolutionized multiple myeloma therapy in the last 20 years, with median survival that has doubled to up to 8-10 years. The introduction of therapeutic strategies, such as consolidation and maintenance after autologous stem cell transplants, has also ameliorated clinical results. The goal of modern therapies is becoming not only complete remission, but also the deepest possible remission. In this context, the evaluation of minimal residual disease by techniques such as next-generation sequencing (NGS) and next-generation flow (NGF) is becoming part of all new clinical trials that test drug efficacy. This review focuses on minimal residual disease approaches in clinical trials, with particular attention to real-world practices.
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http://dx.doi.org/10.3390/jpm10030120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565263PMC
September 2020

Flow Cytometry Assessment of CD26 Leukemic Stem Cells in Peripheral Blood: A Simple and Rapid New Diagnostic Tool for Chronic Myeloid Leukemia.

Cytometry B Clin Cytom 2019 07 3;96(4):294-299. Epub 2019 Feb 3.

Hematology Unit, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Background: Recent investigations in chronic myeloid leukemia (CML) have focused on the identification and characterization of leukemic stem cells (LSCs). These cells reside within the CD34 /CD38 /Lin fraction and score positive for CD26 (dipeptidylpeptidase IV) a marker, expressed in both bone marrow (BM) and peripheral blood (PB) samples, that discriminates CML cells from normal hematopoietic stem cells (HSCs) or from LSCs of other myeloid neoplasms. CD26 evaluation could be a useful tool to improve the identification of CML LCSs by using flow-cytometry assay.

Methods: CD26 LSCs have been isolated from EDTA PB and BM samples of patients with leucocytosis suspected for CML. Analysis of LSCs CML has been performed by using custom-made lyophilized pre-titrated antibody mixture test and control tube and a CD45 /CD34 /CD38 /CD26 panel as a strict flow cytometric gating strategy.

Results: The expression of CD26 on CD34 /CD38 population was detectable in 211/211 PB and 84/84 BM samples of subsequently confirmed BCR-ABL CP-CML patients. None of the 32 samples suspicious for CML but scoring negative for circulating CD26 LSCs were diagnosed as CML after conventional cytogenetic and molecular testing. To validate our results, we checked for PB CD26 LSCs in patients affected by other hematological disorders and they all scored negative for CD26 expression.

Conclusions: We propose flow cytometry evaluation of CD26 expression on PB CD34 /CD38 population as a new rapid, reproducible, and powerful diagnostic tool for the diagnosis of CML. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767040PMC
July 2019

Study of gene polymorphisms as predictors of treatment efficacy and toxicity in patients with indolent non-hodgkin lymphomas and mantle cell lymphoma receiving bendamustine and rituximab.

Br J Haematol 2019 01 11;184(2):223-231. Epub 2018 Sep 11.

Unit of Haematology, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Bendamustine is used in combination with rituximab (BR) to treat indolent non-Hodgkin lymphomas (iNHL) and mantle cell lymphoma (MCL). The variability in treatment efficacy and toxicity could be related to single nucleotide polymorphisms (SNPs) in immune response genes. We would like to show a correlation between SNPs and treatment outcome in iNHL and MCL patients receiving BR. We investigated some SNPs that had already been associated with NHL outcome. Samples were genotyped for the IL2 (rs2069762), IL10 (rs1800890, rs10494879), VEGFA (rs3025039), IL8 (rs4073), CFH (rs1065489) and MTHFR (rs1801131) SNPs by allelic discrimination assays. We enrolled 70 patients that received rituximab 375 mg/m and bendamustine 90 mg/m every 28 days, both as first-line treatment and ≥ second-line regimens. Overall response rate was 97·1% (complete response [CR] rate 73·9%). Treatment toxicity included grade 3-4 neutropenia (24/70 patients), infections (21/70 patients; 1/70 grade 3), skin rash (26/70 patients; 2/70 grade 3). After a median follow-up of 24 months we did find any correlation between the analysed SNPs, CR rate and PFS. However, we demonstrated an association between the SNP in IL2 (rs2069762) and the onset of skin rash (P = 0·0001). Our study suggests a role for cytokine SNPs in bendamustine-related toxicity, which could represent a promising research field.
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http://dx.doi.org/10.1111/bjh.15582DOI Listing
January 2019

Residual Peripheral Blood CD26 Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Front Oncol 2018 30;8:194. Epub 2018 May 30.

Hematology Unit, Department of Medicine, Surgery and Neuroscience, Azienda Ospedaliera Universitaria Senese, University of Siena, Siena, Italy.

Chronic myeloid leukemia (CML) patients in sustained "deep molecular response" may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34/CD38 LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26 LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45/CD34/CD38 stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26 LSCs (median 19.20/μL, range 0.27-698.6). PB CD26 LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/μL; range 0.0012-0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/μL; range 0.006-0.76). Notably, no correlation between BCR-ABL/ABL ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that "circulating" CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26 LSCs during TKI treatment and the role of a "stem cell response" threshold to achieve and maintain TFR are ongoing.
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http://dx.doi.org/10.3389/fonc.2018.00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988870PMC
May 2018

Ectopic ILT3 controls BCR-dependent activation of Akt in B-cell chronic lymphocytic leukemia.

Blood 2017 11 20;130(18):2006-2017. Epub 2017 Sep 20.

Department of Life Sciences and.

The high proportion of long-term nonprogressors among chronic lymphocytic leukemia (CLL) patients suggests the existence of a regulatory network that restrains the proliferation of tumor B cells. The identification of molecular determinants composing such network is hence fundamental for our understanding of CLL pathogenesis. Based on our previous finding establishing a deficiency in the signaling adaptor p66Shc in CLL cells, we undertook to identify unique phenotypic traits caused by this defect. Here we show that a lack of p66Shc shapes the transcriptional profile of CLL cells and leads to an upregulation of the surface receptor ILT3, the immunoglobulin-like transcript 3 that is normally found on myeloid cells. The ectopic expression of ILT3 in CLL was a distinctive feature of neoplastic B cells and hematopoietic stem cells, thus identifying ILT3 as a selective marker of malignancy in CLL and the first example of phenotypic continuity between mature CLL cells and their progenitors in the bone marrow. ILT3 expression in CLL was found to be driven by Deltex1, a suppressor of antigen receptor signaling in lymphocytes. Triggering of ILT3 inhibited the activation of Akt kinase upon B-cell receptor (BCR) stimulation. This effect was achieved through the dynamic coalescence of ILT3, BCRs, and phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 into inhibitory clusters at the cell surface. Collectively, our findings identify ILT3 as a signature molecule of p66Shc deficiency in CLL and indicate that ILT3 may functionally contribute to a regulatory network controlling tumor progression by suppressing the Akt pathway.
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http://dx.doi.org/10.1182/blood-2017-03-775858DOI Listing
November 2017

High CD200 expression is associated with poor prognosis in cytogenetically normal acute myeloid leukemia, even in FlT3-ITD-/NPM1+ patients.

Leuk Res 2017 07 4;58:31-38. Epub 2017 Apr 4.

Division of Hematology and Bone Marrow Transplantation, Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy. Electronic address:

Overexpression of CD200, a trans-membrane protein belonging to the immunoglobulin superfamily, has been associated with poor prognosis in patients with acute myeloid leukemia (AML). As few data are available in the subset of cytogenetically-normal (CN) AML, we retrospectively evaluated the correlations between CD200 expression and response to therapy in a series of 139 adults with CN-AML. CD200 was expressed in 67/139 (48%) cases; 18 of them (28%) expressed CD200 at high intensity. No differences in CD200 expression rate were observed according to age, WBC count, type of leukemia, FLT3 or NMP1 mutation, and CD56 expression. A higher incidence of CD200 expression was observed in CD34+ cases (P<0.0001) and in BCL2+ patients (P=0.04). Complete remission (CR) was evaluable achieved in 98 patients (70%): 56/71 (79%) in CD200- and 47/67 (63%) in CD200+ patients (P=0.03), with a lower CR rate in patients with high CD200 intensity (9/18, 50%). CD200 expression had a negative impact on long-term outcome. CD200 expression, per se, did not impact on disease-free survival (DFS), but cases with high CD200 expression had a lower 3-year DFS compared to CD200-negative and low-expressing ones (0% vs 65% vs 68%, P=0.019). Three-year overall survival (OS) was 51% in CD200- and 27% in CD200+ patients (P=0.01), with a significant difference among cases with low or high CD200 expression (35% vs 0%, P=0.001). CD200 high expression defined a group with very poor DFS and OS also among the 37 FLT3-/NPM1+: 3-year DFS and OS were 88% and 60% in CD200-, 50% and 32% in CD200 low and 0% and 0% in CD200 high patients, respectively (P=0.01 for DFS and P=0.05 for OS). Our data suggest a negative impact of CD200 expression in CN-AML, with a further worsening in high-expressing cases, also in the subset of FLT3-/NPM1+ patients.
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http://dx.doi.org/10.1016/j.leukres.2017.04.001DOI Listing
July 2017

Clinical impact of CD200 expression in patients with acute myeloid leukemia and correlation with other molecular prognostic factors.

Oncotarget 2015 Oct;6(30):30212-21

Division of Hematology and Bone Marrow Transplantation, Azienda Ospedaliero-Universitaria di Udine, Udine, Italy.

CD200, a protein belonging to the immunoglobulin superfamily, has been associated with a poor prognosis in lymphoproliferative disorders and in acute leukemia. We studied the expression of CD200 in a series of 244 patients with diagnosis of acute myeloid leukemia (AML), to evaluate its impact on outcome and its possible association with other known prognostic factors. CD200 was found in 136/244 (56%) patients, in 41 of whom (30%) with high intensity of expression (MFI ≥ 11). CD200 was more frequent in secondary compared to de novo leukemia (p = 0.0006), in CD34 positive cases (p = 0.00001), in Bcl2 overexpressing cases (p = 0.01), in those wild-type Flt3 (p = 0.004) and with favorable or unfavorable compared to intermediate karyotype (p = 0.0003). CD200+ patients have a two-fold lower probability to attain complete remission, both in univariate (p = 0.006) and multivariate (p = 0.04) analysis. The negative impact of CD200 was found also in overall survival (p = 0.02) and was correlated with the intensity of expression of the molecule (p = 0.024). CD200 has an additive negative impact on survival in patients with unfavorable cytogenetic (p = 0.046) and in secondary leukemia (p = 0.05), and is associate with a worsening of outcome in patients with favorable biological markers, such as mutated NPM (p = 0.02), wild-type Flt3 (p = 0.034), negativity of CD34 (p = 0.03) and of CD56 (p = 0.03). In conclusion, CD200 is emerging as both a prognostic factor and a potential target of novel therapeutic approaches for AML, aiming to reverse the "do not eat me" signal of CD200 or to manipulate the suppressive immune microenvironment induced by CD200 binding to its receptor.
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http://dx.doi.org/10.18632/oncotarget.4901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745791PMC
October 2015

Chemoimmunotherapy with oral low-dose fludarabine, cyclophosphamide and rituximab (old-FCR) as treatment for elderly patients with chronic lymphocytic leukaemia.

Leuk Res 2014 Aug 2;38(8):891-5. Epub 2014 Jun 2.

Hematology, Azienda Ospedaliera Universitaria, Siena, Italy.

Median age at diagnosis for chronic lymphocytic leukaemia (CLL) patients is now 72 years, thus a consistent number of patients may not tolerate standard doses i.v. of fludarabine, cyclophosphamide and rituximab (FCR), the best available therapy, due to unacceptable myelotoxicity and risk of severe infections. We studied safety and efficacy of the addition of rituximab to the oral low-dose FC regimen (old-FCR) in a selected population of 30 elderly (median age 75, 15 untreated, 15 treated with 1 prior therapy) CLL patients. Complete remission (CR) rate was 80% in the untreated patients (overall response rate, ORR 93%), and 30% in pretreated patients (ORR 74%). Progression free survivals (PFS) were 45 months and 30 months in the untreated and treated patients, respectively. In patients achieving CR, old-FCR led to PFS of 67 months. Moreover, haematological toxicity was mild (grade 3-4: 15%) and patients were treated mostly in outpatient clinic. Old-FCR could be a good therapy option for elderly CLL patients outside clinical trials, larger studies are needed to confirm our findings.
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http://dx.doi.org/10.1016/j.leukres.2014.05.016DOI Listing
August 2014

Identification of a novel p190-derived breakpoint Peptide suitable for Peptide vaccine therapeutic approach in ph+ acute lymphoblastic leukemia patients.

Leuk Res Treatment 2012 15;2012:150651. Epub 2012 Feb 15.

Department of Hematology, University of Siena, 53100 Siena, Italy.

Ph+ acute lymphoblastic leukemia (Ph+ ALL) is a high-risk acute leukemia with poor prognosis, in which the specific t(9;22)(q34;q11) translocation results in a chimeric bcr-abl (e1a2 breakpoint) and in a 190 KD protein (p190) with constitutive tyrosine kinase activity. The advent of first- and second-generation tyrosine kinase inhibitors (TKIs) improved the short-term outcome of Ph+ ALL patients not eligible for allo-SCT; yet disease recurrence is almost inevitable. Peptides derived from p190-breakpoint area are leukemia-specific antigens that may mediate an antitumor response toward p190+ leukemia cells. We identified one peptide named p190-13 able to induce in vitro peptide-specific CD4+ T cell proliferation in Ph+ ALL patients in complete remission during TKIs. Thus this peptide appears a good candidate for developing an immune target vaccine strategy possibly synergizing with TKIs for remission maintenance.
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http://dx.doi.org/10.1155/2012/150651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505930PMC
December 2012

Insight into the behavior of hairy cell leukemia by immunogenetic analysis.

Leuk Lymphoma 2011 Jun 19;52 Suppl 2:103-7. Epub 2011 Apr 19.

Sezione di Ematologia e Trapianti, Dipartimento di Medicina Clinica e Scienze Immunologiche, Università di Siena & AOUS, Siena, Italy.

The B cell receptor (BCR) is the functional distinguishing unit that defines any B cell. Immunoglobulin gene (IG) status is preserved in the neoplastic B cell clone and can provide an indicator of the maturation stage reached by the B cell prior to transformation. In hairy cell leukemia (HCL), several data from IG analysis provide clear hints regarding the cell of origin and the ongoing selective interactions of the tumor BCR with environmental stimuli: HCLs (i) have variable levels of IG somatic mutations, (ii) continue somatic mutations at low levels, (iii) have active processes of IG class switching after transformation, and (iv) have a relatively high frequency of selective events in the light chain of the BCR. It has recently emerged that an unmutated status of the HCL-derived IG can be associated with failure to respond to cladribine, genetic abnormalities indicative of poor outcome, and aggressive disease. These observations suggest that IG gene analysis may have biological and prognostic relevance in HCL and merits further characterization.
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http://dx.doi.org/10.3109/10428194.2011.569620DOI Listing
June 2011

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia.

Hematol Oncol 2011 Mar;29(1):31-7

Sezione and Unità di Ematologia, Università di Siena & AOUS, Italy.

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype-switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour-related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGK(I) /IGK(II) , IGK(I) /IGL(I) and IGL(I) /IGL(II) transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single-cell analysis documented co-expression of the tumour-related IGLs in 5/6 cells (83%). In the IGK/IGL co-expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour-derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re-induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery.
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http://dx.doi.org/10.1002/hon.952DOI Listing
March 2011

A der(1)t(1;21)(p36.3;q22) in a patient with acute myelogenous leukemia M2.

Acta Haematol 2010 3;124(1):44-5. Epub 2010 Jul 3.

Department of Medicine and Immunological Sciences, Division of Hematology and Transplants, University of Siena, Siena, Italy.

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http://dx.doi.org/10.1159/000314075DOI Listing
August 2010

Impaired expression of p66Shc, a novel regulator of B-cell survival, in chronic lymphocytic leukemia.

Blood 2010 May 8;115(18):3726-36. Epub 2010 Jan 8.

Department of Evolutionary Biology, University of Siena, 53100 Siena, Italy.

Intrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc(-/-) mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.
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http://dx.doi.org/10.1182/blood-2009-08-239244DOI Listing
May 2010

Hairy cell leukemias with unmutated IGHV genes define the minor subset refractory to single-agent cladribine and with more aggressive behavior.

Blood 2009 Nov 10;114(21):4696-702. Epub 2009 Aug 10.

Ematologia e Trapianti, Università di Siena, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P < .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P < .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.
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http://dx.doi.org/10.1182/blood-2009-03-212449DOI Listing
November 2009

The prognosis of clinical monoclonal B cell lymphocytosis differs from prognosis of Rai 0 chronic lymphocytic leukaemia and is recapitulated by biological risk factors.

Br J Haematol 2009 Jun 5;146(1):64-75. Epub 2009 May 5.

Division of Haematology, Department of Clinical and Experimental Medicine and BRMA, Amedeo Avogadro University of Eastern Piedmont and Azienda Ospedaliero-Universitaria Maggiore della Carità, Novara, Italy.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5.0 x 10(9)/l circulating CLL-phenotype B-cells. The relationship between MBL and Rai 0 CLL, as well as the impact of biological risk factors on MBL prognosis, are unknown. Out of 460 B-cell expansions with CLL-phenotype, 123 clinical MBL (cMBL) were compared to 154 Rai 0 CLL according to clinical and biological profile and outcome. cMBL had better humoral immune capacity and lower infection risk, lower prevalence of del11q22-q23/del17p13 and TP53 mutations, slower lymphocyte doubling time, and longer treatment-free survival. Also, cMBL diagnosis was a protective factor for treatment risk. Despite these favourable features, all cMBL were projected to progress, and lymphocytes <1.2 x 10(9)/l and >3.7 x 10(9)/l were the best thresholds predicting the lowest and highest risk of progression to CLL. Although IGHV status, CD38 and CD49d expression, and fluorescence in situ hybridization (FISH) karyotype individually predicted treatment-free survival, multivariate analysis identified the presence of +12 or del17p13 as the sole independent predictor of treatment requirement in cMBL (Hazard ratio: 5.39, 95% confidence interval 1.98-14.44, P = 0.001). Overall, these data showed that cMBL has a more favourable clinical course than Rai 0 CLL. Given that the biological profile can predict treatment requirement, stratification based on biological prognosticators may be helpful for cMBL management.
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http://dx.doi.org/10.1111/j.1365-2141.2009.07711.xDOI Listing
June 2009

Higher expression of BDNF receptor gp145trkB is associated with lower apoptosis intensity in T cell lines in multiple sclerosis.

J Neurol Sci 2009 Feb 6;277(1-2):65-70. Epub 2008 Nov 6.

Department of Neurological and Behavioural Sciences, University of Siena, Siena, Italy.

Conflicting data exist on expression of gp145trkB, the high affinity receptor for brain-derived neurotrophic factor (BDNF), on peripheral blood immunocompetent cells in multiple sclerosis (MS). We analyzed expression of gp145trkB by western blotting and flow cytometry in myelin basic protein (MBP)- and ovalbumin (OVA)-T cell lines prepared from 12 patients with relapsing-remitting MS and 12 normal healthy subjects (NHS) and correlated it with activation-induced apoptosis. We found a higher percentage of gp145trkB-expressing MBP-T cells in MS patients than in NHS (p=0.011). gp145trkB was mainly expressed by CD8(+) T cells to a higher extent in MS patients than in NHS (p=0.04). MBP-T cell lines from MS patients showed significantly lower apoptosis intensity than those from NHS (p=0.011). We found also a significant negative correlation between gp145trkB expression and apoptosis intensity in MS patients only (p=0.02). OVA-T cell lines showed a gp145trkB expression similar to that of MBP-T cell lines, with a higher expression in MS patients than NHS, and similar correlations with apoptosis intensity in MS. These findings suggest that gp145trkB is mainly expressed on T cell lines from MS patients and that the BDNF/gp145trkB axis is involved in the regulation of peripheral T cell apoptosis in MS.
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http://dx.doi.org/10.1016/j.jns.2008.10.006DOI Listing
February 2009

Genome-wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion.

Br J Haematol 2008 Nov 24;143(4):532-6. Epub 2008 Aug 24.

Sezione Ematologia e Trapianti, Dipartimento di Medicina Clinica e Scienze Immunologiche, Università di Siena, Siena, Italy.

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p- CLL) with aggressive behaviour. Genome-wide DNA-profiling was performed to investigate 18 patients with 17p- CLL. All cases had multiple copy-number (CN) changes. Among the several recurrent CN changes identified, 8q24.13-q24.1-gain (MYC), 8p-loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1-p14-gain (REL/BCL11A) appeared frequently represented. 8p-loss and 2p16.1-p14-gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p-loss) and overall survival (8p-loss and 2p16.1-p14-gain, P < 0.05). These observations document a highly unstable genome in 17p- CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.
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http://dx.doi.org/10.1111/j.1365-2141.2008.07373.xDOI Listing
November 2008

Low-dose oral fludarabine plus cyclophosphamide in elderly patients with untreated and relapsed or refractory chronic lymphocytic Leukaemia.

Hematol Oncol 2008 Dec;26(4):247-51

Ematologia e Trapianti, Università di Siena, AOUS, Italy.

Fludarabine plus cyclophosphamide (FC) at conventional doses is an effective treatment for chronic lymphocytic leukaemia (CLL). However, FC at standard doses may give hematological and non-hematological toxicity, predominantly in the elderly. Intravenous or oral low-dose FC regimens remain highly effective in elderly patients with Low-Grade Lymphomas other than CLL and are well tolerated. We tested efficacy and toxicity of oral FC at reduced doses in 26 elderly patients (median 71 years) with previously untreated (UT-CLL, n = 14) or relapsed/refractory CLL (R-CLL, n = 12), unfit for conventional treatments. Twentyfour-of-26 (92%) patients (14/14, 100% UT-CLL; 10/12, 83.5% R-CLL) obtained a response, with 12/26 (46%) complete responses (9/14, 64.2% in UT-CLL; 3/12, 25% in R-CLL). Non-hematological toxicity was mild and myelosuppression was documented in 8/26 (31%) patients (4/14, 28% UT-CLL; 4/12, 33% R-CLL). With a median follow-up of 24 months, median event-free survival was 48 months with no differences between UT-CLL and R-CLL and all responders were alive. Low-dose oral FC treatment showed good efficacy in both untreated and refractory/relapsed CLL. The treatment is useful in elderly patients who cannot benefit of more aggressive schedules and is easy to administer on an outpatient basis.
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http://dx.doi.org/10.1002/hon.868DOI Listing
December 2008

High density genome-wide DNA profiling reveals a remarkably stable profile in hairy cell leukaemia.

Br J Haematol 2008 May 7;141(5):622-30. Epub 2008 Apr 7.

Sezione Ematologia e Trapianti, Università di Siena, Italy.

Hairy cell leukaemia (HCL) is a rare B-cell neoplasm for which the molecular mechanisms are largely unknown. High-density genome-wide DNA profiling was performed with Affymetrix 250K arrays to analyse copy number (CN) changes and loss of heterozygosity (LOH) in 16 cases of HCL. Four of 16 cases (25%) demonstrated gross non-recurrent CN deletions. Within the affected regions, we identified genes involved in bone marrow fibrosis (FGF12) and response to treatment (TP53) in individual cases. Large regions (> 5 Mb) of LOH without any concomitant DNA CN changes were identified in 5/16 (31%) HCL and were indicative of uniparental disomy UD. The germline origin of UD was demonstrated in one case for which a matched normal sample was available. Overall analysis of LOH showed that identical loci were recurrently targeted in chromosomes 1, 2 and 6. As a whole, however, HCL showed a remarkably stable genome. This finding adds to several other features that are unique to HCL among mature B-cell tumours.
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http://dx.doi.org/10.1111/j.1365-2141.2008.07106.xDOI Listing
May 2008

Selective influences in the expressed immunoglobulin heavy and light chain gene repertoire in hairy cell leukemia.

Haematologica 2008 May 2;93(5):697-705. Epub 2008 Apr 2.

DM, Sezione di Ematologia e Trapianti, Dipartimento di Medicina Clinica e Scienze Immunologiche, Università di Siena, AOUS, viale Bracci, 53100 Siena, Italy.

Background: We previously reported ongoing mutational and isotype switch events in the immunoglobulin (Ig) heavy chain (H) locus in hairy cell leukemia. Those analyses raised questions on the incidence and type of selective influences occurring on the tumor B-cell receptor of hairy cell leukemia.

Design And Methods: To further investigate this issue, we examined the full IGH and kappa and lambda light chains (IGkappa and IGlambda) variable and constant region transcripts expressed in a large cohort of patients with hairy cell leukemia (n=88).

Results: Multiple IgH isotypes were expressed in 46/56 (82%) cases of hairy cell leukemia. Comparison of tumor with normal B-cell repertoires revealed preferential usage of IGHV3-21, IGHV3-30 and IGHV3-33 in hairy cell leukemia (p=0.001, p=0.003 and p=0.001, respectively). Light chain analysis demonstrated preferential Igl use with an inverted IGk:IGl ratio (0.7:1) and universal usage of IGLJ3. Analysis of LCDR3 junctions revealed highly homologous motifs in 40% of IGL. Parallel analysis of IGH and IGL showed selective pairing of IGHV3-21/30/33 segments to specific LCDR3-J3 subsets (p=0.008). Of 40 cases of hairy cell leukemia, 38 had mutated IGHV and/or IGK/LV, with variations in 13/13 cloned cases, while two had 100% unmutated IGHV and IGK/LV.

Conclusions: Overall, biased IGV usage, preference for Iglambda with universal IGLJ3 usage and a high incidence of LCDR3 homologous motifs suggest selective influences on the B-cell receptor of hairy cell leukemia. Ongoing mutations and isotype switching suggest that influences occur on the tumor B-cell receptor at ectopic sites.
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http://dx.doi.org/10.3324/haematol.12282DOI Listing
May 2008