191 results on '"Katharina Fleischhauer"'
Search Results
152. Characterization of antigenic peptide epitopes by reverse immunology: induction of cytotoxic T lymphocytes specific for exogenous peptide only
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Claudio Bordignon, Peter van Endert, Silvia Tanzarella, Katharina Fleischhauer, Catia Traversari, Tanzarella, S., Fleischhauer, K., van Endert, P., Bordignon, Claudio, and C., Traversari
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chemistry.chemical_classification ,Cytotoxicity, Immunologic ,Cancer Research ,Chemistry ,Peptide ,CD8-Positive T-Lymphocytes ,Epitope ,Neoplasm Proteins ,Oncology ,Antigens, Neoplasm ,Immunology ,Tumor Cells, Cultured ,Cytotoxic T cell ,Humans ,Peptides ,Antigenic peptide ,Melanoma ,Cells, Cultured ,T-Lymphocytes, Cytotoxic - Published
- 1997
153. Modeling leukemia immunoediting in mouse-human chimeras (P2169)
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Giacomo Oliveira, Cristina Toffalori, Jose Manuel Garcia-Manteiga, Barbara Camisa, Lara Crucitti, Dejan Lazarevic, Jacopo Peccatori, Massimo Bernardi, Claudio Bordignon, Attilio Bondanza, Elia Stupka, Fabio Ciceri, Katharina Fleischhauer, Chiara Bonini, and Luca Vago
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Immunology ,Immunology and Allergy - Abstract
Acute Myeloid Leukemia (AML) can be recognized and eliminated by the immune system, as demonstrated by the clinical efficacy of allogeneic hematopoietic stem cell transplantation. Still, immune-resistant variants of AML can outgrow upon the selective pressure of the transplanted immune system and determine clinical relapse, in a process called “leukemia immunoediting”, the biological bases of which remain largely unknown. To unravel novel mechanisms of immunoediting, we engrafted primary human AML in immunocompromised NOD/SCID γ-chainnull mice and modeled immune pressure by serial infusions of human T cells, either autologous or allogeneic to the leukemic cells. HLA-mismatched allogeneic T cells eradicated AML from 6/6 treated mice, whereas HLA-identical T cells granted only temporary control in 3/3 mice and autologous T cells were completely inefficacious in 3/3 mice. To fine-tune immune pressure from HLA-mismatched T cells, and thus model a phase of equilibrium before leukemia immune escape, we genetically modified allogeneic T cells to express the HSV-Tk suicide gene. The activation of the suicidal machinery abated circulating T cell counts in 3/3 mice, halted the ongoing antileukemic response and resulted in leukemia outgrowth. Gene expression profiling of the AML blasts purified from the mice upon escape from immune pressure demonstrated the selective and significant deregulation of genes involved in immune processes, including antigen processing and presentation.
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- 2013
154. Sequencing of a new HLA-A*32 subtype (A*3202)
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Elena Benazzi, Katharina Fleischhauer, Benedetta Mazzi, Claudio Bordignon, Elisabetta Zino, and Giovanni Maria Severini
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Genetics ,Base Sequence ,HLA-A Antigens ,Immunology ,Molecular Sequence Data ,Humans ,Biology ,Human genetics ,Alleles ,HLA-A - Published
- 1996
155. Full Dose Treosulfan Based Reduced Toxicity Conditioning Regimen in Allogeneic Stem Cell Transplantation: Results in 123 Patients
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Fabio Giglio, Massimo Bernardi, Salvatore Gattillo, Jacopo Peccatori, Consuelo Corti, Alessandro Lorusso, Sara Mastaglio, Roberto Crocchiolo, Katharina Fleischhauer, Chiara Bonini, Carlo Messina, Magda Marcatti, Maria Teresa Lupo Stanghellini, Fabio Ciceri, Alessandra Forcina, and Andrea Assanelli
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medicine.medical_specialty ,Neutrophil Engraftment ,business.industry ,Immunology ,Cell Biology ,Hematology ,Treosulfan ,Biochemistry ,Gastroenterology ,Fludarabine ,Surgery ,Transplantation ,Regimen ,Internal medicine ,Toxicity ,medicine ,Rituximab ,Cumulative incidence ,business ,medicine.drug - Abstract
Abstract 3139 Conditioning in allogeneic stem cell transplantation has been widely explored in the last decades, since the introduction of the Reduced Intensity Conditioning (RIC) allowed the extension of the feasibility of allotransplants in previously ineligible patients. Outcome of both treatments remains comparable because of an higher incidence of relapse although reduction of Non Relapse Mortality (NRM) in RIC. Treosulfan is a bifunctional alkylating agent without major extra-haematological toxicities. Here we show the results of a Treosulfan based regimen in use at our Center in patients enrolled in “AlloTreo” trial (Eudract 2005–005182–11). We analyzed 123 pts transplanted between 2004 and 2011 consecutively enrolled at our center. Patient's median age was 50 years (21–69) and median Sorror comorbidity index was 1 (0–7). Diagnosis: acute myeloid leukemia (41 pts), acute lymphoblastic leukemia (14 pts), Myelodysplastic syndrome (17 pts), Non Hodgkin Lymphoma (23 pts), Hodgkin Lymphoma (5 pts), Myeloproliferative neoplasms (11 pts), and others (12 pts). Fourty-three pts were in first CR, 18 pts were transplanted upfront, 46 pts in persistence of disease; 16 pts in CR2 or CR3. Donor was unrelated (UD) in 57% of pts; source of stem cells were BM, PBSC and CB respectively in 5, 106 and 12 pts. Conditioning was: Treosulfan (14 g/msq for 3 days) and Fludarabine (30 mg/msq for 5 days). In vivo T and B-cell depletion was performed by ATG-Fresenius (10 mg/kg for 3 days) and Rituximab (single 500mg dose) only in pts receiving an UD. GvHD prophylaxis was: Cyclosporine A and short course of Methotrexate, excepting CB. Median CD34+ infused for BM-PBSC was 6.6×106/kg (1–17). Median follow-up was 48 months (1–83). At day 60 cumulative incidence (CI) of neutrophil engraftment was 93±2%, with a median time 17 days. In evaluable pts molecular chimerism at day 30 was full-donor in 89% thus confirming myeloablative properties of full dose treosulfan. Regimen was very well tolerated, median grade of regimen-related toxicity (CTC score) was 1 (0–4), mostly a transient rise of bilirubin (74%). Grade 4 toxicity was observed in 2 pts, with significant increase of bilirubin without signs of VOD. Grades II-IV aGvHD occurred in 42 pts (34%), grade III-IV in 21 (17%). Among evaluable pts after day 100, 46% developed chronic GvHD, half of which was extensive. Thirty days, 100 days and 4 years CI of NRM were respectively 2%, 11% and 21%; in multivariate analysis (MVA) an advanced stage of disease (HR=6.4, p=0.01) was associated with increased TRM. 4 years PFS was 44%, in MVA factor associated with a decreased PFS was advanced stage disease at transplant (HR=2.31, p=0.004). Thirty days, 100 days and 4 years CI of relapse were respectively 3%, 15% and 35%; no risk factors found. OS at 4 years was 51%, in MVA once again advanced stage of disease was a risk factor (HR=2.62, p=0.003). Importantly, even if a trend of increased NRM was found in UD transplants, it does not meet the significance criteria; in MVA, indeed, UD were not associated with increased risk of NRM, PFS, RI or worst OS. 57 pts died after transplant, 26 for relapse and 31 for transplant related causes (GvHD 32%, infection 45%). Treosulfan is a safe and effective myeloablative drug with low toxicities. Use of ATG as in-vivo T-cell depletion guarantees similar results in unrelated donor settings without enhancing NRM or worsening OS. Anti-leukemic effect, rapid engraftment as well as relatively low toxicity and NRM configure this regimen as a possible paradigm of the so called Reduced Toxicity Conditioning. However strategies to ameliorate outcome in advanced diseases are warranted. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
156. Haploidentical Transplantation Outcome Is Not Inferior to Standard Matched Related and Unrelated Donor Transplantation: An Intention-to-Treat Analysis of 241 Patients with Acute Myeloid Leukemia
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Francesca Lunghi, Elena Guggiari, Andrea Assanelli, Milena Coppola, Magda Marcatti, Laura Bellio, Massimo Bernardi, Claudio Bordignon, Chiara Bonini, Matteo Carrabba, Fabio Ciceri, Carlo Messina, Consuelo Corti, Elisa Sala, Salvatore Gattillo, Jacopo Peccatori, Sarah Marktel, Michela Tassara, Katharina Fleischhauer, Maria Teresa Lupo-Stanghellini, and Sara Mastaglio
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medicine.medical_specialty ,Intention-to-treat analysis ,Haploidentical transplantation ,business.industry ,medicine.medical_treatment ,Incidence (epidemiology) ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Gastroenterology ,Surgery ,Transplantation ,Leukemia ,Unrelated Donor ,Internal medicine ,medicine ,business - Abstract
1920 Background. Understanding of leukemia biology and advances in the transplant field have improved the safety as well as access of allogeneic hematopoietic cell transplantation (HCT) for a larger number of patients (pts) affected by acute myeloid leukemia (AML). The trend of growth of HCTs in adult pts with AML can be expected to continue based on acceptance and availability of alternative donor. Few data are available for: i) the reliable estimates of the number of HCT for AML and the total number of AML pts for whom HCT is appropriate, ii) the choice of the different donor sources. A risk-adapted treatment strategy is crucial to improve the outcome of pts with AML. Our policy is to offer a haploidentical HCT to adult pts lacking a matched donor in the appropriate time according to clinical indications ([www.leukemianet.org][1], [www.ebmt.org][2]). This policy is integrated in ongoing protocols for primary disease ([www.nilg.it][3]). Methods. Here we are reporting the intention-to-treat (ITT) analysis of HCT in all consecutive AML pts referred to our Institution between January 2004 and April 2012. Classification, prognostic evaluation, response criteria and survival outcomes for AML were defined according to the standard recommendation of the European LeukemiaNet (Dohner H et al, Blood. 2010: 115:453–474). Results. Indication to HCT was given to 241 pts (median age 52y, r17-72, 66 pts over-60y; male 138). HCT was performed in 201/241 pts (median time from diagnosis to HCT 222 days, median time from HCT-indication to HCT 80 days), 24/201 pts received a second HCT due to disease relapse. Pts distribution according to ITT algorithm is reported in [figure 1][4]. In ITT analysis, 83,4% of candidate pts received an HCT. Characteristic related to pts/disease/transplant are reported in [table 1][5]. Noteworthy, pts in 1st complete remission (CR1) are evenly distributed according to donor source (namely HLA-matched sibling donor – MSD, unrelated donor – URD, haploidentical related donor - haplo-HCT). The overall survival (OS) analysis for pts transplanted in CR is 71% at 1y and 56% at 3y, for pts in morphologic leukemia free state (MLFS) 32% and 21%, for pts in relapse 23% and 11% (p/2 | 4 (9%) | 12 (29%) | 2 (28%) | 24 (18%) | | - MLFS | 1 (2%) | 3 (7%) | 2 (28%) | 7 (5%) | | - relapse | 14 (31%) | 3 (7%) | 3 (43%) | 74 (57%) | Table 1. Patients, disease and transplant characteristics. ![Figure][7] ![Figure][7] Disclosures: Bordignon: MolMed SpA: Employment. [1]: http://www.leukemianet.org [2]: http://www.ebmt.org [3]: http://www.nilg.it [4]: #F1 [5]: #T1 [6]: #F2 [7]: pending:yes
- Published
- 2012
157. Loss of Mismatched HLA At Leukemia Relapse After Hematopoietic Stem Cell Transplantation Is Significantly Associated with Clinical and Immunogenetic Hallmarks of Donor-Versus-Host Alloreactivity
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Chiara Bonini, Cristina Toffalori, Matteo Carrabba, Katharina Fleischhauer, Benedetta Mazzi, Roberto Crocchiolo, Claudio Bordignon, Magda Marcatti, Sarah Marktel, Maria Teresa Lupo Stanghellini, Jacopo Peccatori, Lara Crucitti, Massimo Bernardi, Luca Vago, Andrea Assanelli, Elisabetta Zino, Consuelo Corti, and Fabio Ciceri
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Myeloid ,business.industry ,Lymphocyte ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,medicine ,Bone marrow ,business ,Myelofibrosis - Abstract
Abstract 1957 Background: In spite of the considerable progress achieved during the last decade in the field of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), disease relapse remains a crucial unsolved issue, due to our limited insights into the biology of the interplay between leukemia and donor immunity. We and others have shown that genomic loss of patient-specific mismatched HLA is a frequent mechanism by which residual leukemic cells can evade donor T cell-mediated immune control and determine a clinical relapse. HLA loss relapses have been reported both after family haploidentical and after volunteer unrelated donor HSCT, but the actual incidence and risk factors for these peculiar relapses are to date unknown. Methods: We retrospectively evaluated 230 consecutive transplants performed over the last ten years in a single institution. All donors were partially HLA mismatched (family mismatched: 170; volunteer unrelated: 60). All patients were affected by high-risk myeloid malignancies (Acute Myeloid Leukemia (AML): 179; Myelodysplastic Syndrome (MDS): 27; Myeloproliferative Neoplasms: 17; Others: 7), received a fully myeloablative conditioning regimen, and infusion of donor T cells, either as part of the graft or as post-transplantation add-back. In addition to standard bone marrow morphological examination, post-transplantation follow-up comprised genomic HLA typing of bone marrow aspirate samples, aimed to identify HLA loss relapses. Results: A total of 83 relapses occurred, 72 and 11 after related and unrelated donor HSCT, respectively. We documented 21 relapses with genomic loss of the patient-specific mismatched HLA (25% of relapses). HLA loss occurred predominantly in patients with AML (n=19), but was also observed in patients with MDS (n=1) and primary myelofibrosis (n=1). None of the 11 cases of relapse after volunteer unrelated donor HSCT displayed HLA loss, whereas it was evident in 21 out of 72 (29%) relapses after family mismatched donor HSCT. Accordingly, the presence of multiple (more than 4) HLA mismatches between donor and recipient represented a significant risk factor for HLA loss (HR: 4.17, 95% CI: 0.88–19.82, p=0.07). None of the disease-related factors tested (morphological subtype, presence of dysplasia, hyperleukocytosis, cytogenetic and molecular profile) correlated significantly with HLA loss. In line with the hypothesis that selection of these leukemic variants may be elicited more frequently in the presence of a strong alloreactive immune pressure, HLA loss occurred more frequently in patients with clinical Graft-versu-Host Disease (GvHD), either acute (p=0.02) or chronic (p=0.007). Interestingly, HLA loss relapses occurred later than their “classical” counterparts (median time to relapse 307 vs 116 days, range 56–784 vs 10–579, p Conclusions: Loss of the mismatched HLA is a frequent mechanism of relapse for patients with high-risk myeloid malignancies, and is associated with donor-versus-host alloreactivity, as demonstrated by its significant correlation with the number of donor-recipient HLA mismatches and with the occurrence of GvHD. Ultimately, given the documented ineffectiveness of lymphocyte infusions from the original stem cell donor in patients with HLA loss relapses, efforts should be aimed towards the optimization of early detection tools and targeted therapeutic strategies specific for these peculiar and frequent relapse variants. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed S.p.A.: Consultancy.
- Published
- 2012
158. Complete generic and extensive fine-specificity typing of the HLA-B locus by the PCR-SSOP method
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E. Benazzi, Elisabetta Zino, Katharina Fleischhauer, Claudio Bordignon, Fleischhauer, K., Zino, E., Bordignon, Claudio, and E., Benazzi
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Molecular Sequence Data ,Immunology ,Dot blot ,Locus (genetics) ,Human leukocyte antigen ,Biology ,Polymerase Chain Reaction ,Biochemistry ,Exon ,Tumor Cells, Cultured ,Genetics ,Humans ,Immunology and Allergy ,Amino Acid Sequence ,Typing ,Serotyping ,Allele ,Gene ,Alleles ,Cell Line, Transformed ,DNA Primers ,Base Sequence ,Histocompatibility Testing ,Reproducibility of Results ,General Medicine ,Molecular biology ,Hypervariable region ,HLA-B Antigens ,Oligonucleotide Probes - Abstract
This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA-B locus genes. A single HLA-B specific PCR-product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty-seven chemiluminescent labeled oligonucleotide probes. Thirty-one of these probes were derived from four hypervariability zones in exon 3 of HLA-B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty-six distinct hybridization patterns that could be used to unequivocally define all WHO-designated serological HLA-B specificities except for HLA-B54 in all homo- and heterozygous combinations. Furthermore, sixty-six out of ninety-seven molecularly defined HLA-B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty-four Workshop reference cell lines and of seventy-eight randomly chosen samples (one-hundred forty-seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty-five samples (one-hundred-thirty-three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety-one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA-B locus can be used as an alternative to biochemical methods such as one-dimensional isoelectric focusing for assignment of serologically cross-reacting HLA-B molecules as well as for subtype characterization of a large variety of HLA-B alleles.
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- 1995
159. Characterization of natural peptide ligands for HLA-B*4402 and -B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA-B44 heavy chain
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H.-J. Wallny, Katharina Fleischhauer, D. Avila, Claudio Bordignon, Catia Traversari, and F. Vilbois
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Immunology ,Molecular Sequence Data ,Spectrometry, Mass, Secondary Ion ,Peptide ,Biology ,Biochemistry ,Epitope ,Mass Spectrometry ,Cell Line ,HLA-B44 Antigen ,Protein sequencing ,Antigen ,Genetics ,Immunology and Allergy ,Humans ,Amino Acid Sequence ,Allorecognition ,Peptide sequence ,chemistry.chemical_classification ,Binding Sites ,Edman degradation ,General Medicine ,Amino acid ,chemistry ,HLA-B Antigens ,Peptides - Abstract
This study describes the characterization of endogenous peptides associated with the two major subtypes of HLA-B44. The two subtypes differ for a single amino acid substitution from Asp (HLA-B*4402) to Leu (HLA-B*4403) in position 156 of the alpha 2 domain, causing strong alloreactivity in vivo. In order to study the involvement of peptides in this phenomenon, the peptide motifs of the two subtypes were determined from natural peptide pools using Edman degradation. The motif was found to be essentially identical for HLA-B*4402 and -B*4403, with a strong predominance for Glu at position 2, Tyr or Phe at positions 9 and 10 and hydrophobic residues, especially Met, at position 3. Two individual naturally processed ligands of HLA-B*4403 were sequenced and shown to be derived from intracellularly expressed proteins found in protein sequence databases. The sequence of these natural peptide ligands conform well to the determined motif. These data will allow the prediction of HLA-B44 restricted peptide epitopes from viral and tumor antigens of known amino acid sequences. Moreover, they indicate that the peptide repertoire presented by HLA-B*4402 and -B*4403 is very similar, suggesting that the strong alloresponse between these two subtypes is not due to presentation of a different set of self peptides.
- Published
- 1994
160. Genomic and Transcriptional Immunoediting of Acute Myeloid Leukemia in Response to Allogeneic Hematopoietic Stem Cell Transplantation
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Chiara Bonini, Cristina Toffalori, Cristina Barlassina, Fabio Ciceri, Orietta Spinelli, Barbara Camisa, Andrea Calabria, Claudio Bordignon, Attilio Bondanza, Dejan Lazarevic, Katharina Fleischhauer, Alessandro Rambaldi, Massimo Bernardi, Luca Vago, and Elia Stupka
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education.field_of_study ,medicine.medical_treatment ,Immunology ,Population ,Myeloid leukemia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biology ,medicine.disease ,Donor Lymphocytes ,Biochemistry ,Gene expression profiling ,Transplantation ,Leukemia ,medicine ,education - Abstract
Abstract 329 BACKGROUND: The curative potential of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Acute Myeloid Leukemia (AML) is mostly based on the ability of donor lymphocytes to actively control the outgrowth of host residual malignant cells. We demonstrated in the specific context of HLA-haploidentical HSCT that this equilibrium is frequently broken by de novo genomic mutations, causing loss of the mismatched HLA haplotype in leukemic cells, in turn leading to immune evasion and clinical disease relapse (Vago et al., N Engl J Med, 2009). This finding led us to hypothesize that different de novo genomic or transcriptional alterations may be at the base of other cases of post-transplantation AML relapse, prompting us to verify this hypothesis by high-throughput techniques in the present study. METHODS: Paired samples of AML harvested at diagnosis and at relapse after non T cell-depleted allogeneic HSCT (either from HLA-identical, matched unrelated or haploidentical donors) were FACS-purified and analyzed by whole-genome Single Nucleotide Polymorphysm (SNP) profiling (by the Illumina Human660W-Quad BeadChip) and gene expression analysis (by the Illumina HumanHT-12 v3.0 Expression Bead chip). The presence and allelic burden of the Internal Tandem Duplication (ITD) in the FLT3 gene was validated by a locus-specific PCR followed by capillary electrophoresis. RESULTS: In 6 out of the 11 patients without genomic loss of HLA analyzed to date (54.5%), SNP profiling demonstrated the acquisition of de novo genomic alterations at post-transplantation relapse, preferentially occurring in well-characterized leukemia-associated genes (WT1, FLT3). Uniparental Disomy (UPD) was the most common alteration (45.5% of cases at diagnosis and 63.6% at relapse), frequently involving chromosome 13 in regions encompassing the FLT3 gene. Interestingly, in 5/11 patients (45.4%) we observed clonal evolution of leukemia at relapse from an oligoclonal population at diagnosis, with preferential expansion of the clones carrying genomic alterations. A total of 5 patients showed at relapse either de novo appearance of chromosome 13 UPD or the enrichment in a preexisting clone carrying the alteration: interestingly 4 out of these 5 patients gained in the process a substantial increase in the allelic burden of the ITD form of the FLT3 gene, which tightly correlates with aggressive behavior of leukemia. Gene expression profiling of paired diagnosis-relapse samples from 7 patients revealed deregulation by at least three-fold in an average of 3% (0.8–5.1%) genes at post-transplantation relapse as compared to diagnosis. An unsupervised Gene Ontology analysis of these genes evidenced a significant enrichment in immune-related processes (p A relevant observation was made in a patient who relapsed early after haploidentical HSCT without any documented genomic alteration in the HLA locus. In this patient, we could demonstrate at the time of post-transplantation relapse the specific transcriptional downregulation of the HLA class II antigen presentation pathway, whereas class I expression was preserved. Moreover, HLA class II expression was completely recovered in the absence of T cell immune pressure when the leukemic blasts harvested at relapse were transferred in immunodeficient NOD/SCID mice, suggesting plasticity of this mechanism of immune evasion. CONCLUSIONS: Our data demonstrate that both genomic and transcriptional alterations occur frequently at leukemia relapse after allogeneic HSCT. The frequent selection of leukemic clones displaying specific unfavorable mutations (such as FLT3-ITD) from initially mixed populations grants a biological rationale for the post-transplantation use of targeted inhibitors to block the outgrowth of the more aggressive subsets. Moreover, the observation that not only genomic, but also transcriptional HLA loss can be at the basis of relapse after haploidentical HSCT warrants further optimization of current protocols of post-transplantation monitoring and treatment of disease relapse. Disclosures: Bonini: MolMed SpA: Consultancy.
- Published
- 2011
161. Isoelectric focusing subtypes of HLA-A can be defined by oligonucleotide typing
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Soo Young Yang, Katharina Fleischhauer, and Seung‐Hoon Oh
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Linkage disequilibrium ,Immunology ,Molecular Sequence Data ,Human leukocyte antigen ,Biology ,Biochemistry ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Cell Line ,Gene Frequency ,Sequence Homology, Nucleic Acid ,Consensus Sequence ,Genetics ,Immunology and Allergy ,Humans ,Typing ,Amino Acid Sequence ,Base Sequence ,HLA-A Antigens ,Isoelectric focusing ,Histocompatibility Testing ,General Medicine ,DNA Probes, HLA ,Molecular biology ,HLA-A ,Hypervariable region ,genomic DNA ,Isoelectric Focusing ,Oligomer restriction ,Sequence Alignment ,Pseudogenes - Abstract
This study describes a simple and direct method for sequence-specific oligonucleotide probe (SSOP) typing of the A locus of HLA class I genes. Genomic DNA from a panel of over 200 cells which have been characterized by the methods of serology and isoelectric focusing (IEF) for the HLA class I antigens was used for locus-specific PCR amplification of HLA-A sequences. Dot blot hybridization of the amplified products was performed with 28 SSOPs derived from hypervariable regions in exon 2 and 3. Co-amplification of three alleles of HLA-H pseudogene in apparent linkage disequilibrium with HLA-A2 and A10 was observed but did not interfere with the typing of HLA-A alleles. Using short SSOPs (15 nucleotides each) in single temperature tetramethylammonium chloride (TMAC) hybridization and washing steps, 30 IEF-definable isotypes of HLA-A antigens could be unambiguously defined by their hybridization patterns. Moreover, comparison of the typing results with available nucleotide sequences of HLA-A alleles showed that the conditions used allowed faithful detection of single codon mismatches between probe and template. Thus, these alleles can be identified by their unique hybridization patterns generated by the SSOPs. Nucleotide sequence analysis of any new HLA-A allele will further permit its rapid and unambiguous characterization by SSOP typing.
- Published
- 1993
162. Thymic Renewal and Anti-Leukemic Effect In Adults After Haploidentical Transplantation and Donor T Cell Suicide Gene Therapy
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Luca Vago, Maria Teresa Lupo Stanghellini, Raffaella Greco, Giacomo Oliveira, Claudio Bordignon, Fabio Ciceri, Jacopo Peccatori, Maddalena Noviello, Sara Mastaglio, Chiara Bonini, Domenico Ghio, Roberto Nicoletti, Attilio Bondanza, Alessandro Aiuti, Corrado Soldati, Antonio Lambiase, Immacolata Brigida, and Katharina Fleischhauer
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business.industry ,Lymphocyte ,medicine.medical_treatment ,T cell ,Immunology ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,Suicide gene ,medicine.disease ,Donor Lymphocytes ,Biochemistry ,Leukemia ,medicine.anatomical_structure ,Immune system ,medicine ,business - Abstract
Abstract 833 Introduction: Hematopoietic Stem Cell Transplantation (HSCT) from partially HLA-matched (haploidentical) family donors represents a promising therapy for high-risk leukemia, but requires appropriate strategies to control the adverse reactions mediated by the partially incompatible, transplanted immune system. In a recent phase II study (TK007 study), we demonstrated that the infusion of donor lymphocytes transduced with the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows to control Graft-versus-Host Disease (GvHD) and to rapidly provide an effective and polyclonal anti-infective T cell repertoire (Ciceri and Bonini et al., Lancet Oncology, 2009). Even though their engraftment is necessary to achieve these effects, HSV-Tkpos cells represent the minority of lymphocytes circulating in treated patients. Therefore, in the present study, we investigated the putative role of HSV-Tkpos cells in promoting thymic activity and T cell development from graft progenitors. Methods: Twenty-eight adult patients underwent haploidentical HSCT and infusion of purified suicide gene-modified donor T cells for high-risk hematologic malignancies in the TK007 study. Thymic function was investigated in a selected cohort of this study (n=14) and in a control group who underwent unselected T cell-replete haploidentical transplantation with an ATG-rapamycin-mycophenolate-based GvHD prophylaxis (n=31), after validation in healthy pediatric and adult controls. T cell subsets and the proportion of CD31+ recent thymic emigrants (RTEs) amongst CD4+ naïve T cells were measured by immunophenotypic analysis. Single joint T cell Receptor Excision Circles (sjTREC) were quantified by qPCR. Thymic output was correlated with thymic volume, as assessed by CT scans. Post-transplantation pathogen-specific immune response was quantified by ELISpot. Alloreactivity against leukemic blasts was studied by mixed lymphocyte cultures. Results: Post-transplantation recovery of naïve CD45RA+CD62L+ T cells occurred in patients treated with gene modified T cells, reaching values of healthy controls in approximately one year. At the time of immune reconstitution (median 76 days after HSCT, defined as CD3+ cells > 100/ml peripheral blood), 76.5% of circulating T cells did not carry the HSV-Tk suicide gene, and the CD4+ naïve subset was largely comprised of cells recently originated from the thymus (90.5±3.2%). This observed frequency of CD31+ RTEs in these patients was significantly higher than that measured in the same patients before HSCT (60.7±6%, p=0,0087) or in patients analyzed 90 days after T cell-replete haploidentical HSCT (31.0±6.3%, p Conclusions: These data show that the infusion of suicide gene-modified T cells prompts the renewal of thymic activity, which contributes to the recovery of a polyclonal T cell repertoire protective against pathogens. Contextually, the infused transduced cells mediate also a direct antitumor effect through their recognition of allogeneic determinants on leukemic cells. A phase III clinical trial (TK008 study) to assess the efficacy of HSV-Tkpos cells in the context of haploidentical HSCT for leukemia started in 2010 in Italy, and is currently expanding to multiple centers throughout Europe. Disclosures: Bonini: MolMed S.p.A.: Consultancy.
- Published
- 2010
163. Permissive HLA-DPB1 Mismatching Compared to a Non-Permissive Mismatching Significantly Improves Overall Survival Following Allogeneic Transplantation In Patients with Both 10/10 and 9/10 Matched Unrelated Donors
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Bronwen E. Shaw, Elisabetta Zino, Yasuo Morishima, Stephen R. Spellman, Jean-Denis Bignon, Mari Malkki, J. Alejandro Madrigal, Effie W. Petersdorf, Katharina Fleischhauer, Andrea Velardi, Peter G Bardy, and Ted Gooley
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Permissiveness ,Oncology ,medicine.medical_specialty ,HLA-DPB1 ,Donor selection ,business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biochemistry ,Histocompatibility ,Transplantation ,Internal medicine ,medicine ,Permissive ,business - Abstract
Abstract 227 It is well established that the use of a donor matched for 9–10/10 alleles at HLA-A,-B,-C,-DRB1,-DQB1 significantly improves overall survival (OS) after unrelated donor (UD) haematopoietic stem cell transplantation (HSCT). Whilst the matching status for HLA-DPB1 alleles has been shown to influence transplant complications (relapse and graft-versus-host disease (GVHD), its impact on survival has not been well defined. The current unmet need in clinical practice is an approach to stratify selection criteria when a clinician is confronted with the choice between several 10/10 or 9/10 matched unrelated donors. There is now considerable interest in exploring different types of matching criteria to define permissive HLA-DPB1 mismatches which may be associated with an improved outcome. We have previously shown that HLA-DPB1 permissiveness can be functionally defined by the characterization of shared T cell epitopes (TCE) recognized by alloreactive T cells. In this model, allelic HLA mismatches are classified as permissive if they do not involve TCE disparities, and as non-permissive if they do. Using this concept, we developed two overlapping algorithms of permissivity for allelic HLA-DPB1 mismatches, on the basis of 3 (TCE3) or 4 (TCE4) groups of DPB1 alleles encoding immunogenic TCE. Data from relatively small prospective studies has shown a worse outcome to be associated with non-permissive DPB1 TCE disparities. Here, we present outcomes in 9123 UD-HSCT pairs, collected through the International Histocompatibility Working Group (IHWG). The cohort was comprised of 5809 10/10 matched transplant pairs and 3314 9/10 matched pairs. Within the 10/10 and 9/10 matched pairs three groups of patients were identified: 1. Zero DPB1 mismatches (i.e. allele matched), 2. Permissive DPB1 mismatch, 3. Non-permissive DPB1 mismatch. The model was adjusted for disease severity, source of stem cells, conditioning regimen, use of T-cell depletion, patient/donor gender and patient age. In line with DPB1 allele frequencies in worldwide populations, the number of transplants scored as permissive was higher for TCE3 (4398/7270 [60.4%]) than for TCE4 (2577/7270 [35.4%]). Using the DPB1 permissive mismatch transplants as the reference group (either 10/10 or 9/10 matched), we showed that DPB1 allelic matches resulted in similar survivals to DPB1 permissive mismatches, both in the 10/10 (HR 0.96, p=0.498 for TCE3 and HR 0.99, p=0.85 for TCE4) and the 9/10 setting (HR 0.97, p=0.70 for TCE3 and HR 0.99, p=0.96 for TCE4). In contrast, survival was significantly worse in the presence of a non-permissive TCE3 or TCE4 mismatch, both in the 10/10 (HR 1.15, p=0.0005 for TCE3 and HR 1.13, p=0.0035 for TCE4) and in the 9/10 matched setting (HR 1.13, p=0.0140 for TCE3 and HR 1.11, p=0.0448 for TCE4). The survival detriment appeared to be due to a significantly increased non-relapse mortality (TCE3: 10/10 HR 1.27, p In conclusion, our results suggest that extending donor selection to include HLA-DPB1 both allelic and functional TCE matching may result in better prediction of survival for patients. These findings provide an attractive new algorithm to stratify donor choice when several well-matched UD are identified. Disclosures: No relevant conflicts of interest to declare.
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- 2010
164. Effect of Related and Unrelated Donor Haemopoietic Stem-Cell Transplantation on Outcome In Adults with High Risk Acute Leukemia: An Intention-to-Treat Analysis at a Single Center Institution
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Maria Teresa Lupo-Stanghellini, Elena Guggiari, Fabio Ciceri, Francesca Lunghi, Alessandro Crotta, Valeria Calbi, Barbara Forno, Jacopo Peccatori, Milena Coppola, Magda Marcatti, Lionello Camba, Chiara Bonini, Cinzia Bitetti, Francesca Matteazzi, Sarah Marktel, Andrea Assanelli, Daniela Clerici, Katharina Fleischhauer, Michela Tassara, Massimo Bernardi, Raffaella Greco, Elisa Sala, Consuelo Corti, Simona Malato, Matteo Carrabba, Claudio Bordignon, Lupo Stanghellini, Mt, Marcatti, M, Coppola, M, Forno, B, Bitetti, C, Sala, E, Assanelli, A, Greco, R, Lunghi, F, Crotta, A, Tassara, M, Calbi, V, Matteazzi, F, Guggiari, E, Carrabba, M, Camba, L, Clerici, D, Malato, S, Marktel, S, Fleischhauer, K, Bonini, MARIA CHIARA, Bordignon, Claudio, Corti, C, Bernardi, M, Peccatori, J, and Ciceri, Fabio
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Acute leukemia ,medicine.medical_specialty ,Intention-to-treat analysis ,Myeloid ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Single Center ,Biochemistry ,Chemotherapy regimen ,Transplantation ,Leukemia ,surgical procedures, operative ,medicine.anatomical_structure ,hemic and lymphatic diseases ,High Risk Acute Leukemia ,Internal medicine ,medicine ,business - Abstract
Abstract 2385 Background. Allogeneic haemopoietic stem cell transplantation (HSCT) is the best therapeutic option for high-risk hematological disease (HRHD), particularly acute myeloid and lymphoblastic leukemia (AML/ALL). A widely application of HSCT is limited by lack of availability of a suitable donor for every candidate patients (pts). In order to offer a donor to every candidate pts, several centers had developed in the last decade alternative strategy of HSCT, such as umbilical cord blood (UCB) or family haploidentical HSCT (haplo-HSCT). With the aim to treat high-risk leukemia in the ideal appropriate time by allogeneic HSCT, we adopted a policy relying upon HLA typing at entry of every pts diagnosed with HRHD, followed, in absence of a MRD, by the prompt activation of the MUD search. Patients lacking a MRD or a MUD at the appropriate timing are proposed for haplo-HSCT. Methods. Here we report an intention-to-treat analysis of alternative donor HSCT at our Institution for pts diagnosed with high risk AML or ALL. Data were obtained from local institutional database. Results. Between Jan-2004 and July-2010 241 pts (median age 48y, r 15–72) with diagnosis of ALL (60 pts; median age 33y, r 15–64; male 39) or AML (181 pts, median age 51y, r 19–72; male 83) were defined as “high risk status” and received an indication to HSCT according to EBMT (European Group for Blood and Marrow Transplantation) and Northern Italy Leukemia Group (NILG, www.nilg.it) recommendations. In the ALL group 3 pts died before proceed to HSCT, 5 pts are waiting for the identification of a donor. Overall, 52/60 (86%) pts underwent HSCT, the status of disease at transplant was of complete remission (CR) for 27 pts, persistence of disease (PD) for 25. In the AML group 6 pts died before proceed to HSCT, 15 pts are waiting for the identification of a donor. Overall, 160/181 (88%) pts underwent HSCT, the status of disease at transplant was of CR for 97 pts, PD 63. Overall 92 pts activated the research of a MUD, 42 proceed to transplant, 7 received a UCB, 26 received a haplo-HSCT due to absence of a suitable donor in the appropriate time frame or failure to met the criteria to engage a MUD donor. The median time from diagnosis to registry activation was 69 days (r 5–876), the median time from activation to transplant 84 days (r 28–348). In the group of pts in CR at transplant, 37 underwent HSCT from a MRD and 36 from a MUD, 4 pts received a UCB and 47 a haplo-HSCT. Seventy-two pts are alive and in CR at last follow-up, 3/72 after a second transplant from a different donor (haplo-HSCT) and 3/72 after chemotherapy and adoptive immunotherapy to treat hematological relapse. Fifty-two pts died and the causes of death were: relapse (27), infection-related (19), graft-versus-host-disease (GvHD; 5), acute myocardial infarction (1). In the group of pts in PD at transplant, 16 underwent HSCT from a MRD and 6 from a MUD, 3 pts received UCB and 63 a haplo-HSCT. Fifteen pts are alive, 14 in CR, 1 pts under adoptive immunotherapy; 1/14 pts received a second transplant from a different donor (haplo-HSCT) to treat hematological relapse. Seventy-three pts died and the causes of death were: relapse (43), infection (23), GvHD (7). Overall, the median survival is 382 days and the median follow-up for pts alive is 548 days. The 1-year overall survival (OS)/transplant related mortality (TRM)/relapse incidence (RI) are 50,76% 26,96% and 40% respectively. For pts transplanted in CR the 1y OS/TRM/RI are 77,2%, 20,01% and 25% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) is comparable (p=ns). For pts transplanted in PD the 1y OS/TRM/RI are 19,7%, 41% and 67% respectively. The outcome analysis per donor source (MRD vs MUD vs haplo-HSCT) shows a trend of lower RI and TRM in the haplo-SCT vs MRD. Conclusion. The policy adopted provided an allogeneic HSCT in > 80% of candidate high-risk acute leukemia patients. No significant differences were registered in outcome for patients transplanted from matched-related, unrelated or family haploidentical donors. Further evaluation and a long-term follow-up will add important evaluation in term of long-term disease control and long-term toxicities. Disclosures: Bonini: MolMed: Consultancy. Bordignon: Molmed: Employment.
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- 2010
165. 91-P: Characterization of Common and Well-Documented HLA Alleles in Italy: A Multicenter Study on 24.000 Unrelated Individuals
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Roberta Morici, Elisabetta Zino, Giovanna Mongelli, Elisabetta Sironi, Annamaria Pasi, Benedetta Mazzi, M. Guarene, Katharina Fleischhauer, Miryam Martinetti, Biagio Favoino, D. Mininni, and Cristina Capittini
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Genetics ,Multicenter study ,Immunology ,Immunology and Allergy ,General Medicine ,Human leukocyte antigen ,Allele ,Biology - Published
- 2010
166. 111-P: Loss of Mismatched HLA in Family Haploidentical and Unrelated HSCT
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Magda Marcatti, Chiara Bonini, Cristina Toffalori, Jacopo Peccatori, Maria Teresa Lupo Stanghellini, Daniela Clerici, Katharina Fleischhauer, Benedetta Mazzi, Claudio Bordignon, Alessandro Crotta, Massimo Bernardi, Fabio Ciceri, Luca Vago, and Consuelo Corti
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business.industry ,Immunology ,Immunology and Allergy ,Medicine ,General Medicine ,Human leukocyte antigen ,business - Published
- 2010
167. 63-OR: Significant Correlation Between Donor-Recipient HLA-DPB1 T Cell Epitope Matching and Survival in 4490 Unrelated 10/10 Matched Hematopoietic Stem Cell Transplants Analyzed Within the 15th International Histocompatibility Workshop
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Elisabetta Zino, Andrea Velardi, Jean-Denis Bignon, Yasuo Morishima, Stephen R. Spellman, Bronwen E. Shaw, Mari Malkki, Alejandro Madrigal, Effie W. Petersdorf, Peter Brady, Katharina Fleischhauer, and Ted Gooley
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Matching (statistics) ,HLA-DPB1 ,T cell ,Immunology ,Hematopoietic stem cell ,General Medicine ,Biology ,Epitope ,Histocompatibility ,Correlation ,medicine.anatomical_structure ,medicine ,Immunology and Allergy - Published
- 2010
168. Quantitative Real-Time PCR Detection of Wilms' Tumor Gene (WT1) Transcript in Autologous Peripheral Blood Stem Cell (PBSC) Products Predict the Risk of Acute Myeloid Leukaemia (AML) Relapse After Autologous Transplantation (ASCT)
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Carlo Messina, Consuelo Corti, Claudio Bordignon, Katharina Fleischhauer, Massimo Bernardi, Alessandro Crotta, Cristina Tresoldi, Michela Tassara, Barbara Forno, Jacopo Peccatori, Simona Malato, Magda Marcatti, and Fabio Ciceri
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,CD34 ,Cell Biology ,Hematology ,Leukapheresis ,Treosulfan ,Biochemistry ,Minimal residual disease ,Fludarabine ,medicine.anatomical_structure ,Internal medicine ,medicine ,Cytarabine ,Autologous transplantation ,Bone marrow ,business ,medicine.drug - Abstract
Abstract 4690 Introduction when allogeneic transplantation is not feasible, ASCT is an alternative option as post-remission therapy for patients (pts) with AML, as it can prolong their disease-free survival (DFS). Relapse is the main cause of AML treatment failure after initial complete response; AML relapse after ASCT is partly due to contamination with leukemic blasts of the PBSC products, although neither morphological nor genetic evidence of disease are detected in the bone marrow before leukapheresis. Thus, identification and quantification of a reliable minimal residual disease (MRD) marker in the collected PBSC could be relevant in determining the relapse risk after ASCT. The WT1 gene is overexpressed in leukemic blasts of most AML cases; several studies have shown that quantification with RT-PCR of WT1 in the bone marrow and peripheral blood of AML pts in complete remission has prognostic value. We have determined the WT1 transcript levels in autologous PBSC of AML pts autografted for complete remission (CR) consolidation; preliminary results and data interpretation are here presented. Aim to evaluate quantitative WT1 transcript levels in autologous PBSC collections and to compare results with outcome in AML patients who received an ASCT as consolidation of CR, at our Institute. Patients and Methods 9 pts, period 06/2006-03/2009, median age 68 years (range 41-76). At diagnosis cytogenetic prognostic risk was intermediate for all pts. All pts were in morphological and genetic CR at the time of PBSC collection and before ASCT. Eight patients were in first and 1 in second CR. PBSC collection by leukapheresis (COBE Spectra cell separator): median count of CD34+ 9.75 ×106/kg (3.79-32). Conditioning regimen: Treosulfan 30 mg/sqm, Fludarabine 150 mg/sqm and Cytarabine 5 g/sqm. Median count of CD34+ cells infused: 5×106/kg (range 3.3-8.5×106/kg). RT-PCR quantification of WT1 transcript was performed using TaqMan technology starting from 1 μg of RNA extracted from mononucleated cells of fresh (4) or cryopreserved (5) PBSC samples. The housekeeping gene ABL was used as the control gene for these quantifications with WT1 level being normalised to 104 copies of ABL per sample. We used the Mann-Whitney-U-test to determine if median WT1 levels in the PBSC products of relapsed and not-relapsed pts was statistically different. Than we used the log-rank test to compare RFS and median WT1 value in the PBSC products. Results at last follow-up 4 pts relapsed and 5 were still in CR. The WT1 levels in the autologous PBSC of the 4 relapsed pts were 89.96, 193.87, 779.43 and 839.63, of the 5 CR pts were 8.40, 16.96, 36.45, 74.89 and 82.49. Overall median WT1 in the PBSC products was 82,49 copies. The median WT1 levels in the PBSC products of relapsed and not-relapsed pts were 486.65 (89.96–839.63) and 36.35 (8.40–82.49) copies, respectively; this difference was statistically significant (p 82,49 copies (n=4), and has not been reached for pts with a WT1 level ' 82,49 (n=5) (p=ns). Conclusions these results suggest that RT-PCR quantification of the WT1 transcript in autologous PBSC could predict AML relapse in pts who receive ASCT in CR. Higher WT1 levels should reflect higher PBSC product contamination with leukemic blasts, indicating an increased risk of relapse after ASCT. These last pts could probably benefit of other strategies than ASCT. We conclude that, if our preliminary data will be confirmed in a larger number of pts, RT-PCR quantification of WT1 transcripts should be used for MRD detection and quantification in autologous PBSC, before proceeding to ASCT; according to our preliminary data the cut-off level could be about 80 WT1 copies to discriminate which pts should receive the ASCT and which should not. Disclosures: No relevant conflicts of interest to declare.
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- 2009
169. WT1 Transcripts Is a Powerful Leukemia Marker to Predict Early Relapse After Allogeneic Haematopoietic Stem Cell Transplantation
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Francesca Lunghi, Cristina Tresoldi, Chiara Bonini, Magda Marcatti, Simona Malato, Fabio Giglio, Elena Guggiari, Matteo Carrabba, Alessandro Crotta, Barbara Forno, Jacopo Peccatori, Maria Teresa Lupo Stanghellini, Claudio Bordignon, Stefania Girlanda, Milena Coppola, Massimo Bernardi, Katharina Fleischhauer, Raffaella Greco, Marta Bruno Ventre, Simona Piemontese, Fabio Ciceri, Andrea Assanelli, Daniela Clerici, Consuelo Corti, Carlo Messina, and Sara Mastaglio
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Oncology ,medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Treosulfan ,medicine.disease ,Biochemistry ,Donor lymphocyte infusion ,Fludarabine ,Transplantation ,Leukemia ,medicine.anatomical_structure ,Graft-versus-host disease ,Internal medicine ,medicine ,Bone marrow ,business ,medicine.drug - Abstract
Abstract 4488 Introduction Definition of leukemia remission requires the lack of disease markers at sub-microscopic level. This is highly important after a potentially curative approach as allogeneic hematopoietic stem cell transplant (HSCT), where early detection of relapse at molecular level may lead to modulation of immunosuppressive therapy or donor lymphocyte infusion (DLI). In AML various approaches has been used to define MRD, but still the majority of AML cases do not have a useful and sensitive MRD marker. Over-expression of Wilms' tumor gene 1 (WT1) in leukemic blasts has been reported in >80% of AML and >40% of MDS. Physiologic hematopoietic stem cell compartments also express WT1, however, a ‘malignant’ WT1 expression can be clearly distinguished based on quantitative detection methods such as semiquantitative RT-PCR. Quantification of WT1 expression levels can detect frequencies of leukemic cells in bone marrow (BM) and peripheral blood (PB) as low as 10-3 and 10-5, respectively. Therefore WT1 expression levels provide a new marker for leukemic blast cells regardless of the type of leukemia especially in the relevant percentage of patients that lacks a specific molecular marker of their disease and thus may be a useful marker MRD and may predict the relapse after allogeneic HSCT. Materials and methods We measured quantitative expression of WT1 at diagnosis, before and after allogeneic transplant monthly for the first six months and then every three months. The quantitative assessment of the WT1 transcript amount was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Results Our study included 19 AML and 6 MDS pts who underwent allogeneic HSCT in our Institute between 12/2007 and 6/2009. Median age at diagnosis was 49 years (range 22-68). Bone marrow samples at diagnosis showed a WT1 median expression level of 5851.66 copies (range 77.98-31203.57). Fifteen pts (60%) had a specific cytogenetic marker that could allow MRD monitoring. At HSCT 17 pts (68%) were in CR, 5 (20%) had a refractory or relapsed disease, while 3 (12%) were transplanted upfront. Six pts (24%) received grafts from a matched sibling donors, 6 (24%) were transplanted from a matched unrelated donor (MUD), 10 (40%) from a familiar haploidentical donor and 3 (12%) received a cord blood unit. Myeloablative conditioning regimen consisted of Treosulfan 42 g/sqm, Fludarabine 150 mg/sqm, ALG 30 mg/kg and Rituximab 500 mg (last two drugs only for alternative donors). After HSCT a rapid decline of WT1 expression levels was observed in all pts that obtained or maintained a condition of CR. Two pts (8%) relapsed and both had an increase in WT1 expression before relapse. In the first relapsed pt, analysis of WT1 showed a dramatically increase between pre transplant level and day +28, while STR showed 100% donor chimerism. Relapse occurred on day +43, still on IST, with 69% of blast at AM evaluation and 40% donor chimerism. This pt was successfully reinduced with chemotherapy followed by allogeneic PBSC infusion without GvHD profilaxys obtaining a rapid reduction of WT1 (43.66). The pt developed GvHD that required a new IST, confirming a strong immune-surveillance of HSCT. The second pt was still on immunosuppressive treatment (IST) at the time of relapse (+136). WT1 in BM on day +30 was 30 cp and then increased gradually to 167 and 190 cp on day +67 and +102, respectively. Cytogenetic analysis and STR chimerism still showed a cCR with 100% donor chimerism. At relapse AM showed 10% blasts, WT1 expression level was 7447 cp with >95% donor chimerism. IST was discontinued and one month later WT1 expression level decreased to 1640 with >95% donor chimerism. In this situation we reasoned that DLI was the best available treatment. The total dose of donor T cell infused was 5 ×105 CD3/Kg. This procedure allowed an immune-mediated leukemia control with reduction of WT1 (1.58), cCR and 100% donor chimerism. Conclusions These data show that WT1 may be useful as a non-specific leukemia marker for monitoring MRD in AML and MDS after allogeneic HSCT and should enable the detection of early relapse allowing intervention at a more favourable stage than at overt relapse. We observed a complete concordance between WT1 expression levels and status of AML before and after allogeneic HSCT. Based on these results cases with increase of WT1 levels after HSCT and without GVHD may be candidate to immune intervention such as discontinuation of immunosuppression and/or DLI. Disclosures: Bonini: MolMed S.p.A.: Consultancy.
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- 2009
170. Interleukin-10 Anergized Donor T Cell Infusion Improves Immune Reconstitution without Severe Graft-Versus-Host-Disease After Haploidentical Hematopoietic Stem Cell Transplantation
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Fabio Ciceri, Elisabetta Zappone, Maria Teresa Lupo Stanghellini, Patrick Miqueu, Massimo F. Martelli, Raffaella Greco, Andrea Velardi, Barbarella Lucarelli, Katharina Fleischhauer, Jacopo Peccatori, Claudia Sartirana, Massimo Bernardi, Rosa Bacchetta, Silvia Gregori, Maria Grazia Roncarolo, and Franco Aversa
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Adoptive cell transfer ,business.industry ,medicine.medical_treatment ,T cell ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Cell therapy ,Graft-versus-host disease ,medicine.anatomical_structure ,Immune system ,Antigen ,medicine ,Antigen-presenting cell ,business - Abstract
Abstract 45 Background: Adoptive transfer of regulatory T cells is a potentially attractive alternative to conventional immunosuppressive therapy in allogeneic hematopoietic stem cell transplantation (HSCT) (Roncarolo MG., Nat Rev Immunol 2007). Among CD4+ T cells, the subset known as Type 1 regulatory T (Tr1) cells are induced in an antigen specific manner by interleukin-10 (IL-10) and suppress via production of high levels of IL-10 (Groux H., Nature 1997). The aim of this phase I/II study was to establish the safety and efficacy of a new cellular therapy with Tr1 cells in a non-randomized study. Patients and Methods: A cellular therapy protocol for the adoptive transfer of IL-10 induced alloantigen specific donor-derived Tr1 cells in patients transplanted with CD34+ selected cells from haploidentical donor, has been applied to patients with high risk hematopoietic malignancies (www.risetfp6.org). Donor T cells, anergized ex vivo toward host alloantigens, presented by monocytes (original protocol) or tolerogenic dendritic cells (modified protocol) as host antigen presenting cells, in the presence of IL-10, are infused post-transplant into the host (IL-10 DLI). The infusion is made in the absence of immunosuppression for graft-versus-host-disease (GVHD) prophylaxis, with the ultimate goal to provide immune reconstitution without severe GVHD. The infused donor T cells, at the dose of 105 CD3+ cells/kg or 3 × 105 CD3+ cells/kg, are anergic towards host-HLA antigens and contain precursors of host-specific Tr1 cells but, at the same time, comprise memory T cells able to respond to nominal and viral antigens. Results: Eighteen patients have been enrolled, sixteen received CD34+ selected cells from haploidentical donor after myeloablative conditioning. Twelve patients have been treated with IL-10 anergized cell therapy at day +30 post transplant, at the dose of 105 CD3+ cells/kg with the exception of two patients who received 3 × 105 CD3+ cells/kg. No severe immediate reactions post infusion were registered. Five patients died from infections by day +30 after Tregs cell infusion and two patients dropped out for graft rejection. Five patients achieved immune reconstitution at a median of 30,5 days (range 15–46 days) after IL-10 DLI, followed by progressive normalization of TCR repertoire, memory/naïve phenotype and T cell functions in vitro and in vivo. Acute GVHD grade III was observed in one patient who received 3 × 105 CD3+ cells/kg; GVHD grade II was observed in 4 patients who received 105 CD3+ cells/kg and were successfully immune reconstituted. The median follow-up of the IL-10 DLI treated patients is 980 days (range 291–1624); 4 patients are alive and disease free and they do not require immunosuppressive treatment. Conclusions: Cellular therapy with IL-10 anergized donor T cells has proven to be safe and feasible, and to sustain immune reconstitution associated with a reduced severity of GVHD and no occurrence of relapse. This trial represents the first step towards an extended use of Tr1 cells as adjuvant treatment in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.
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- 2009
171. Leukemic Dendritic Cells Expand Central Memory T Lymphocytes From HCT Donors Able to React against the Original Leukemia in Vitro and In Vivo
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Serena K. Perna, Elena Provasi, Attilio Bondanza, Monica Casucci, Zulma Magnani, Katharina Fleischhauer, Massimo Bernardi, Claudio Bordignon, Fabio Ciceri, Alessandro Crotta, Cristina Tresoldi, Maurilio Ponzoni, Alessandro Cignetti, Chiara Bonini, and Federico Caligaris-Cappio
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Myeloid ,Immunology ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Haematopoiesis ,Leukemia ,medicine.anatomical_structure ,Immune system ,Antigen ,medicine ,Stem cell - Abstract
Abstract 4090 Poster Board III-1025 Allogeneic hematopoietic transplantation (allo-HCT) is the only curative option for patients affected by high-risk acute myeloid leukemia (AML). This is largely due to the ability of allogeneic immune system to eradicate leukemic stem cells (LSC). However, the fact that some patients still relapse after allo-HCT, suggests that strategies to increase LSC targeting by donor T cells are needed. For this purpose, we exploited the unique ability of myeloid blasts to differentiate into leukemic dendritic cells (LDC). We observed that a short (48h) exposure to calcium ionophore A23187 and IL-4 is able to induce LDC differentiation in 14/16 (86%) of AML that we studied, both de novo and secondary. Importantly, despite phenotypic and functional changes indicative of differentiation into DC-like cells, the process was accompanied by the maintenance of disease markers such as CD34 and CD117. Moreover, LDC maintained the expression of the oncogenic protein WT1, which is a putative LSC antigen. Thanks to these favourable characteristics, LDC proved to be superior to the original blasts in expanding leukemia-reactive T lymphocytes both in the autologous and allogeneic HCT setting (on average, 5-fold expansion of blasts-stimulated T cells vs 95-fold expansion of LDC-stimulated T cells, SEM=2,7 and 67,7 respectively, p=0,01). We observed that the level of T-cell expansion directly correlate with the percentage of LDC obtained upon treatment with A23187 and IL-4. Most importantly, LDC proved to be more potent than blasts in expanding central memory T lymphocytes (TCM), which are known to confer superior anti-tumor immunity (on average, 29% of TCM upon stimulation with blasts vs 53% TCM upon stimulation with LDC, SEM=7,2 and 5,7 respectively, p=0,01). LDC-expanded T lymphocytes were able to efficiently recognize and kill leukemic blasts in vitro (on average, 953 specific spots of IFN-g/50'000 effectors at E:T ratio of 10:1 -SEM=120- and 29% of specific killing at E:T ratio of 50:1 -SEM=7,4-). Importantly, analysis of different HLA-settings and different targets of patient origin, suggests that LDC can expand T lymphocytes with specificities against multiple antigens expressed by the original leukemia. In particular, we observed the expansion of WT-1 specific T cells upon LDC stimulation. Finally, when infused in NOD/Scid mice transplanted with the original leukaemia, LDC-stimulated T lymphocytes were able to induce long-term complete remissions (>16 weeks) in all mice analyzed, suggesting that this approach may be active against leukemic stem cells. These results show for the first time that LDC-stimulated human T cells could exert a strong GvL activity in vivo. Disclosures: Bordignon: Molmed Spa: Employment.
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- 2009
172. Immune Reconstitution and Immune Correlates of Clinical Outcome IN Acute LEUKEMIA PATIENTS Treated with Haploidentical STEM CELL TRANSPLANTATION
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Alessandra Forcina, Maddalena Noviello, Massimo Bernardi, Luca Vago, Chiara Bonini, Consuelo Corti, Jacopo Peccatori, Veronica Valtolina, Attilio Bondanza, Sara Mastaglio, Claudio Bordignon, Fabio Ciceri, Maria Teresa Lupo-Stanghellini, Katharina Fleischhauer, and Daniela Clerici
- Subjects
business.industry ,ELISPOT ,medicine.medical_treatment ,Immunology ,Immunosuppression ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Transplantation ,Immune system ,Graft-versus-host disease ,Medicine ,IL-2 receptor ,business ,CD8 - Abstract
Abstract 46 The broader application of haploidentical stem cell transplantation (haplo-HCT), is limited by the delayed immune reconstitution (IR) secondary to the procedures for GvHD prophylaxis. This ultimately results in a high-rate of infectious complications and non-relapse mortality. We dynamically analyzed immunoreconstitution (IR) in patients undergoing haplo-HCT for acute leukemias enrolled in two different phase I-II clinical trials aimed at improving IR. In the first trial (TK007), 28 patients (out of 50 enrolled) received suicide-gene transduced donor T cells at day +42 after a T-cell depleted graft, in the absence of post-transplant immunosuppression. In the second trial (TrRaMM), 40 patients received an unmanipulated graft and a rapamycin-based GvHD prophylaxis. T-cell immune reconstitution was more rapid in TrRaMM than in TK007 patients, with a threshold of CD3 cells>100/μl reached at days +30 and +90, respectively. In both trials IR was mainly composed of Th1/Tc1 lymphocytes with an inverted CD4/CD8 ratio. While in TrRaMM patients we observed an early expansion of naïve and central memory T cells, producing high amounts of IL-2, in TK patients IR was mainly composed of activated effectors. Furthermore, in TrRaMM patients we detected high levels of CD4+CD25+CD127- T regulatory cells (up to 15% of circulating T lymphocytes) that persisted after rapamycin withdrawal, and was significantly superior to that observed in TK patients and in healthy controls. Interestingly, in contrast to the different kinetics of T-cell reconstitution, no differences were observed in time required to gain protective levels of CMV-specific T cells, as shown by ψIFN ELISPOT analysis. Protective frequencies of CMV-specific lymphocytes were observed 3 months after HCT in both groups, a time-point that in TrRaMM patients corresponds to the average time of rapamycin withdrawal. In both trials the number of circulating CMV-specific T cells was inversely correlated to the number and severity of subsequent CMV reactivations and days of antiviral therapy. GvHD was diagnosed in 16 TrRaMM patients (40%) and in 10 TK patients (35% of patients who received TK cells). Severity of GvHD was different in the two cohort of patients with 5 TrRaMM patients (12,5%) and only 2 TK patients (7%) with grade III-IV GvHD. Of interest, in the TrRaMM group CMV-specific immunity was significantly hampered by the immunosuppressive treatment required to treat GvHD. On the contrary, in the TK group, the administration of ganciclovir was able to activate the suicide machinery and control GvHD without impairing viral-specific T-cell immunocompetence. These results matched with the kinetics of CMV reactivations. We observed that while in TrRaMM patients 80% of viral reactivations occurred after the immunosuppressive therapy, in TK patients no significant differences could be assessed before and after therapy. IFN-ψ ELISPOT might thus be a relevant and predictive test to guide patient-specific clinical monitoring and antiviral treatment. Overall, these results show that early immune reconstitution can be promoted in haplo-HCT by different strategies associated with a wide range of alloreactive potential. The risks and benefits associated with alloreactivity should guide the therapeutic choice tuned on patient disease status and co-morbidities. Disclosures: Bordignon: Molmed Spa: Employment.
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- 2009
173. Loss of Mismatched HLA as a Mechanism of Leukemia Immune Escape in Family Haploidentical and Unrelated HSCT: Analysis of 103 Transplants From Alternative Donors
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Consuelo Corti, Monica Zanussi, Katharina Fleischhauer, Maurizio Ferrari, Magda Marcatti, Silvano Rossini, Andrea Assanelli, Massimo Bernardi, Maria Teresa Lupo Stanghellini, Daniela Clerici, Sarah Marktel, Benedetta Mazzi, Luca Vago, Carlo Messina, Chiara Bonini, Claudio Bordignon, Barbara Forno, Jacopo Peccatori, Alessandro Crotta, and Fabio Ciceri
- Subjects
business.industry ,medicine.medical_treatment ,Immunology ,Chronic myelomonocytic leukemia ,Myeloid leukemia ,Cell Biology ,Hematology ,Human leukocyte antigen ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Histocompatibility ,Transplantation ,Leukemia ,medicine.anatomical_structure ,medicine ,Bone marrow ,business - Abstract
Abstract 203 INTRODUCTION: Occurrence of a robust Graft versus Leukemia (GvL) effect is at the basis of the curative efficacy of Hematopoietic Stem Cell Transplantation (HSCT). Although leukemia-specific antigens have been characterized, most of the antileukemic potential of transplantation resides in alloreactivity towards patient-specific antigens, such as minor and major histocompatibility antigens. This is particularly relevant in the context of transplantation from related haploidentical and matched unrelated donors (MUD), in which mismatched HLA molecules are potent targets for alloreactive donor T cells. Still, upon in vivo selective pressure by donor T cells, acute myeloid leukemia can undergo genomic rearrangements which result in loss of the patient-specific HLA haplotype, a mechanism which our group recently demonstrated to be frequently responsible for leukemia relapse after haploidentical HSCT (Vago et al. NEJM, 2009). METHODS: 103 patients who underwent a partially HLA-matched transplantation for Acute Myeloid Leukemia (AML), MyeloDysplastic Syndrome (MDS), or Chronic Myelomonocytic Leukemia (CMML) at the San Raffaele Hospital from 2002 to present, were included in our analysis. For 67 patients, 26 of whom transplanted in complete remission, the stem cell donor was related haploidentical. For the remaining 35 patients, 23 of whom transplanted in complete remission, the donor was unrelated and mismatched for an average of 2/12 HLA alleles. All patients received donor T cells as part of the transplantation protocol. Post-transplantation follow-up comprised monthly bone marrow examination, with Short Tandem Repeat (STR) chimerism analysis and HLA typing performed in parallel on the marrow aspirate samples. The same analyses were performed also for an additional patient, referred to our attention for relapse of AML after two MUD transplantations performed in another center, both from HLA-C and DPB1-mismatched donors, and several donor lymphocyte add-backs. In cases of relapse with suspected loss of the mismatched HLA alleles, STR chimerism and HLA typing were performed also on purified leukemic blasts. RESULTS: Disease relapse occurred in 28/67 and 8/35 patients after haploidentical or MUD transplantation performed at our center, respectively. After haploidentical transplantation, 10/28 relapses (35.7%) were due to mutated leukemic blasts which had lost the patient-specific HLA haplotype. Median time to relapse in these patients was 281 days after HSCT (range 67–390), and all patients had received a high dose of donor T cells (median 289×106 CD3+ cells/kg, range 90–583). Upon detection of the mutated leukemic blasts, five of these ten patients were enrolled to receive a subsequent transplantation from a different stem cell donor, mismatched for the remaining HLA haplotype. None of the 8 relapses after MUD transplantations performed at our center displayed loss of the mismatched HLA. However, in the additional patient referred to our attention after two transplantations from partially HLA-mismatched unrelated donors, we documented loss of the HLA haplotype carrying mismatched alleles in the leukemic blasts responsible for leukemia relapse. CONCLUSIONS: With respect to our original series of five relapses with leukemic loss of the mismatched HLA haplotype, we describe here five additional cases based on the same mechanism, further consolidating the utmost clinical relevance of this escape mechanism from the GvL effect mediated by haploidentical donor T cells. Prompt detection of the mutant leukemic blasts allowed us to avoid infusion into the patients of donor T cell add-backs, predictably inefficacious in controlling these relapses, and to quickly enroll these patients into a salvage transplant from a different donor, mismatched for the remaining HLA haplotype. Moreover, we demonstrate that even in the case of fewer HLA mismatches, such as in MUD transplants, the genomic rearrangement we described can lead to clinical relapse. Given the constant increase in the number of transplants performed from partially HLA-mismatched donors worldwide, we recommend that post-transplantation HLA typing of bone marrow samples should be routinely performed at disease relapse to detect mutant leukemic blasts, and to guide therapeutic strategies targeted against them. Disclosures: Bonini: MolMed S.p.A.: Consultancy.
- Published
- 2009
174. 43-OR: Genomic loss of mismatched HLA in leukemia is a major mechanism of in vivo escape from T cell immunosurveillance following haploidentical hematopoietic stem cell transplantation
- Author
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Monica Zanussi, Jacopo Peccatori, Chiara Bonini, Benedetta Mazzi, Claudio Bordignon, Katharina Fleischhauer, Luca Vago, Fabio Ciceri, Maria Teresa Lupo Stanghellini, and Cristina Barlassina
- Subjects
Mechanism (biology) ,medicine.medical_treatment ,T cell ,Immunology ,General Medicine ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biology ,medicine.disease ,CXCR4 ,Immunosurveillance ,Leukemia ,medicine.anatomical_structure ,In vivo ,Cancer research ,medicine ,Immunology and Allergy - Published
- 2009
175. 170-P: Non-permissive HLA-DPB1 disparity is a significant independent risk factor for mortality after unrelated hematopoietic stem cell transplantation
- Author
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Nicoletta Sacchi, Luca Vago, Katharina Fleischhauer, Roberto Crocchiolo, Giuseppe Bandini, Andrea Bacigalupo, and Elisabetta Zino
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HLA-DPB1 ,business.industry ,medicine.medical_treatment ,Immunology ,medicine ,Immunology and Allergy ,General Medicine ,Hematopoietic stem cell transplantation ,Risk factor ,Permissive ,business - Published
- 2009
176. Genomic Loss of the Mismatched HLA Locus in Leukemia Is a Major Mechanism of in Vivo Escape from T Cell Immunosurveillance Following Haploidentical HSCT
- Author
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Benedetta Mazzi, Consuelo Corti, Claudio Bordignon, Fabio Ciceri, Katharina Fleischhauer, Chiara Bonini, Massimo Bernardi, Maurizio Ferrari, Luca Vago, Serena K. Perna, Nicola Flavio Perrelli, Maria Grazia Roncarolo, Maria Teresa Lupo Stanghellini, Barbara Forno, Silvano Rossini, Jacopo Peccatori, and Monica Zanussi
- Subjects
Adoptive cell transfer ,medicine.medical_treatment ,T cell ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Biology ,medicine.disease ,Biochemistry ,Minimal residual disease ,Leukemia ,medicine.anatomical_structure ,Graft-versus-host disease ,medicine ,Cytotoxic T cell - Abstract
Hematopoietic Stem Cell Transplantation (HSCT) from haploidentical family donors is a promising therapeutic option for nearly all patients suffering from high-risk leukemia. Until now, its application has been limited by the prolonged immunodeficiency that patients suffer as a consequence of graft T cell depletion, used to prevent severe Graft versus Host Disease (GvHD). When efficient strategies to control GvHD are applied, adoptive immunotherapy with donor T cells grants a significant advantage for immune reconstitution. However, direct evidence for the role of haploidentical donor T cells in controlling leukemia relapse is still missing. Here we report on the in vivo selection of de novo mutant variants of acute myeloid leukemia (AML), accounting for relapse after haploidentical HSCT and adoptive transfer of donor T cells. These novel variants of AML were observed in 5 out of 17 (29%) patients suffering from disease relapse in a series of 43 patients transplanted at the San Raffaele Hospital in Milan from 2002 to 2008. All patients received a myeloablative conditioning regimen and high doses of haploidentical donor stem cells (median 10.2×106 CD34+ cells/kg, range 4.6–15.5). Donor T lymphocytes were infused as part of the graft (n=21, median 438×106 CD3+ cell/kg, range 179–796) or as post-transplant add-backs (n=22, median 111×105 CD3+ cell/kg, range 1–900). Human Leukocyte Antigen (HLA) genomic typing was routinely used for post-transplant donor-recipient chimerism assessment. The five patients with de novo mutant variants of the original leukemia came to our attention because patient-specific HLA alleles could not be detected in bone marrow samples harvested at disease relapse, nor in subsequently sorted AML blasts. A Loss of Heterozygosity (LOH) study was performed on purified blasts from these patients, and demonstrated that patient-specific HLA alleles were lost due to extensive events of homologous recombination, encompassing a region of chromosome 6 comprising the entire HLA locus. We show that donor T cells capable of recognizing the original, HLA-heterozygous, leukemia were efficiently transferred from the haploidentical donor to the patient, granting an in vivo cytotoxic, cytokine and proliferative anti-tumor response by specific recognition of the mismatched HLA molecules. However, consistent with genomic loss of the patientspecific HLA locus in disease recurrence, the same alloreactive T cells were unable to recognize the mutant variant of the leukemia, harvested at the time of relapse. This observation strongly suggests that the genomic rearrangements we identified granted the disease an in vivo selective advantage in escaping from an established donor T cell response. Taken together, our data show that adoptive transfer of alloreactive donor T cells in haploidentical HSCT is efficient in providing a patient-specific antileukemic effect, and that the loss of this effect is an important mechanism underlying the outgrowth of relapsing disease. The frequency we documented for this phenomenon calls for routine assessment of the leukemia HLA genotype in the post-transplant follow-up and for careful consideration in the choice of a putative second haploidentical donor in case of leukemia relapse. Ultimately, our data provide the first direct evidence for the role of donor T cell alloreactivity in controlling minimal residual disease after haploidentical HSCT, favoring the use of donor T cell-based immunotherapeutic strategies to exploit alloreactivity for the cure of high-risk leukemia.
- Published
- 2008
177. NON-Permissive HLA-DPB1 T CELL Epitope Disparities Correlate with Engraftment and Survival after Unrelated Stem Cell Transplantation
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Roberto Crocchiolo, Andrea Bacigalupo, Elisabetta Zino, Giuseppe Bandini, Simona Pollichieni, Jessica Marcon, Nicoletta Sacchi, Katharina Fleischhauer, Rosi Oneto, and Fabio Ciceri
- Subjects
Myeloid ,HLA-DPB1 ,business.industry ,T cell ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Epitope ,Lymphoma ,Transplantation ,medicine.anatomical_structure ,Medicine ,Bone marrow ,Stem cell ,business - Abstract
Over 80% of stem cell transplantations (SCT) from unrelated donors (UD) are performed across allelic HLA-DPB1 disparities which have been associated with acute graft-versus-host disease (aGvHD) and, in 10/10 matched pairs, protection from disease relapse, while no significant correlation with overall survival (OS) could be revealed so far. We have previously developed an algorithm for non-permissive HLA-DP disparities involving an immunogenic T cell epitope (TCE) encoded by DPB1*0901, *1001 and *1701 (group 1), and, in a weaker form, by DPB1*0301, *1401 and *4501 (group 2) (Zino et al, Blood 2004). Here we report on the analysis of this algorithm in 627 UD SCT facilitated through the Italian Bone Marrow Donor Registry (IBMDR) and the Gruppo Italiano Trapianto di Midollo Osseo (GITMO) between 1999 and 2006, performed for malignant disorders including acute myeloid or lymphoid leukaemia (n=320; 51%) and Hodgkin’s or non-Hodgkin’s lymphoma (n=86; 13.7%). 242 pairs (38.5%) were matched at the allelic level for HLA-A, B, C, DRB1 and DQB1 (10/10 allele-matched pairs), while the remaining 385 pairs (61.5%) presented at least one mismatch at either of these loci (
- Published
- 2008
178. Optimal Thalassemia Free Survival and Minimal Regimen Related Toxicity in 50 Consecutive Transplants of High Risk Beta Thalassemia Pediatric Patients Using Myelablative Therapy with Intravenous Busulphan
- Author
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Costanza Evangelio, Sara Napolitano, Barbara Cappelli, Erika Biral, Laura Cursi, Anna Noè, Fabio Ciceri, Rossana Fiori, Roberto Miniero, Roberto Crocchiolo, Federica Cattaneo, Laura Zito, Clara Soliman, Tito Roccia, Katharina Fleischhauer, Marco Fossati, Sarah Marktel, Ilaria Frugnoli, Robert Chiesa, and Maria Grazia Roncarolo
- Subjects
Hepatitis ,medicine.medical_specialty ,Liver Iron Concentration ,Blood transfusion ,business.industry ,medicine.medical_treatment ,Thalassemia ,Immunology ,Beta thalassemia ,Cell Biology ,Hematology ,ThioTEPA ,medicine.disease ,Biochemistry ,Gastroenterology ,Surgery ,Transplantation ,Regimen ,Internal medicine ,Medicine ,business ,medicine.drug - Abstract
In the developed world, the survival and quality of life of patients with beta thalassemia (Bthal) has dramatically improved with optimization of blood transfusions and iron chelation. By contrast, in countries with limited resources most affected children die before the age of 20 because of the unavailability of safe blood products, expensive iron chelating drugs and inadequate management of co-morbidities. For these patients allogeneic stem cell transplantation (SCT) from matched donors offers a cure with low morbidity and mortality. Between June 2005 and May 2008, 47 consecutive Bthal patients underwent SCT from an HLA identical sibling in our center, among these, 3 patients underwent 2 SCTs. Median age was 8 years (2–15), country of origin: Lebanon (9), Iraq (19), Palestine (3), Syria (16). One pt was classified as Lucarelli class I, 24 as class II and 22 as class III. Most patients had severe iron overload evidenced by irregular iron chelation (83%), median ferritin 2973 (956–14280), median liver iron concentration 22 mg Fe/g (6.9–95.7). Most patients had liver toxicity due to iron overload and hepatitis evidenced by median ALT 71 (12–545), AST 59 (18–371), liver fibrosis Ishak 3 (0–5), HepC pos 16/47 (34%). Patients had inadequate transfusional management evidence by a median pre-transfusion Hb of 8 g/dL and anti HLA antibodies in 81% pts. Class I-II patients were conditioned with a regimen based on iv busulphan (iv Bu, Busilvex®, dosage according to weight, adjusted from the 5th dose to a target AUC 1200 umol/min) and cyclophosphamide (Cy) 200mg/kg (n19) with the addition of thiotepa (TT 10mg/kg) if
- Published
- 2008
179. Impaired GvL Potential of Natural Killer Cells Early Reconstituting Following Haploidentical HSCT
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Maria G. Roncarolo, Benedetta Mazzi, Claudio Bordignon, Silvano Rossini, Luca Vago, Chiara Bonini, Katharina Fleischhauer, Maria Teresa Lupo Stanghellini, Elisabetta Zino, Barbara Forno, Jacopo Peccatori, Serena K. Perna, Simona Di Terlizzi, and Fabio Ciceri
- Subjects
Acute leukemia ,business.industry ,Immunology ,CD34 ,Cell Biology ,Hematology ,CD16 ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,medicine ,Neural cell adhesion molecule ,IL-2 receptor ,Progenitor cell ,business - Abstract
Alloreactive NK cells have been suggested to be important functional players in GvL activity after haploidentical HSCT for high risk leukemia. In this study we have characterized NK cells differentiating from purified haploidentical CD34+ cells after transplantation into 16 patients who did (n=8) or did not (n=8) suffer acute leukemia relapse in a long term follow-up (median 208 days). The incidence of relapse in these patients was not correlated with the presence (n=9) or absence (n=7) of predicted donor NK alloreactivity (p=0.94). NK cells in the first month after transplantation were, regardless of the occurence of relapse, NKG2A+ (>95%) and KIR− (13%), thus resembling CD56bright NK cells from healthy donors. However, in contrast to mature CD56bright cells, the patients’ NK cells expressed heterogeneous intensities of CD56, were only partly positive for the lymph node homing markers CD62L and CCR7, and expressed a higher amount of Fcγ receptor III (CD16). Importantly, in contrast to mature CD56bright cells, which constitrutively express the high affinity αβγ IL-2 receptor, thus releasing γ-IFN in response to low dose IL2, the patients’ NK cells lacked IL-R α (CD25) and did not release cytokines in response to low-dose IL2, nor, most importantly, when challenged with leukemic blasts. γ-IFN release induced by leukemic blasts could be restored by inhibition of NKG2A while cytotoxicity, which was consistently lower as compared to that of mature CD56+ cells, could not. Our data suggest that NK cells differentiating in patients from CD34+ progenitors after haploidentical HSCT have important phenotipical and functional differences from both subsets of mature NK cells, accounting for an impaired in vivo GvL potential.
- Published
- 2006
180. Epitope-Specific Typing (EST) for HLA-DPB1 Matching: Proof of Principle for an Innovative Approach to Unrelated Hematopoietic Stem Cell Donor Selection
- Author
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Fabio Ciceri, Katharina Fleischhauer, Silvano Rossini, Elisabetta Zino, Luca Vago, Luigi D’ Amato, Simona Di Terlizzi, Maria G. Roncarolo, Benedetta Mazzi, and Claudio Bordignon
- Subjects
Genetics ,HLA-DPB1 ,medicine.diagnostic_test ,Donor selection ,Immunology ,Cell Biology ,Hematology ,Human leukocyte antigen ,Biology ,Biochemistry ,Epitope ,Transplantation ,medicine ,Typing ,Allele ,Tissue typing - Abstract
Background. Conventional matching of patients and their unrelated donor (UD) hematopoietic stem cells (HSC) by 4-digit molecular HLA typing is associated with lengthy donor searches and elevated social costs. 80% of UD transplants are performed across DPB1 mismatches which, if involving disparity in host versus graft (HvG) direction for an immunogenic T cell epitope, have been shown to be associated with poor clinical outcome of transplantation for hematopoietic malignancies and beta-thalassemia. In this study we have developed an innovative approach of DPB1 epitope- rather than allele-specific matching, by only two PCR reactions (epitope-specific typing; EST). Moreover, we have determined allelic DPB1 frequencies in Italy, confronted them with the ones previously reported for other ethnic groups, and calculated the probability of finding non-permissive DPB1 mismatches in unrelated HSC donor searches. Methods. High resolution genomic DPB1 typing and EST were performed in parallel on blood samples taken from 112 healthy unrelated Italian blood donors. Results. EST of DPB1 alleles encoding the immunogenic T cell epitope yielded 100% concordant results with high resolution DPB1 typing in all 112 samples studied, and is therefore suitable to univocally determine the presence or absence of non-permissive DPB1 disparities. The overall frequency of DPB1 alleles encoding the shared T cell epitope in the Italian population was 23.15%. Importantly, we show that based on DPB1 allelic polymorphism in the four ethnic groups representative of the world-wide UD registries, over 75% of UD matched for the other HLA loci will not present a DPB1 epitope disparity in HvG direction, demonstrating that prospective UD-recipient DPB1 matching by EST does not significantly limit the number of suitable donors, and has a negligible impact on the time and cost of the search. Conclusions. EST is a challenging alternative to conventional tissue typing which, if applied more broadly to HLA loci other than DPB1, could fundamentally change current approaches to UD searches.
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- 2006
181. Erratum
- Author
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Silvia Gregori, Megan K. Levings, Rosa Bacchetta, M. G. Roncarolo, Katharina Fleischhauer, and Manuela Battaglia
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Interleukin 10 ,Immunology ,Immunology and Allergy ,Physiology ,Biology - Published
- 2006
182. Receptor Repertoire Reconstitution Suggests Acquisition of Patient-Specific Tolerance by Natural Killer Cells Arising from Hematopoietic Progenitor Stem Cells after Haploidentical Transplantation
- Author
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Fabio Ciceri, Chiara Bonini, Chiara F. Magnani, Benedetta Mazzi, Katharina Fleischhauer, Zulma Magnani, Claudio Bordignon, Silvano Rossini, Simona Di Terlizzi, Maria Teresa Lupo Stanghellini, Marco Bregni, Elisabetta Zino, Daniela Clerici, Attilio Bondanza, Luca Vago, and Jacopo Peccatori
- Subjects
T cell ,KIR Ligand ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Human leukocyte antigen ,Biology ,Biochemistry ,Transplantation ,Interleukin 21 ,medicine.anatomical_structure ,Interleukin 12 ,medicine ,Stem cell - Abstract
Donor-recipient incompatibility for human leukocyte antigen (HLA) ligands of killer cell immunoglobulin-like receptors (KIRs) in haploidentical hematopoietic stem cell transplantation (HSCT), has been associated with a selective graft versus leukemia (GvL) effect mediated by donor-derived alloreactive natural killer (NK) cells expressing KIRs whose ligands are missing in the recipient. In this study, we show that NK cells arising from hematopoietic stem cell progenitors after transplantation into haploidentical recipients, acquire a receptor repertoire that is compatible with patient-specific tolerance due to engagement of patient HLA ligands by inhibitory NK receptors. Using four-color immunofluorescence with monoclonal antibodies (mAbs) specific for the receptors CD94/NKG2A, KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2 and KIR3DL1, we have analyzed NK receptor reconstitution kinetics in eleven adult patients affected by acute myeloid (n=9) or lymphoblastic (n=2) leukemia, who underwent HSCT from a KIR ligand matched (n=5) or mismatched (n=6) haploidentical family donor, using high doses (median 12.5x106/kg) of purified CD34+ progenitors. Nine patients achieved long-term (>150 days) complete remission of disease, independently from disease status at time of transplantation, and, importantly, from the presence (n=5) or absence (n=4) of donor NK alloreactivity. Within the first two months after transplantation, the vast majority (96% at 30 days, 86% at 60 days; SD 2% and 11%, respectively) of NK cells arising in the patients expressed the inhibitory receptor CD94/NKG2A, whose ligand HLA-E is ubiquitously expressed by cells positive for classical HLA class I molecules including leukemic blasts. As shown by mAb inhibition studies, lysis of patient-derived phytohemagglutinin-activated T cell blasts by these early arising NK cells was specifically inhibited by engagement of CD94/NKG2A. KIR expression was restored with variable kinetics in the later post-transplantation phase (3–9 months). Interestingly, however, during this period, NK cells devoid of CD94/NKG2A were found to express at least one KIR specific for an HLA ligand present in the patient, suggesting functional silencing of NK cells arising in the later phases after transplantation by acquisition of specific KIRs. Taken together, these data challenge current broad view on putative antileukemic effect of alloreactive NK cells reconstituting from haploidentical donor CD34+ cells and suggest that optimal exploitation of NK alloreactivity for GvL requires the presence of NK cells matured in the context of the donor’s rather than the recipient’s HLA repertoire. Ultimately, these findings provide a rationale for emerging clinical evidence in favor of efficacy of NK-based immunotherapy with mature donor NK cells.
- Published
- 2005
183. Suicide Gene Therapy of Graft-Versus-Host Disease Induced by Central Memory Human T Lymphocytes
- Author
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Chiara Bonini, Claudio Bordignon, Fabio Ciceri, Katharina Fleischhauer, Simona La Seta-Catamancio, Marina Radrizzani, Salvatore Toma, Catia Traversari, Mark Bonyhadi, Francesca Sanvito, Maurilio Ponzoni, Shin Kaneko, Zulma Magnani, Veronica Valtolina, and Attilio Bondanza
- Subjects
Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.
- Published
- 2005
184. Characterization of peptides with binding activity to HLA-B44 encoded by tumor associated antigen Mage-3
- Author
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Doriana Fruci, Catia Traversari, Pierre Coulie, H J Wallny, Jacques Herman, Claudio Bordignon, Silvia Tanzarella, P van Endert, and Katharina Fleischhauer
- Subjects
Chemistry ,Macrophage-1 antigen ,Immunology ,T-cell receptor ,Immunology and Allergy ,General Medicine ,Molecular biology ,Tumor associated antigen - Published
- 1996
185. Fine-specificity analysis of HLA-A*02 restricted cytotoxic T lymphocytes specific for peptides from tumor associated antigens Mage-3 and Melan-A/MART-1
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Catia Traversari, P. van der Bruggen, Claudio Bordignon, Silvia Tanzarella, and Katharina Fleischhauer
- Subjects
Antigen ,Immunology ,Cancer research ,Immunology and Allergy ,Cytotoxic T cell ,General Medicine ,Biology ,Pan-T antigens ,Tumor associated antigen ,HLA-A - Published
- 1996
186. HLA-A rare alleles in Caucasian bone marrow donors identified by molecular analysis
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Benedetta Mazzi, G. Ghidini, Claudio Bordignon, E. Benazzi, Katharina Fleischhauer, R. Carpanelli, and I. Colombini
- Subjects
Immunology ,Bone marrow donors ,Immunology and Allergy ,General Medicine ,Allele ,Biology ,Molecular analysis ,HLA-A - Published
- 1996
187. Molecular analysis of HLA-A*02 subtype frequencies in the population of northern Italy
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Benedetta Mazzi, Claudio Bordignon, E. Benazzi, Katharina Fleischhauer, and Elisabetta Zino
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education.field_of_study ,Evolutionary biology ,Immunology ,Population ,Immunology and Allergy ,General Medicine ,Biology ,education ,Molecular analysis ,HLA-A ,Northern italy - Published
- 1996
188. Detection of HLA-A*66 in linkage disequilibrium with HLA-B41 in a significant number of individuals from northern Italy
- Author
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Benedetta Mazzi, Katharina Fleischhauer, R. Carpanelli, G. Ghidini, E. Benazzi, and I. Colombini
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Genetics ,Linkage disequilibrium ,Immunology ,Immunology and Allergy ,General Medicine ,Human leukocyte antigen ,Biology ,Northern italy ,HLA-A - Published
- 1996
189. The Impact of Amino Acid Variability on Alloreactivity Defines a Functional Distance Predictive of Permissive HLA-DPB1 Mismatches in Hematopoietic Stem Cell Transplantation
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Martin Maiers, Luigi Naldini, Arend Mulder, Elisabetta Zino, Pietro Crivello, Laura Zito, Luca Vago, Cristina Toffalori, Katharina Fleischhauer, Federico Sizzano, Fabio Ciceri, Crivello, P, Zito, L, Sizzano, F, Zino, E, Maiers, M, Mulder, A, Toffalori, C, Naldini, Luigi, Ciceri, Fabio, Vago, L, and Fleischhauer, K.
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CD4-Positive T-Lymphocytes ,medicine.medical_treatment ,T cell ,Medizin ,Epitopes, T-Lymphocyte ,Gene Expression ,Hematopoietic stem cell transplantation ,Human leukocyte antigen ,Biology ,Epitope ,Cell Line ,medicine ,Humans ,Protein Isoforms ,Transplantation, Homologous ,Amino Acids ,Allele ,Allorecognition ,Alloreactivity ,Alleles ,HLA-DP beta-Chains ,B cell ,Genetics ,B-Lymphocytes ,Transplantation ,HLA-DPB1 ,Histocompatibility Testing ,Hematology ,Clone Cells ,medicine.anatomical_structure ,Mutation ,Immunology ,Amino acid mutation analysis ,Unrelated Donors ,T cell epitope ,Permissive HLA mismatches - Abstract
A major challenge in unrelated hematopoietic stem cell transplantation (HSCT) is the prediction of permissive HLA mismatches, ie, those associated with lower clinical risks compared to their nonpermissive counterparts. For HLA-DPB1, a clinically prognostic model has been shown to be matching for T cell epitope (TCE) groups assigned by cross reactivity of T cells alloreactive to HLA-DPB1*09:01; however, the molecular basis of this observation is not fully understood. Here, we have mutated amino acids (aa) in 10 positions of HLA-DPB1*09:01 to other naturally occurring variants, expressed them by lentiviral vectors in B cell lines, and quantitatively measured allorecognition by 17 CD4(+) T cell effectors from 6 unrelated individuals. A significant impact on the median alloresponse was observed for peptide contact positions 9, 11, 35, 55, 69, 76, and 84, but not for positions 8, 56, and 57 pointing away from the groove. A score for the "functional distance" (FD) from HLA-DPB1*09:01 was defined as the sum of the median impact of polymorphic aa in a given HLA-DPB1 allele on T cell alloreactivity. Established TCE group assignment of 23 alleles correlated with FD scores of = 2 for TCE groups 1, 2, and 3, respectively. Based on this, prediction of TCE group assignment will be possible for any given HLA-DPB1 allele, including currently 367 alleles encoding distinct proteins for which T cell cross reactivity patterns are unknown. Experimental confirmation of the in silico TCE group classification was successfully performed for 7 of 7 of these alleles. Our findings have practical implications for the applicability of TCE group matching in unrelated HSCT and provide new insights into the molecular mechanisms underlying this model. The innovative concept of FD opens new potential avenues for risk prediction in unrelated HSCT. (C) 2015 American Society for Blood and Marrow Transplantation. OI Maiers, Martin/0000-0002-0198-2064
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190. A combined DPA1∼DPB1 amino acid epitope is the primary unit of selection on the HLA-DP heterodimer
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Susan Flesch, Bronwen E. Shaw, Martin Maiers, Loren Gragert, Abeer Madbouly, Elizabeth Trachtenberg, Marcelo Fernandez-Vina, Neng Yu, Stephen R. Spellman, Ann B. Begovich, Cynthia Vierra-Green, Thomas M. Williams, Harriet Noreen, Katharina Fleischhauer, and Jill A. Hollenbach
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Models, Molecular ,Linkage disequilibrium ,Genotype ,Immunology ,Locus (genetics) ,HLA-DP ,HLA-DP alpha-Chains ,Biology ,Linkage Disequilibrium ,White People ,Epitope ,Epitopes ,Gene Frequency ,DPA1 ,Haplotype ,Genetics ,Humans ,Amino Acids ,Allele ,Allele frequency ,HLA-DP beta-Chains ,Heterodimer ,Original Paper ,Polymorphism, Genetic ,Hematopoietic Stem Cells ,Amino acid ,Transplantation ,Haplotypes ,DPB1 ,Epitope Mapping - Abstract
Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context. Electronic supplementary material The online version of this article (doi:10.1007/s00251-012-0615-3) contains supplementary material, which is available to authorized users.
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191. Follow-up study on PCR-SSOP typing of the HLA-A locus: Improved resolution of A-10 and A-19 splits
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Benedetta Mazzi, Elisabetta Zino, Claudio Bordignon, Katharina Fleischhauer, E. Benazzi, M. Berti, and Elisabetta Sironi
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Genetics ,HLA-A Antigens ,business.industry ,Immunology ,Molecular Sequence Data ,Follow up studies ,Pcr ssop ,Chromosome Mapping ,Locus (genetics) ,General Medicine ,Biochemistry ,Polymerase Chain Reaction ,HLA-A ,Immunology and Allergy ,Medicine ,Humans ,Typing ,Amino Acid Sequence ,business ,Oligonucleotide Probes ,Alleles ,Follow-Up Studies
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