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P089 HLA microsatellites analysis: implications for unrelated donor matching in hematopoietic stem cell transplantation
S104
Poster Sessions
stage of the analysis. Finally, for 20 patients we analyzed chimerism status in different cell populations. Conclusion: STR-PCR method proved to be a reliable and useful tool in prediction of graft rejection, disease relapse and occurrence of GVHD thus enabling an early clinical intervention.
P089 HLA microsatellites analysis: implications for unrelated donor matching in hematopoietic stem cell transplantation K. Stingl, Z. Grubic, E. Cecuk-Jelicic, R. Zunec, R. Serventi-Seiwerth, B. Labar, V. Brkljacic-Kerhin. Tissue Typing Centre and Department of Medicine, Clinical Hospital Center Zagreb and School of Medicine, University of Zagreb, Zagreb, Croatia Microsatellites located within HLA region demonstrate considerable polymorphism and very strong linkage disequilibrium with HLA loci. Recently, an approach which uses HLA microsatellites for identification of non-HLA markers that could influence the transplantation outcome and also for the selection of potential donor has been proposed. Patients and methods: Twenty patients who underwent alloHSCT for treatment of various hematologic malignancies were retrospectively studied. Fifteen of them received transplant from an unrelated donor (HLA-A, B, DRB1 and DQB1 matched), while for the remaining 5 patients, donor was a parent (HLA-A, B, DRB1 and DQB1 matched in 2 cases; HLA-B allele mismatched in one case; HLA-B and DRB1 allele mismatched in 2 cases). All pairs were tested for 12 HLA microsatellites (D6S1014, D6S265, D6S273, D6S2793, D6S291, D6S2927, D6S2939, MICA, MIB, TNFa, TNFb and TNFd). Analysis was performed using electrophoresis on a polyacrylamide gel in an ALFexpress sequencer (Pharmacia Biotech). Results: According to the percentage of allele matching, microsatellite loci were divided in 3 groups: microsatellites with high matching level (>80%, D6S2927, MICA, MIB and TNFb), intermediate matching level (50-80%, D6S1014, D6S273 and TNFd), and low matching level (<50%, D6S265, D6S2793, D6S291, D6S2939 and TNFa). This was expected since the level of matching is proportional to the distance between microsatellites and HLA loci, their linkage disequilibrium and level of microsatellite polymorphism. Association of matching level and degree of GvHD could not be determined since 12 patients (60%) had GvHD grade 3 or higher, and out of the remaining 8 patients, 2 showed signs of transplant rejection and disease relapse. Conclusion: A larger group of patients is necessary and therefore this type of investigations should be undertaken in collaborative international studies to obtain conclusive results about the role of microsatellites in better definition of unrelated donors.
P090 Molecular detection of Epstein-Barr virus in patients with bone marrow transplantation A. Vince, S. Židovec-Lepej, S. Kozi´c, I. Ba´ca-Vrakela, R. Serventi-Seiwerth, L. Grkovi´c, B. Labar. University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, and Department of Medicine University Hospital Center, Zagreb, Croatia Aim: Posttransplant lymphoproliferative disease (PTLD) is the cause of significant morbidity and mortality in transplanted patients. The onset of PTLD in transplanted patients is associated with Epstein-Barr virus (EBV)-induced proliferation of infected B-cells during the suppression of specific CD8+ T-cell immunity. The aim of this study was to investigate the kinetics of EBV viremia in the peripheral blood of patients following bone marrow transplantation and compare it with patients that were treated with chemotherapy and/or radiation. Patients and Methods: We enrolled 29 hematologic patients that were treated with bone marrow transplantation (allogenic, autologous, mini) (17 women, median age 20 years, 12 men, median age 47 years) and 27 patients (16 women, median age 30 years, 11 men, median age 38 years) with Hodgkin’s (n= 12) or non-Hodgkin’s lymphoma (n=16) that were treated with chemotherapy and/or radiation. Clinical diagnoses in transplant patients were: Clinical diagnoses in transplant patients were: aplastic anemia (n=4), acute lymphoblastic leukemia (n=7), acute myeloid leukemia (n=7), chronic lymphocytic leukemia (n=2), chronic myelogenous leukemia (n=1), Hodgkin’s disease (n=2), non-Hodgkin’s lymphoma (n=2), multiple myeloma (n=3) and plasmacytoma (n=1). EBV viremia in the peripheral blood was determined before and after transplantation by using LightCycler EBV Quantification Kit“ (Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany). Lymphocyte subpopulations in the blood of patients treated with chemotherapy and/or radiation were determined by flow cytometry (Cytomics FC500, Beckman Coulter, Inc., Fullerton, CA, USA). Results: EBV viremia before transplantation was undetectable in all patients. Monitoring of EBV viremia during follow-up (10, 25, 40, 55, 62, 66, 75, 81, 127 and 365 days following transplantation) showed detectable viremia in 5 transplant patients. Three transplanted patients had a single episode of detectable viremia that was followed by undetectable viremia untill the end of follow-up. The highest measured viremia in single episodes of detectability was 275 000 copies of EBV DNA/ml. Two transplanted patients had two consecutive measurements of detectable viremia. In one AML patient, EBV viremia 40 days after transplantation was 8 410 copies of EBV DNA/ml, six days later it was 2 200 copies/ml and on day 66 become undetectable again. In the patient with CML, on day 62 after transplantation we detcted 7 298 copies of EBV DNA/ml and on day 66 the viremia decreased to 5 330 copies/ml. EBV viremia in that patient become undetectable on days 81 and 127 after transplantation. None of the patients enrolled in this study
Poster Sessions
stage of the analysis. Finally, for 20 patients we analyzed chimerism status in different cell populations. Conclusion: STR-PCR method proved to be a reliable and useful tool in prediction of graft rejection, disease relapse and occurrence of GVHD thus enabling an early clinical intervention.
P089 HLA microsatellites analysis: implications for unrelated donor matching in hematopoietic stem cell transplantation K. Stingl, Z. Grubic, E. Cecuk-Jelicic, R. Zunec, R. Serventi-Seiwerth, B. Labar, V. Brkljacic-Kerhin. Tissue Typing Centre and Department of Medicine, Clinical Hospital Center Zagreb and School of Medicine, University of Zagreb, Zagreb, Croatia Microsatellites located within HLA region demonstrate considerable polymorphism and very strong linkage disequilibrium with HLA loci. Recently, an approach which uses HLA microsatellites for identification of non-HLA markers that could influence the transplantation outcome and also for the selection of potential donor has been proposed. Patients and methods: Twenty patients who underwent alloHSCT for treatment of various hematologic malignancies were retrospectively studied. Fifteen of them received transplant from an unrelated donor (HLA-A, B, DRB1 and DQB1 matched), while for the remaining 5 patients, donor was a parent (HLA-A, B, DRB1 and DQB1 matched in 2 cases; HLA-B allele mismatched in one case; HLA-B and DRB1 allele mismatched in 2 cases). All pairs were tested for 12 HLA microsatellites (D6S1014, D6S265, D6S273, D6S2793, D6S291, D6S2927, D6S2939, MICA, MIB, TNFa, TNFb and TNFd). Analysis was performed using electrophoresis on a polyacrylamide gel in an ALFexpress sequencer (Pharmacia Biotech). Results: According to the percentage of allele matching, microsatellite loci were divided in 3 groups: microsatellites with high matching level (>80%, D6S2927, MICA, MIB and TNFb), intermediate matching level (50-80%, D6S1014, D6S273 and TNFd), and low matching level (<50%, D6S265, D6S2793, D6S291, D6S2939 and TNFa). This was expected since the level of matching is proportional to the distance between microsatellites and HLA loci, their linkage disequilibrium and level of microsatellite polymorphism. Association of matching level and degree of GvHD could not be determined since 12 patients (60%) had GvHD grade 3 or higher, and out of the remaining 8 patients, 2 showed signs of transplant rejection and disease relapse. Conclusion: A larger group of patients is necessary and therefore this type of investigations should be undertaken in collaborative international studies to obtain conclusive results about the role of microsatellites in better definition of unrelated donors.
P090 Molecular detection of Epstein-Barr virus in patients with bone marrow transplantation A. Vince, S. Židovec-Lepej, S. Kozi´c, I. Ba´ca-Vrakela, R. Serventi-Seiwerth, L. Grkovi´c, B. Labar. University Hospital for Infectious Diseases “Dr. Fran Mihaljevi´c”, and Department of Medicine University Hospital Center, Zagreb, Croatia Aim: Posttransplant lymphoproliferative disease (PTLD) is the cause of significant morbidity and mortality in transplanted patients. The onset of PTLD in transplanted patients is associated with Epstein-Barr virus (EBV)-induced proliferation of infected B-cells during the suppression of specific CD8+ T-cell immunity. The aim of this study was to investigate the kinetics of EBV viremia in the peripheral blood of patients following bone marrow transplantation and compare it with patients that were treated with chemotherapy and/or radiation. Patients and Methods: We enrolled 29 hematologic patients that were treated with bone marrow transplantation (allogenic, autologous, mini) (17 women, median age 20 years, 12 men, median age 47 years) and 27 patients (16 women, median age 30 years, 11 men, median age 38 years) with Hodgkin’s (n= 12) or non-Hodgkin’s lymphoma (n=16) that were treated with chemotherapy and/or radiation. Clinical diagnoses in transplant patients were: Clinical diagnoses in transplant patients were: aplastic anemia (n=4), acute lymphoblastic leukemia (n=7), acute myeloid leukemia (n=7), chronic lymphocytic leukemia (n=2), chronic myelogenous leukemia (n=1), Hodgkin’s disease (n=2), non-Hodgkin’s lymphoma (n=2), multiple myeloma (n=3) and plasmacytoma (n=1). EBV viremia in the peripheral blood was determined before and after transplantation by using LightCycler EBV Quantification Kit“ (Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany). Lymphocyte subpopulations in the blood of patients treated with chemotherapy and/or radiation were determined by flow cytometry (Cytomics FC500, Beckman Coulter, Inc., Fullerton, CA, USA). Results: EBV viremia before transplantation was undetectable in all patients. Monitoring of EBV viremia during follow-up (10, 25, 40, 55, 62, 66, 75, 81, 127 and 365 days following transplantation) showed detectable viremia in 5 transplant patients. Three transplanted patients had a single episode of detectable viremia that was followed by undetectable viremia untill the end of follow-up. The highest measured viremia in single episodes of detectability was 275 000 copies of EBV DNA/ml. Two transplanted patients had two consecutive measurements of detectable viremia. In one AML patient, EBV viremia 40 days after transplantation was 8 410 copies of EBV DNA/ml, six days later it was 2 200 copies/ml and on day 66 become undetectable again. In the patient with CML, on day 62 after transplantation we detcted 7 298 copies of EBV DNA/ml and on day 66 the viremia decreased to 5 330 copies/ml. EBV viremia in that patient become undetectable on days 81 and 127 after transplantation. None of the patients enrolled in this study