Supported by Czech Govt. Funding MSM 6198959205 MADCAM1 GENE POLYMORPHISMS AND THE OUTCOME OF THE ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION Z. Ambruzova1, F. Mrazek1, L. Raida2, A. Stahelova1, E. Faber2, K. Indrak2, M. Petrek1 1Laboratory of Immunogenomics and Proteomics, Department of Immunology, 2Department of Haematooncology Palacky University and University Hospital Olomouc, Czech Republic Supported by Czech Govt. Funding MSM 6198959205 and IGA MZ CR NR 9099

INTRODUCTION various non-HLA gene polymorphisms are intensively studied for their possible relevance for allogeneic haematopoietic stem cell transplantation (aHSCT) outcome migration and distribution of activated donor T cells to the recipient mucosal sites and parenchymal target organs is important for development of graft-versus-host disease (GVHD) organ-specific „homing“ of donor cells is mediated via interaction between adhesion molecules and their ligands

MUCOSAL ADDRESSIN CELL ADHESION MOLECULE-1 (MAdCAM-1) glycoprotein expressed on the surface of the endothelial cells (high level of expression in Peyer´s patches and mesenteric lymph nodes) ligands: α4β7 integrins (LPAM-1, CD49d/β7) L-selectin (CD62L) function: lymphocyte traffic to mucosal sites adhesion of leucocytes on the endothelium aHSCT - donor T cells „homing“ into the recipient mucosal tissue Eksteen et al, Clin Med 2004; 4:173-80

MADCAM1 GENE POLYMORPHISM many single nucleotide polymorphisms (SNPs) were described throughout the MADCAM1 gene (chromosome 19) particular variants of the MADCAM1 gene may affect structure and/or expression of MAdCAM-1 molecule SNPs selected to our study: rs 758502 T/C promotor rs 2302217 G/A exon 3 synonymous (Pro/Pro) rs 3745925 G/T exon 4 non-synonymous (His/Pro)

AIM OF THE STUDY to investigate whether the selected MADCAM1 gene SNPs are the risk factors for development of the serious complications after aHSCT MADCAM1 gene SNPs rs 758502 T/C rs 2302217 G/A rs 3745925 G/T acute or chronic GVHD overall survival

INVESTIGATED SUBJECTS aHSCT pairs 87 Age – median (range) Donor type Patients 44 (18-61) Related 70 Donors 40 (18-69) Unrelated 17 Recipient gender Cell source Female 37 PBSC 86 Male 50 Bone Marrow 1 Diagnosis Acute GVHD Acute leukaemia (AML, ALL) 43 Grade 0-I 53 Chronic leukaemia (CML, CLL) 15 Grade II 23 Non-Hodgkin lymphoma 14 Grade III 4 Other 15 Grade IV 8 Conditioning regimen Chronic GVHD Non-myeloablative 48 None 56 Myeloablative 39 Limited 17 Donor HLA compatibility Extensive 14 Identical 87 Mismatched 0

MADCAM1 genotyping (rs758502) METHODS Genotyping Polymerase Chain Reaction with Sequence Specific Primers (PCR-SSP) Primers designed according to the reference MADCAM1 gene sequence: rs 758502 T/C rs 2302217 G/A rs 3745925 G/T 2. Statistics Conformity to the Hardy-Weinberg equilibrium: Chi-square test Differences between allele and genotype frequencies: Pearson´s Chi-squared test, Bonferroni correction for multiple comparisons, binary logistic regression model Overall survival analysis: Kaplan-Meier, log-rank test and Cox regression analysis (SPSS software, version 15.0) TT TC CC MADCAM1 genotyping (rs758502)

MADCAM1 SNPs GENOTYPE FREQUENCIES IN PATIENTS AND DONORS Patients Donors n = 87 n = 85 MADCAM1 T/C (rs758502) Genotype CC 0.43 (37) 0.53 (44) Genotype TC 0.47 (41) 0.37 (31) Genotype TT 0.10 (9) 0.10 (8) MADCAM1 G/A (rs2302217) Genotype AA 0.26 (22) 0.35 (29) Genotype GA 0.51 (43) 0.40 (33) Genotype GG 0.24 (20) 0.24 (20) MADCAM1 G/T (rs3745925) Genotype GG 0.64 (54) 0.77 (63)* Genotype GT 0.33 (28) 0.18 (15) Genotype TT 0.04 (3) 0.05 (4) ______________________________________________________________________ *MADCAM1 (rs3745925) GG genotype frequency: p = 0.03, pcorr >0.05 for comparison patients with donors

FREQUENCIES OF MADCAM1 GENOTYPES IN PATIENTS WITH AND WITHOUT ACUTE GVHD Patients aGVHD+ Patients aGVHD- p* MADCAM1 T/C (rs758502) n = 35 n = 49 Genotype CC 0.37 (13) 0.45 (22) 0.477 Genotype TC 0.54 (19) 0.43 (21) Genotype TT 0.09 (3) 0.12 (6) 0.592 MADCAM1 G/A (rs2302217) n = 34 n = 48 Genotype AA 0.15 (5) 0.31 (15) 0.086 Genotype GA 0.62 (21) 0.46 (22) Genotype GG 0.23 (8) 0.23 (11) 0.948 MADCAM1 G/T (rs3745925) n = 34 n = 48 Genotype GG 0.59 (20) 0.69 (33) 0.354 Genotype GT 0.35 (12) 0.29 (14) Genotype TT 0.06 (2) 0.02 (1) 0.367 ___________________________________________________________________________ * comparison of particular homozygous genotype versus carriage of an alternative allele

FREQUENCY OF CHRONIC GVHD IN RECIPIENTS ACCORDING TO THEIR MADCAM1 rs2302217 GENOTYPES pcorr = NS

MULTIVARIATE ANALYSIS OF RISK FACTORS FOR OVERALL SURVIVAL AFTER aHSCT Factor p HR aGVHD 0,00001 4,35 cGVHD <0,00005 5,7 MADCAM1 rs2302217 AA genotype 0,001 2,99 Factors included to the analysis: Diagnosis (malignancy vs. non-malignancy), type of the donor (related vs. unrelated), stem cell source, conditioning regimen, GVHD prophylaxis, donor-recipient gender combination (female donor in male recipient vs. others), aGVHD, cGVHD, MADCAM1 gene variants

CONCLUSION preliminary data suggest that MADCAM1 rs2302217 AA genotype in recipients may be associated with the risk of chronic GVHD and decreased overall survival after aHSCT possible impact of MADCAM1 SNP variants for aHSCT outcome has to be confirmed in substantially larger cohorts of donor-recipient aHSCT pairs

Dept. of Immunology, Medical Faculty Palacky University and University Hospital Olomouc Prof. Martin Petrek Frantisek Mrazek Anna Stahelova Jana Onderkova Silva Zachova Dept. of Haematooncology, Medical Faculty Palacky University and University Hospital Olomouc Prof. Karel Indrak Ludek Raida Edgar Faber Thank you for your attention…