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Molecular biology class II typing may not always be sufficient to show discrepancies between bone marrow donors and recipients
Abstracts
126 ABSTRACTS SELECTED FOR GROUP DISCUSSION: ABSTRACTS 17-75 Abstracts 17-27 Group discussion I: Bone marrow transplantation 17
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H L A -A, -B, -DR H A P L O T Y P E F R E Q U E N C I E S IN F R A N C E : I M P L I C A T I O N S F O R L I S T S OF P O T E N T I A L B O N E M A R R O W D O N N E R S C Lonjou, J Clayton, A Carnbon-Thomsen, C Raffoux. CNRS-CRPG, Htpital Purpan, Toulouse et France Greffe de MOelle, Hfpital Saint Louis, Paris, France A study o f haplotype frequencies has been undertaken with the file o f potential bone marrow donors held by "France Graffe de Moelle". This file contains 40156 individuals, from 20 regions o f France, who have been typed for H L A -A, -B and DR. A maximum likelihood estimator was used in order to calculate haplotype frequaneies, their support limits (confidence intervals) for each region and for the whole o f France. The observed differences between the regions were statistically significant. For each region, the minimum number o f haplotypes necessary to explain 5 0 % and 90% o f the total frequency were calculated, and compared with the equivalent values, and their confidence intervals, obtained by repeated random samplings from the overall file. This approach shows that some regions (eg Provence) appear to be richer in terms o f the numbers o f haplotypes observed, and others (eg Bretagne) poorer.In the latter ease however, the frequeeeies o f the commoner haplotypas are greater. The haplotype frequencies o f the whole sample were used to calculate the probability o f finding a match for the next potential reeipiant for given sizes o f the donner file, assuming random selection o f donors. They were also used to eahiulate expected numbers o f the major phenotypes, assuming I-Iardy-Wanberg equilibrium, and these were compared with those observed in the real data file. In this way, a large number o f under-represented and non represented phenotypes were identified (eg A2,A9 B 1 2 , B l g DP,5,DR7 : observed 1, expected 8,20). For each o f these phenotypes, the most probable haplotypes (A9-B 12-DR7 and A2-B 18-DR5 for the example) and the regions in which these have the greatest frequencies have been identified (Aquitaine and Langudoc respectively). A search for a donor with this particular phenotype would be much more fruitful if directed towards these regions.
THE I M P A C T OF GENETIC M I S M A T C H I N G ON B M T O U T C O M E B.A. Bradley ~, T R . Downie ~, J.M. Hews ~, S.M. Gore 2, G.J. Laundy ~, C.N. Hume ~ ~University of Bristol, Department Transplantation Sciences, U K 2MRC Cambridge, U K , for the I M U S T Study Strict FILA matching severely limits the numbers of unrelated donor B M T (UDB M T ) performed. But some mismatched grafts are successful for example paediatric and TCD transplants. The effect of mismatching was analysed in a prospective multicentre study involving transplants performed between 1989-1993 in 42 centres worldwide. 305 U D - B M T were compared with 628 HLA genetically identical B M T ( I D - B M T ) collected as controls. 33 (11%) and 4 (1%) o f U D - B M T were mismatched (MM) for one or two HLA-A, B, DR antigens respectively (37 patients in total) and 268 were matched ( M ) Kaplan Meier life table analysis showed the following % probabilities (95% CI): Ouhiome: ID-BMT (628) M-UD.BMT (268) MM-UD-BMT (37) Survival (12m) 63 (61-65) 45 (38-51) 24 (7-4 ] ) AGVHD (lOOd) 38 (35-42) 45 (39-51) 50 (33-66) Engraftment (100d) 98 (97-99) 87 (83-91) 84 (71-98) In muhifactorial analysis HLA-A, B, DR mismatching failed to reach significance as an independent risk factor, but donor type (ID versus UD) and interestingly sex mismatch reached significance after correction for all major clinical and therapeutic variables suggesting an influence o f unidentified HLA and non HLA genes. In summary, results of B M T are universally poorer wiah UD than with ID due to genetic mismatches that cannot be identified by routine HLA-A, B, DR typing and a prospective study is undenvay to elucidate this vital difference.
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20
EVIDENCE FOR PERMISSIVEAND NONPERMISSIVE ItLA MOLECULAR MISMATCHINGIN MLC : STUDY IN 39 PAIRS SELECTED FOR UNRELATEDBONE MARROW TRANSPLANTATION. M M= Marfinetti, C Daielli, F. Locatelli,L. Prete, E.P Alessandrino, M. Bonfichi, A. Pastormerlo, L Sahianeschi. Sere. Immunoematol. Trasf, IRCCS, Pavia-ltaly;Dip. pediatria,IRCCS, Pavia;Die Ematol.IRCCS, Pavia Twenty-one patients (9 adults and 12 children), who were found to be HLA serologicalidenticalto at least one unrelated bone man'ow donor, underwent HLA molecular typing to select the best matching for BMT. Thirtynine patient-donor pairs were evaluated in mixed lymphocyte culture (MLC). The DRBI, DQB1 and DPB1 mismatches were evaluated by using ECR-fingerprinting, whereas the genomi¢ polymorphism wa~ defined by using PCR-SSP for DRBI and DQBI and by ECR-heteroduplex for DPBI. At the geno~lie level, 9 out of 39 couples wer~ fully matched; 15 were one locus mismatched (all for DPBI locus),6 were diffe~nt at two loci and 9 were completelyrmsmatched The largestdiscrepancies were at DPBI locus (28 mismatched pairs) followed by those at DQB1 and DRBI loci (14 and 12 cases respectively).An MLC relative response index (RR]) lower than 10 in both GVHD and rejectiondirections was considered as negative. The mean RRt was higher in pediatric patients than in adults: in the rejection direction it was 72.8 vs 18.5 respectively,p--O.007;in the GVHDdirection it was 44.8 vs 14.3,p=0.014. The mismatching at the DPBI l~us seems to be permissive in the adult patients both in rei~ilon and GVHD sense (mean RRIs always <10). In children, the incompatibility at the DPBI locus seems to be implicated in positive MLC parileul~ly in the rejection direction (mean RILl value = g7.s). The addition of DRBI and DQBI mismatching to the DPBI discrepancies negatively influences MLC responses mainly in GVHD direction (the mean RR for 3 loci incompatibilitiesvs DPB1 alone was : 28 1 vs 3.5, p=n.s, in adults and 79.7 vs 28.0, p=0.057 in children). As some negativeMLCs were present in pairs with 4 loci mismatching and, on the contrary, there were some positive MLCs in fully matched pairs, the MLC reactivity could be considered as an allele
DETECTION OF HLA CLASS I INCOMPATIBILITIES IN SEROLOGICALLYMATCHED PATIENTS AND UNRELATEDBONE NARROWDONORSBY OLIGOTYPING FOR A2, A3. AND B44 ALLELIC SUBTYPES. J.~M. Tiercy. N. Djavad. N. Rufer, D. Speiser, M. Jeannet, E. Roosnek. Transplantation Immunology Unit, Division of Immunology and Allergology, H6pital Cantonal. Geneva HLA A2, A3, and B44 are among the most frequent class I antigens in Caucasoids. They are typed as individual HLA antigens by routine serology but numerous subtypes can be discriminated by other techniques such as CTL assays. IEF or DNA sequencing. Since essentially all of the polymorphism resides in the alphal and alpha2 domains responsible for peptide binding and TCR recognition, subtype incompatibilities should form important transplantation b a r r i e r s We show that by using a simple group-specific PCR/SSO-oligotyping strategy (4 primer combinations/12 probes), ten A2. two A3 and three B44 alleles can be discriminated. A*0201 and A*0301 are the dominant subtypes in a large panel of Caucasians. whereas B'4402 occurs twice as frequently as B'4403. Oligotyping of a group of 30 patients and their 116 unrelated serologically ABDRmatched donors showed that 23% of the patients had at least one donor mismatched for a subtype of A2 or B44. In all cases pre transplant in v i t r o CTLp frequency test was positive, All of these class I mismatches occurred in the group of D/R pairs that consisted in more than one DR genotype. When high stringency matching (i.e. DRB1/B3/B5 DNA typing) was taken into account the number of A2/B44-mismatched combinations decreases to 7%. This most l i k e l y results from the predominance of conserved hapletypes in a population, Particularly when donors are recruited from registries from different geographical areas, class I matching should be tested by more sophisticated methods such as DNA typing.
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MOLECULAR BIOLOGY CLASS II TYPING MAY NOT ALWAYS BE SUFFICIENT TO SHOW DISCREPANCIES BETWEEN BONE MARROW DONORS AND RECIPIENTS. M. Laforet, A. Oflacher, M. Jeras, M.-M. Tongio. Laboratoire d'histocompatibilitt, Centre R t g i o n a l de Transfusion Sanguine, 10 rue Spielmann, 6 7 0 8 5 Strnsbourg, France Today molecular biology typing is tending to replace functional tests to define the compatibility between bone marrow donors and recipients. Nevertheless by doing so, some class II differences may pass unnoticed. This is the case o f the newly described D R B I * 1 6 0 4 allele (§) when compared to the well k n o w n D R B I * I 6 0 1 allele. DRB 1" 1604 differs from DRB 1* 1601 by one nucleotide located in codon 72 and two nucleotides in codon 74. Use o f D R generic and D R 2 - D R B 1 specific primers attributes the same pattern by PCR-SSO to both alleles, when using the uligoprobes of the XI th International Histocompatibility Workshop. To investigate i f these alleles behaved similarly at the functional level the following stimulating - responding cell combinations were used: (cell type 1: Fern: D R B 1 ' 1604, 0404; DQB 1"0302, 0502; DPB 1"0301, 2001) (cell type 2: Doff: DRB 1" 1601, 0404; DQB 1 *0302, 0502; DPB 1"0601, 1001 ) The mixed lymphocyte reaction showed a w e a k proliferative response when type 1 was used as stimulator and type 2 as responder and no proliferation in the reverse situation. Analysis of the allomactive helper T cell precursor ( H ' l ~ p ) frequency was more sensitive. When type 1 B-lymphoblastoid cell line ( B - L C L ) was taken as stimulator, H T L p frequency was: 1 cell on 974 peripheral blood mononuelear cells (PBMC). In the reverse situation it was: I cell on 1748 P B M C . These results show that: 1) new alleles (like D R B I * 1 6 0 4 ) may not always be detectable using routine molecular biology techniques; 2) even the M L C test may not give the solution; 3) study of die precursor frequency seems much more accurate and might thus become the ultimate test to perform before bone marrow transplantation. (§) Laforet et al. Tissue Antigens: in pros
THE USE OF CTLP FREQUENCIES IN THE SELECTION OF MATCHED UNRELATED BONE MARROW DONORS. M. Oudshoorn, M.B, Ruigrok, J.L.W.T. Lie, W.E. Fibbe, A. Haraldsson, K. Sintnicolans, J.L van Rood, D.L. Roelan, F.H.L Clans, Department of Immunohematology & Blood Bank, University Hospital Leiden, The Netherlands Previously, Kaminski et at. have shown a significant correlation between high cytotoxic T lymphocyte precursor (C'TLp) frequency of the donors against the recipient and the severity of acute GvHD after "matched" unrelated BMTs. To date, 40 matched unrelated bone marrow donors have been tested by us for 20 recipients and 8 potential nongenotypically identical family donors for 6 recipients. A positive CTLp outcome was defined as those with a CTLp frequency of ;~ 8 : 1,000,000. Results: 1) Of the observed HLA differences [HLA-C, -DQ, -DP and/or a positive MLC test] in donor/recipiant pairs, only the HLA-C locus differences showed a significant correlation (p = 0,04) with a positive CTLp test. Presumably, the C locus antigen mismatch serves as a marker for a mismatched haplotype including other Class I antigens that are not detectable by serological methods. 2) Statistical analysis of 12 donor/recipient pairs revealed a correlation between high CTLp frequencies of the donor before transplantation and the development of clinically significant aGvHD post-transplantation (p = 0.07), thus confirming the data of Kaminski et at. 3) Several case histories illustrating the potential importance of the CTLp test in selecting the best donor if more than one is available, will be discussed. Examples will include selection of unrelated donors as well as selecting the best of the non-genotypically identical related donors found through extended family searching.
126 ABSTRACTS SELECTED FOR GROUP DISCUSSION: ABSTRACTS 17-75 Abstracts 17-27 Group discussion I: Bone marrow transplantation 17
18
H L A -A, -B, -DR H A P L O T Y P E F R E Q U E N C I E S IN F R A N C E : I M P L I C A T I O N S F O R L I S T S OF P O T E N T I A L B O N E M A R R O W D O N N E R S C Lonjou, J Clayton, A Carnbon-Thomsen, C Raffoux. CNRS-CRPG, Htpital Purpan, Toulouse et France Greffe de MOelle, Hfpital Saint Louis, Paris, France A study o f haplotype frequencies has been undertaken with the file o f potential bone marrow donors held by "France Graffe de Moelle". This file contains 40156 individuals, from 20 regions o f France, who have been typed for H L A -A, -B and DR. A maximum likelihood estimator was used in order to calculate haplotype frequaneies, their support limits (confidence intervals) for each region and for the whole o f France. The observed differences between the regions were statistically significant. For each region, the minimum number o f haplotypes necessary to explain 5 0 % and 90% o f the total frequency were calculated, and compared with the equivalent values, and their confidence intervals, obtained by repeated random samplings from the overall file. This approach shows that some regions (eg Provence) appear to be richer in terms o f the numbers o f haplotypes observed, and others (eg Bretagne) poorer.In the latter ease however, the frequeeeies o f the commoner haplotypas are greater. The haplotype frequencies o f the whole sample were used to calculate the probability o f finding a match for the next potential reeipiant for given sizes o f the donner file, assuming random selection o f donors. They were also used to eahiulate expected numbers o f the major phenotypes, assuming I-Iardy-Wanberg equilibrium, and these were compared with those observed in the real data file. In this way, a large number o f under-represented and non represented phenotypes were identified (eg A2,A9 B 1 2 , B l g DP,5,DR7 : observed 1, expected 8,20). For each o f these phenotypes, the most probable haplotypes (A9-B 12-DR7 and A2-B 18-DR5 for the example) and the regions in which these have the greatest frequencies have been identified (Aquitaine and Langudoc respectively). A search for a donor with this particular phenotype would be much more fruitful if directed towards these regions.
THE I M P A C T OF GENETIC M I S M A T C H I N G ON B M T O U T C O M E B.A. Bradley ~, T R . Downie ~, J.M. Hews ~, S.M. Gore 2, G.J. Laundy ~, C.N. Hume ~ ~University of Bristol, Department Transplantation Sciences, U K 2MRC Cambridge, U K , for the I M U S T Study Strict FILA matching severely limits the numbers of unrelated donor B M T (UDB M T ) performed. But some mismatched grafts are successful for example paediatric and TCD transplants. The effect of mismatching was analysed in a prospective multicentre study involving transplants performed between 1989-1993 in 42 centres worldwide. 305 U D - B M T were compared with 628 HLA genetically identical B M T ( I D - B M T ) collected as controls. 33 (11%) and 4 (1%) o f U D - B M T were mismatched (MM) for one or two HLA-A, B, DR antigens respectively (37 patients in total) and 268 were matched ( M ) Kaplan Meier life table analysis showed the following % probabilities (95% CI): Ouhiome: ID-BMT (628) M-UD.BMT (268) MM-UD-BMT (37) Survival (12m) 63 (61-65) 45 (38-51) 24 (7-4 ] ) AGVHD (lOOd) 38 (35-42) 45 (39-51) 50 (33-66) Engraftment (100d) 98 (97-99) 87 (83-91) 84 (71-98) In muhifactorial analysis HLA-A, B, DR mismatching failed to reach significance as an independent risk factor, but donor type (ID versus UD) and interestingly sex mismatch reached significance after correction for all major clinical and therapeutic variables suggesting an influence o f unidentified HLA and non HLA genes. In summary, results of B M T are universally poorer wiah UD than with ID due to genetic mismatches that cannot be identified by routine HLA-A, B, DR typing and a prospective study is undenvay to elucidate this vital difference.
19
20
EVIDENCE FOR PERMISSIVEAND NONPERMISSIVE ItLA MOLECULAR MISMATCHINGIN MLC : STUDY IN 39 PAIRS SELECTED FOR UNRELATEDBONE MARROW TRANSPLANTATION. M M= Marfinetti, C Daielli, F. Locatelli,L. Prete, E.P Alessandrino, M. Bonfichi, A. Pastormerlo, L Sahianeschi. Sere. Immunoematol. Trasf, IRCCS, Pavia-ltaly;Dip. pediatria,IRCCS, Pavia;Die Ematol.IRCCS, Pavia Twenty-one patients (9 adults and 12 children), who were found to be HLA serologicalidenticalto at least one unrelated bone man'ow donor, underwent HLA molecular typing to select the best matching for BMT. Thirtynine patient-donor pairs were evaluated in mixed lymphocyte culture (MLC). The DRBI, DQB1 and DPB1 mismatches were evaluated by using ECR-fingerprinting, whereas the genomi¢ polymorphism wa~ defined by using PCR-SSP for DRBI and DQBI and by ECR-heteroduplex for DPBI. At the geno~lie level, 9 out of 39 couples wer~ fully matched; 15 were one locus mismatched (all for DPBI locus),6 were diffe~nt at two loci and 9 were completelyrmsmatched The largestdiscrepancies were at DPBI locus (28 mismatched pairs) followed by those at DQB1 and DRBI loci (14 and 12 cases respectively).An MLC relative response index (RR]) lower than 10 in both GVHD and rejectiondirections was considered as negative. The mean RRt was higher in pediatric patients than in adults: in the rejection direction it was 72.8 vs 18.5 respectively,p--O.007;in the GVHDdirection it was 44.8 vs 14.3,p=0.014. The mismatching at the DPBI l~us seems to be permissive in the adult patients both in rei~ilon and GVHD sense (mean RRIs always <10). In children, the incompatibility at the DPBI locus seems to be implicated in positive MLC parileul~ly in the rejection direction (mean RILl value = g7.s). The addition of DRBI and DQBI mismatching to the DPBI discrepancies negatively influences MLC responses mainly in GVHD direction (the mean RR for 3 loci incompatibilitiesvs DPB1 alone was : 28 1 vs 3.5, p=n.s, in adults and 79.7 vs 28.0, p=0.057 in children). As some negativeMLCs were present in pairs with 4 loci mismatching and, on the contrary, there were some positive MLCs in fully matched pairs, the MLC reactivity could be considered as an allele
DETECTION OF HLA CLASS I INCOMPATIBILITIES IN SEROLOGICALLYMATCHED PATIENTS AND UNRELATEDBONE NARROWDONORSBY OLIGOTYPING FOR A2, A3. AND B44 ALLELIC SUBTYPES. J.~M. Tiercy. N. Djavad. N. Rufer, D. Speiser, M. Jeannet, E. Roosnek. Transplantation Immunology Unit, Division of Immunology and Allergology, H6pital Cantonal. Geneva HLA A2, A3, and B44 are among the most frequent class I antigens in Caucasoids. They are typed as individual HLA antigens by routine serology but numerous subtypes can be discriminated by other techniques such as CTL assays. IEF or DNA sequencing. Since essentially all of the polymorphism resides in the alphal and alpha2 domains responsible for peptide binding and TCR recognition, subtype incompatibilities should form important transplantation b a r r i e r s We show that by using a simple group-specific PCR/SSO-oligotyping strategy (4 primer combinations/12 probes), ten A2. two A3 and three B44 alleles can be discriminated. A*0201 and A*0301 are the dominant subtypes in a large panel of Caucasians. whereas B'4402 occurs twice as frequently as B'4403. Oligotyping of a group of 30 patients and their 116 unrelated serologically ABDRmatched donors showed that 23% of the patients had at least one donor mismatched for a subtype of A2 or B44. In all cases pre transplant in v i t r o CTLp frequency test was positive, All of these class I mismatches occurred in the group of D/R pairs that consisted in more than one DR genotype. When high stringency matching (i.e. DRB1/B3/B5 DNA typing) was taken into account the number of A2/B44-mismatched combinations decreases to 7%. This most l i k e l y results from the predominance of conserved hapletypes in a population, Particularly when donors are recruited from registries from different geographical areas, class I matching should be tested by more sophisticated methods such as DNA typing.
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22
MOLECULAR BIOLOGY CLASS II TYPING MAY NOT ALWAYS BE SUFFICIENT TO SHOW DISCREPANCIES BETWEEN BONE MARROW DONORS AND RECIPIENTS. M. Laforet, A. Oflacher, M. Jeras, M.-M. Tongio. Laboratoire d'histocompatibilitt, Centre R t g i o n a l de Transfusion Sanguine, 10 rue Spielmann, 6 7 0 8 5 Strnsbourg, France Today molecular biology typing is tending to replace functional tests to define the compatibility between bone marrow donors and recipients. Nevertheless by doing so, some class II differences may pass unnoticed. This is the case o f the newly described D R B I * 1 6 0 4 allele (§) when compared to the well k n o w n D R B I * I 6 0 1 allele. DRB 1" 1604 differs from DRB 1* 1601 by one nucleotide located in codon 72 and two nucleotides in codon 74. Use o f D R generic and D R 2 - D R B 1 specific primers attributes the same pattern by PCR-SSO to both alleles, when using the uligoprobes of the XI th International Histocompatibility Workshop. To investigate i f these alleles behaved similarly at the functional level the following stimulating - responding cell combinations were used: (cell type 1: Fern: D R B 1 ' 1604, 0404; DQB 1"0302, 0502; DPB 1"0301, 2001) (cell type 2: Doff: DRB 1" 1601, 0404; DQB 1 *0302, 0502; DPB 1"0601, 1001 ) The mixed lymphocyte reaction showed a w e a k proliferative response when type 1 was used as stimulator and type 2 as responder and no proliferation in the reverse situation. Analysis of the allomactive helper T cell precursor ( H ' l ~ p ) frequency was more sensitive. When type 1 B-lymphoblastoid cell line ( B - L C L ) was taken as stimulator, H T L p frequency was: 1 cell on 974 peripheral blood mononuelear cells (PBMC). In the reverse situation it was: I cell on 1748 P B M C . These results show that: 1) new alleles (like D R B I * 1 6 0 4 ) may not always be detectable using routine molecular biology techniques; 2) even the M L C test may not give the solution; 3) study of die precursor frequency seems much more accurate and might thus become the ultimate test to perform before bone marrow transplantation. (§) Laforet et al. Tissue Antigens: in pros
THE USE OF CTLP FREQUENCIES IN THE SELECTION OF MATCHED UNRELATED BONE MARROW DONORS. M. Oudshoorn, M.B, Ruigrok, J.L.W.T. Lie, W.E. Fibbe, A. Haraldsson, K. Sintnicolans, J.L van Rood, D.L. Roelan, F.H.L Clans, Department of Immunohematology & Blood Bank, University Hospital Leiden, The Netherlands Previously, Kaminski et at. have shown a significant correlation between high cytotoxic T lymphocyte precursor (C'TLp) frequency of the donors against the recipient and the severity of acute GvHD after "matched" unrelated BMTs. To date, 40 matched unrelated bone marrow donors have been tested by us for 20 recipients and 8 potential nongenotypically identical family donors for 6 recipients. A positive CTLp outcome was defined as those with a CTLp frequency of ;~ 8 : 1,000,000. Results: 1) Of the observed HLA differences [HLA-C, -DQ, -DP and/or a positive MLC test] in donor/recipiant pairs, only the HLA-C locus differences showed a significant correlation (p = 0,04) with a positive CTLp test. Presumably, the C locus antigen mismatch serves as a marker for a mismatched haplotype including other Class I antigens that are not detectable by serological methods. 2) Statistical analysis of 12 donor/recipient pairs revealed a correlation between high CTLp frequencies of the donor before transplantation and the development of clinically significant aGvHD post-transplantation (p = 0.07), thus confirming the data of Kaminski et at. 3) Several case histories illustrating the potential importance of the CTLp test in selecting the best donor if more than one is available, will be discussed. Examples will include selection of unrelated donors as well as selecting the best of the non-genotypically identical related donors found through extended family searching.