In the present study we analyzed for the first time HLA class I and class II polymorphisms defined by high-resolution typing methods in the Bulgarian population. Comparisons with other populations of common historical background were performed. Most HLA-A, -B, -DRB alleles and haplotypes observed in the Bulgarian population are also common in Europe. Alleles and haplotypes considered as Mediterranean are relatively frequent in the Bulgarian population. Observation of Oriental alleles confirms the contribution of Asians to the genetic diversity of Bulgarians. The use of high-resolution typing methods allowed to identify allele variants rare for Europeans that were correlated to specific population groups. Phylogenetic and correspondence analyses showed that Bulgarians are more closely related to Macedonians, Greeks, and Romanians than to other European populations and Middle Eastern people living near the Mediterranean. The HLA-A,-B,-DRB1 allele and haplotype diversity defined by high-resolution DNA methods confirm that the Bulgarian population is characterized by features of southern European anthropological type with some influence of additional ethnic groups. Implementation of high-resolution typing methods allows a significantly wider spectrum of HLA variation to be detected, including rare alleles and haplotypes, and further clarifies the origin of Bulgarians.

Summary:We evaluated the efficacy and toxicity of different doses of donor T cells given with donor leukocyte infusions (DLI) as treatment for relapse of various hematologic malignancies after allogeneic bone marrow transplantation (BMT). We also studied whether DLI treatment was more effective if circulating T cells were exclusively of donor origin (complete donor T cell chimeras) as compared with T cells originating from both donor and recipient (mixed T cell chimeras). Twenty-eight patients were studied of whom 24 had a complete donor T cell chimerism. The malignancies were as follows: chronic myeloid leukemia (CML) in chronic phase (CP) (n = 9); more advanced CML (n = 5); multiple myeloma (MM) (n = 5); acute leukemia (AL) (n = 9). T cell doses varied from 0.1 ؋ 10 7 to 33 ؋ 10 7 T cells/kg. Eight patients received two to four DLI courses because they failed to respond to one course. Thirteen of 14 patients with CML, including four patients with more advanced CML, achieved complete remission (CR). All five patients with MM responded, including three CRs. Six patients (three with CML, three with MM) responded only after two to four DLI courses. Patients with CML-CP were likely to respond to as few as 1 ؋ 10 7 T cells/kg whereas patients with MM generally responded when they received у10 ؋ 10 7 T cells/kg. However, despite the infusion of high T cell doses (up to 32 ؋ 10 7 T cells/kg), practically all patients with AL failed to respond. The likelihood of response was strongly related to the occurrence of graft-versus-host disease (GVHD) in patients with CML and MM (P = 0.0002), although GVHD was not helpful for patients with AL. Higher T cell doses (у10 ؋ 10 7 /kg) induced serious GVHD (n = 17) and marrow aplasia (n = 5), and GVHD was directly or indirectly the cause of death for six patients. Finally, there were no obvious differences in responses between complete donor T cell chimeras and mixed T cell chimeras. Keywords: allogeneic BMT; donor leukocyte infusions; recurrent haematologic malignancies; T cell chimerism The ability of donor leukocyte infusions (DLI) to induce responses in recurrent hematologic malignancies after allogeneic bone marrow transplantation (BMT) seems to be dependent on at least two variables: (1) the kind of malignancy and (2) the number of T cells present in the DLI. Evidence for the first variable comes from a multicenter study reporting that DLI for recurrent leukemia after BMT can be effective in 80% of patients with chronic myeloid leukemia (CML) whereas this happens in only 20% of patients with acute myeloid leukemia or myelodysplastic syndrome. 1 Evidence for the second variable comes from a study of escalating doses of donor T cells in DLI for patients with recurrent CML after BMT. 2 This is, of course, in line with the strong correlation between the number of donor T cells infused with the BMT procedure and the occurrence of both acute graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect. [3][4][5] It is presently unknown whether patients whose circulating ...

Summary. In this study, we analysed the chimaeric status of peripheral blood leucocytes (PBLs) in recipients of allogeneic bone marrow transplantation (BMT) with the use of short tandem repeat (STR) microsatellite markers for monitoring the efficacy of BMT and donor leucocyte infusions (DLIs). A set of four STR markers was used with a highly discrimative capacity between individuals. STRs were detected by polymerase chain reaction (PCR) and were analysed by gene scanning (STR-GS). Between June 1990 and December 1998, 52 patients treated with BMT for chronic myeloid leukaemia (CML) were analysed. Seventeen patients relapsed after BMT and two patients never achieved remission after BMT. Fourteen of the 17 patients achieved a complete donor chimaerism after BMT, as detected by the presence of only donor STR-GS fragments, and in three cases a weak recipient STR-GS signal remained persistently detectable after BMT. A reappearance or increase of recipient STR-GS signals was indicative of relapse, which was mostly detected by STR-GS several months before relapse was diagnosed clinically. Nineteen patients were treated with DLI for reappearance of CML after BMT which resulted in complete remission in 17 patients, concordant with the disappearance of recipient STR-GS signals. More importantly, DLI treatment could be guided based upon the STR-GS data, which prevented unnecessary extra DLI courses that could cause toxicity. This study indicates that STR-GS is an effective and reliable method for monitoring BMT recipients.

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta2m). The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta2m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.

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The goal was to establish the best typing techniques and strategies for donors selected as potential donors for a specific patient. Complete sequence of exons 2 and 3 for class I and exon 2 for class II molecules by sequencing-based typing was deemed the best technique in reducing problems with ambiguities and null alleles as well as the possibility to establish a completely automated system. Focusing on multiple approaches for typing was regarded as a better strategy than continued usage of serological methods although the latter technique was still considered to be useful by some for detection of null alleles. The patient and finally the selected donor, including the back-up donor, should be typed for at the highest level and typing ambiguities should be resolved. In very difficult cases, race, haplotype information and allele frequency in the population should help to decide whether or not to resolve the ambiguity. Cellular techniques such as the cytotoxic T lymphocyte precursor test may help to choose between single class I mismatched donors in experienced laboratories only.

The use of direct sequencing as a typing strategy is well acknowledged. Direct sequencing identifies all sequence motifs including new polymorphisms in heterozygous sequences. The earlier protocols for human leukocyte antigen HLA-DQB1 Sequencing-Based Typing (SBT) frequently encounter preferential amplification of one of the alleles that can lead to unreliable sequences or even to allelic dropout. In our new approach, the quality of the exon 2 sequences, now including both alleles to the same extend, was achieved by amplifying the HLA-DQB1*05/06 group into two groups by changing the common 3' amplification primer. In combination with exon 3 this updated HLA-DQB1 protocol provides a reliable approach for heterozygous sequencing.

We describe a new HLA-A allele, A*3306, which was identified by sequencing based typing (SBT) in an individual of Indian origin. A*3306 is similar to A*3303, with a difference at position 228 (A to G). This difference leads to an amino-acid change at codon 52 from Ile (ATA) to Met (ATG). Until now this position has been considered conserved.