Long-term follow-up of chimerical state of the patients transplanted for different haematologic diseases.

Long-term follow-up of peripheral cellular chimerism in patients treated with BMT or PBSCT revealed the usefulness of their continuous monitoring at molecular level. Our results are based on monitoring of 120 patients, who were followed for at least 24 months. Comparison of the patients treated for chronic myelogenous leukemia (CML), acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS) and aplastic anaemia (AA) revealed that mixed chimerism was practically absent in MDS and relatively long-lasting in ALL and AA (regardless to substantially different post-transplantation treatment). The first disease relapses signalized by molecular checking of mixed peripheral chimerism were observed also after a period of remission lasting for several years. Molecular watching enables us to detect relapses at their very beginning that would remain hidden to less sensitive methods. We believe that all of the transplanted patients ought to be monitored for residual disease i.e. cellular chimerism using molecular methods without time limits. On the other hand low level of mixed cellular chimerism is not necessarily a sign of disease progression and can remain unchanged as "status quo" for a very long period.

Location of the BCR/ABL fusion genes on both chromosomes 9q34 in Ph negative chronic myeloid leukemia.

We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.

Loss of heterozygosity and heterogeneity of its appearance and persisting in the course of acute myeloid leukemia and myelodysplastic syndromes.

Screening for loss of heterozygosity (LOH) of the panel of 18 highly polymorphic microsatellite markers, especially from the region 11p15, was carried out on 154 samples from 26 patients with acute myeloid leukemia and eight with myelodysplastic syndromes (MDS). LOH was detected at the majority (72%) of the loci tested: 47% of informative patients displayed LOH for at least one of the microsatellite locus from the region 11p15 and 23.5% of patients displayed LOH among the other markers tested within the study. A longitudinal follow-up of patients showed a remarkable heterogeneity of LOH appearance and its persistance during the course of the disease suggesting an intratumor clonal heterogeneity, or alternatively, presence of LOH in more than one cell clone. The data revealed two regions of high loss of one allele in 11p15.5, defined by markers D11S1363 and D11S1338, indicating that LOH at the subtelomeric region of the short arm of chromosome 11 is a much common event in hematological malignancies than it was previously reported.

Detailed monitoring of hematopoietic chimerism in a child treated by adoptive immunotherapy for high risk of relapse after BMT for acute myeloid leukemia.

We report a case of a 13-year-old boy who was transplanted for relapse of acute myeloid leukemia (AML). A detailed study of hematopoietic chimerism was performed using polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) at very short time intervals. We used discontinuation of post-transplant immunosuppression and donor lymphocyte infusions (DLI) in order to prevent leukemia relapse that was indicated by a progressive increase in autologous hematopoiesis. Despite the fact that the boy relapsed 10 months after BMT, we could see a significant influence of adoptive immunotherapy on the mixed chimerism status during the post-transplant period.

Prediction and reversion of post-transplant relapse in patients with chronic myeloid leukemia using mixed chimerism and residual disease detection and adoptive immunotherapy.

In the prospective study, we examined hematopoietic mixed chimerism (using polymerase chain reaction (PCR) of variable number of tandem repeat-VNTR sequences) and minimal residual disease (MRD) status (using qualitative and in the case of positivity quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the BCR/ABL fusion mRNA) in serial peripheral blood samples taken from 25 patients after bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Increasing mixed chimerism in correlation with increasing signal of MRD was detected in 10 patients. In two patients mixed chimera status and BCR/ABL rearrangement led to hematologic relapse, in five patients molecular relapse was followed by reappearance of Ph chromosome and three patients developed molecular relapse only. Adoptive immunotherapy-donor lymphocyte infusion (DLI), interferon (INF) and discontinuation of post-transplant immunosupression-separately or in different combinations was used in nine patients with molecular, cytogenetic or hematologic relapse of CML. The results demonstrate that significant response at the molecular level can be achieved for a majority of CML patients and that using of all forms of adoptive immunotherapy controlled by MC and MRD is more efficient in patients treated in early molecular relapse-with minimal disease burdens.

Frequencies of HLA-DRB1, -DQB1 and -DPB1 alleles in Czech population.

The frequencies of phenotypes, alleles and allelic subtypes of DRB1 and DQB1 HLA loci in 420 unrelated individuals from the Czech population were determined. The frequencies of DPB1 alleles of the HLA locus were determined in 92 individuals. The assays were performed using the polymerase chain reaction (PCR) method or the restriction fragment length polymorphism (RFLP) analysis. The most frequent DRB1 allele was *07, the most frequent DQB1 allele was *03 and the most frequent DPB1 allele detected was *04. These assays define the extent of polymorphism of the HLA system and are useful for determining the selection strategy of HLA-identical donor-recipient pair suitable for bone marrow transplantation.

[DNA polymorphism, allogenic bone marrow transplantation and peripheral cell chimerism]. / DNA polymorfismy, alogenní transplantace kostní drene a periferní bunecný chimérismus.

BACKGROUND: Bone marrow transplantation or transplantation of peripheral stem cells is an effective treatment of a number of diseases. Its increasing success and expanding use in associated with the development of molecular diagnostic methods which enable to follow up the graft from its engraftment in a recipient and then during the whole posttransplantation period at the level extremely small numbers of cells. METHODS AND RESULTS: In peripheral blood of patients, genotypes of the following loci were examined by polymerase chain reaction (PCR): APOB, COL2A1, D17S20, D1S80, HVR/1G, SRY and AMXY. Technique of restriction analysis was used for loci DXYS20 and DXYS75. 1. The first signs of donor bone marrow activity were observed in 50% of patients already at the beginning of the second week after transplantation, while in the second half of patients increasing number of donor cells in peripheral blood was noticed in the second and third week. 2. Engraftment with full and permanent substitution of own bone marrow without presence of recipients cells in peripheral blood--complete chimerism--was achieved only in a part of patients (cca 50%). 3. Peripheral blood of other patients did not contain only donor cells but also recipients cells--mixed chimerism. With regard to its onset, the authors have divided mixed chimerism into early and late, taking into account that some patients can develop both types. In patients under study, early chimerism was found more frequently, which apparently resulted from a shorter period of observation of lately transplanted patients. 4. In cases of oncohaematologic patients, which allowed to study specifically the presence of a pathologic clone, the follow-up of chimerism enabled to distinguish between relapse of the original disease and "biologic" recovery--resurrection of original disease-free haematopoiesis. 5. Regression of mixed chimerism was supposed to be the result of treatment focused at the original disease (CML), in some patients, however, it was a spontaneous process. CONCLUSIONS: Follow-up of cellular chimerism in transplanted patients by means of molecular genetic methods provides substantial information about patient's shape which can be utilized it is necessary to decide on treatment procedures. For this reason it is desirable that examination of chimerism by molecular methods should form integral part of care of these patients.

Transplantation of allogeneic peripheral blood progenitor cells--a single centre experience.

13 patients have been transplanted at Institute of Hematology and Blood Transfusion since 1995 using allogeneic PBPC either alone or with bone marrow as a source of progenitor cells. All donors were G-CSF mobilised HLA identical family members. PBPC harvests were performed on D 4,5, (6) of G-CSF administration. The medium content of TNC, CD34+, CD3+, CD4+and CD8+cells/kg b.w. of the recipients in the grafts were: 13,1x10(8)(TNC), 11,4x10(6)(CD34+), 393x10(6)(CD3+) 243x10(6)(CD4+), 125x10(6)(CD8+) The patients received either BuCy2 or CyTBI preparative regimen and Cyclosporin A + short course of Methotrexate for GVHD prophylaxis. Engraftment of ANC >500 was achieved by D+16 and PLT >20.000 by D+19. Three of ten evaluable patients developed acute and three of nine chronic GVHD. 8 patients survive with the longest follow up 776 days.

[Determination of biological paternity yesterday and today]. / Prukaz biologického otcovství vcera a dnes.

The paper deals with methodical development of expertise in cases of disputed paternity. Presently used approaches to paternity testing were introduced following deeper understanding of heredity of human features as well as development of new methods of their studying. While the genetic characterization of individuals tested was till recent time limited to the level of gene products, in the present also direct molecular genetic analysis of their genome is possible. The most frequently used molecular genetic technique is Polymerase Chain Reaction (PCR) which leads to multiplication of chosen hypervariable regions of DNA followed with direct with following direct detection of polymorphic alleles. Examination of several DNA polymorphisms provides an exclusion of all falsely accused men and in opposite cases a practical proof of parenthood. That is the reason why the world-wide trends of forensic medicine tend to consecutive replacement of all procedures use until now with methods of molecular genetics.

HLA class II gene frequency in a Czech Population.

In a sample of 212 healthy, unrelated individuals of a Czech (Central Bohemian) population, the phenotype and allele frequencies of HLA-DRB1 and -DQB1 loci were determined. DNA typing technique used was the restriction fragment length polymorphism (RFLP) analysis. The restriction enzymes were TaqI and HindIII and specific DRB1 and DQB1 probes were applied. The most frequent DRB1 and DQB1 alleles found were DRB1*11 and DQB1*06, their respective frequencies being 0.1698 and 0.2594.

[The human genome--chromosome Y]. / Lidský genom--chromozom Y.

The Y chromosome, the only chromosome present only in the male karyotype, is one of the smallest human chromosomes. Its content of protein-forming genes is, as compared with other chromosomes, greatly reduced and if we omit the part homologous with the X chromosome it is really minimal. For normal development the area occupied by locus TDF or SRY can be considered essential. The antigenic locus HY is also associated with male sex. Well known are also the amelogenin locus which is in the pseudoautosomal area, the locus conditioning Kallmann's syndrome and locus ASMTY for hydroxyindole-O-methyltransferase--the last enzyme of the metabolic route to the pineal hormone melatonin. With regard to the presence of many highly polymorphous areas the Y chromosome is predestined for population studies where it supplements aptly investigations of the mitochondrial genome, and for investigations of cellular chimerism after allogeneic transplantations.

A genetic profile of Romany (Gypsy) subethnic group from a single region in Slovakia.

The data presented here are on population structure and genetic markers (ABO, RH, MN, HP) in two series of so-called Slovak Romany subethnic groups from a single region (Gemer) in the Southeastern part of Slovakia. The results demonstrate that favourable conditions have existed for population genetic mechanisms operating in isolated populations, namely genetic drift and inbreeding. In addition, an attempt was made to compare the observed data with those available for other Romany populations and for Slovaks.

[Polymorphisms in the pseudoautosomal regions of X and Y in DNA diagnosis]. / Polymorfismy pseudoautosomální oblasti X a Y v DNA diagnostice.

New findings on the pseudoautosomal area of X and Y chromosomes potentiate the importance of this part of the human genome for direct and indirect DNA diagnosis. The submitted paper is focused on the characteristic features of the given area with special emphasis on loci used in the Institute of Haematology and Blood Transfusion during DNA monitoring of patients after allogeneic transplantation of bone marrow and which thus provide important information with a major clinical impact. The authors indicate trends of future research focused on investigation of the polymorphism of the receptor of the haematopoietic factor GM CSF in relation to its function and the influence on the pathogenesis of AML.

Molecular follow-up of patients after bone marrow transplantation.

The follow-up of patients after bone marrow transplantation (BMT) revealed some discrepancies between red blood cell and white blood cell origin. In all six patients under study, the DNA analysis showed full engraftment, while red blood cells in some of them indicated persistence of recipient bone marrow activity. Abnormalities detected by the probe p362A (XY homologous region) in electrophoretic patterns observed during the period of graft versus host disease (GVHD) are discussed.