Evaluation of mixed hematopoietic chimerism in pediatric patients with leukemia after allogeneic stem cell transplantation by quantitative PCR analysis of variable number of tandem repeat and testis determination gene

Abstract

Summary:In order to monitor the clinical outcome of pediatric patients with leukemia following allogeneic hematopoietic transplantation, tests of variable number of tandem repeat (VNTR) and sex determination by quantitative polymerase chain reaction (PCR) were performed. PCR results combined with the blast counts from 21 leukemia patients were analyzed. Complete chimerism (100% donor cells) was found in 15 cases with remission, and incomplete chimerism in six cases with relapse. In the majority of cases, complete chimerism was always associated with no detectable blasts, while blasts were often detected in association with incomplete chimerism. There is significant correlation (P Ͻ 0.0001) between the percentage of donor DNA and blast percentage in these patients. Early detection of incomplete chimerism may therefore predict a poor prognosis. In one patient (case 15), a differing percentage of donor DNA was observed between samples of bone marrow and peripheral blood collected on the same day. This may be due to the fact that allogeneic stem cells proliferate at different rates depending on their environment (bone marrow or peripheral blood). In addition, 100% donor cells found in the peripheral blood may not reflect the number of cells in the bone marrow. In case 17, asynchronous engraftment of donor cells was present between the white and red blood cell lineages, indicating that the degree of chimerism may not be the same in all cell lineages. At the time of this report, the significance of this observation is unknown and needs further investigation. Currently, allogeneic hematopoietic stem cell transplantation plays an important role in the treatment of leukemia and other hematological and oncological disorders in children. Evaluation of the outcomes following transplantation includes the evaluation of early engraftment, graft rejection, disease status and the degree of donor chimerism. Historically early chimerism analyses were based on protein or red blood cell polymorphism or karyotyping. [1][2][3][4] Since the 1980s, more sensitive tests have become available including DNA techniques such as restriction fragment length polymorphisms (RFLPs), Southern blotting and VNTR analysis. 5-7 VNTR loci are groups of DNA sequences that represent a source of highly polymorphic markers for individual identification. When compared with RFLPs and Southern blotting, VNTR has many advantages including the use of smaller quantities of DNA, ease of preparation, faster turnaround time, and elimination of restriction enzymes and radioisotopes that are required with the other two methods. The overall cost saving is also substantial. 8 Tests of sex determination are an additional tool for chimerism analysis. A target DNA sequence located in the motif of the human Y-linked testis-determination gene (SRY) was originally amplified by Cui et al 9 in a study of human sex determination at the single cell level. This highly accurate and precise test allows us to apply this technique to chimerism analysis in the case of sex-mismatch bet...

Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods

“…[7][8][9] Since the 1980s, a variety of techniques that employ polymorphic markers have been established to survey chimerism status, 10 such as the following: fluorescent in situ hybridization (FISH) with XY chromosome-specific probes or polymerase chain reaction (PCR), single-nucleotide polymorphism (SNP) analyses, short tandem repeats (STR), restriction fragment length polymorphism (RFLP) and variable number tandem repeats (VNTR). [10][11][12][13] However, several limitations have been reported associated with these techniques namely low sensitivity, time-consuming, limited to sex-mismatched transplantations, high DNA requirement and limited degree of polymorphism. 11,13 For all these clinical applications, the optimal methodological approach needs to be informative, sensitive, quantitatively accurate, reproducible and cost effective.…”

Platelet chimerism by polymerase chain reaction (PCR) utilizing variable number of tandem repeats (VNTR) in allogeneic stem cell transplant in children: a new novel approach to full chimerism analysis

“…2 Developing an optimal method for detecting full chimerism may enhance our ability to identify patients at risk for early relapse. 3,4 Chimerism assay allows for differentiation between recipient and host cells, and is used as an alternative method to identify minimal residual disease, especially in those cases without specific cytogenetic markers.…”

“…3,4 Chimerism assay allows for differentiation between recipient and host cells, and is used as an alternative method to identify minimal residual disease, especially in those cases without specific cytogenetic markers. 2,8,9 Occasionally, it is used in combination with other tests for documenting engraftment. 5,10 Clearly, any method leading to early detection is crucial, since it is now possible to prevent increasing mixed chimerism at an early stage with DLI .…”

“…The recent workshop sponsored by the International Bone Marrow Transplant Registry and National Marrow Donor Program recommended that a complete or full chimerism analysis be performed in all allogeneic lymphohematopoietic transplantation. 1 Traditionally, chimerism analysis is performed in whole blood [2][3][4] or by utilizing two-cell lineage: T-lymphocytes and myelocytes. [5][6][7] These lineage-specific chimerism studies are currently the method of choice in the setting of nonmyeloablative therapy and reduced intensity conditioning regimens.…”

Age-dependent pharmacokinetic profile of single daily dose i.v. busulfan in children undergoing reduced-intensity conditioning stem cell transplant

“…Donor chimerism was assessed using a PCR-based variable number tandem repeat (VNTR) analysis of peripheral blood leukocytes. 40 Polymorphic gene loci used in the VNTR analysis were APO-B, D1S80, D1S111, D17S30, TDG/ZP3, YNZ22 and AMG. The sensitivity for the detection of donor and recipient chimerism was 5 and 1%, respectively.…”