Detection of engraftment and mixed chimerism following bone marrow transplantation using PCR amplification of a highly variable region-variable number of tandem repeats (VNTR) in the von Willebrand factor gene

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...

Section: Discussionmentioning

confidence: 99%

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

Wang

Chou

Gonzalez-Ryan

et al. 2002

“…Chronic GVHD involved skin in all nine patients, mucosa in five, gastroby polymerase chain reaction as described. 15 Karnofsky performance score of at least 90%.…”

Section: Bmt (Years)mentioning

confidence: 99%

Excellent long-term survival after allogeneic marrow transplantation in patients with severe aplastic anemia

Reiter

Keil

Brugger

et al. 1997

Summary:unrelated donors is less successful and still under clinical investigation. 5 One major complication of BMT in patients with SAA is graft rejection which can be prevented by Between 1982 and 1996, 20 patients (10 male, 10 female) with severe aplastic anemia (SAA) with a median age intensive pretransplant immunosuppressive therapy 1,2 and prolonged use of CsA post-transplant. 1,6 Previous blood of 25 years (17-37 years), received grafts from an HLAidentical sibling (n = 17), HLA-identical unrelated transfusions increase the risk of rejection by sensitizing recipients to transplantation antigens on donor cells. 1,7 donor (n = 2) or identical twin (n = 1). The median time from diagnosis to marrow transplantation (BMT) was Donor buffy coat infusions during the first post-transplant days in addition to immunosuppression resulted in a 15 months (range 1-96 months). More than half of the patients had received more than 10 units of red blood reduced rate of graft rejection, but a higher incidence of chronic graft-versus-host disease (GVHD). 8 cells or platelet transfusions prior to BMT. Pretransplant immunosuppression consisted of cyclophosphamAnother complication resulting in significant morbidity and mortality after BMT is GVHD. Increasing age of ide (CY) alone (n = 10), CY in combination with total body irradiation (n = 8), and CY and antithymocyte recipient and donor and parity of donor are known risk factors for GVHD. 1,4,9 Post-transplant immunosuppression globulin (n = 2). For graft-versus-host disease (GVHD) prophylaxis methotrexate (MTX) alone (n = 9) or MTX with the combination of methotrexate (MTX) and CsA lead to a reduced incidence of acute GVHD and improved surwith cyclosporin A (n = 10) were given. One patient died on day 18 after marrow grafting due to infection; all vival. 10 In addition, transfusion with filtered, leukocytedepleted blood products and improved supportive care with other patients had complete and sustained engraftment (95%). Eight patients developed acute GVHD (42%), new antimicrobial and antiviral agents have led to increased survival rates after BMT during the last years. 1 nine patients chronic GVHD (53%) including four with extensive disease manifestation. One patient experiSince 1982, 20 patients with SAA have undergone BMT at the University Hospital of Vienna. Here, we report our enced a secondary malignancy 11 years after BMT. Eighteen patients followed for a median of 9.45 years long-term results. (0.42-14.7 years) have sustained hematological reconstitution and are alive and well with a Karnofsky performance score of at least 90%. Thus, excellent long-term Patients and methods survival and low morbidity make allogeneic or syngeneic BMT the treatment of choice for younger Patients patients with severe aplastic anemia. Keywords: aplastic anemia; allogeneic marrow transBetween January 1982 and April 1996 20 consecutive patients, 10 men and 10 women, aged 17-37 years (median plantation; long-term follow-up 25 years) with severe aplastic anemia as defined by the Internatio...

“…Engraftment of donor cells was further documented by cytogenetic analyses of recipient marrow cells before and after BMT and amplification of the variable number of tandem repeats (VNTR) region within intron 40 of the von Willebrand factor gene by polymerase chain reaction (PCR), as described. 20 …”

Section: Engraftmentmentioning

confidence: 99%

Low transplant-related mortality in patients receiving unrelated donor marrow grafts for leukemia

Kalhs

Brugger

Reiter

et al. 1999

Summary:Transplantation with unrelated donor (UD) marrow has been shown to potentially cure patients with leukemia. Between January 1991 and April 1998, 54 patients with leukemia have received an UD BMT at our institution. Five patients received their UD BMT as a second transplant after a preceding autologous or syngeneic BMT and were excluded from further analysis. Forty-nine patients with leukemia (acute leukemia n ‫؍‬ 26; CML n ‫؍‬ 23) and a median age of 36 years (range 19-51) were analyzed. For conditioning, all patients received a combination of fractionated TBI and CY. GVHD prophylaxis consisted of MTX and CsA in all patients. As of 30 April 1998, 27 of 49 (55%) patients survive after a median observation time of 18 months. The probability of overall survival for standard risk and high risk patients is 54% and 31% (P ‫؍‬ 0.05). Probability of transplant-related mortality (TRM) is 27%, 24% in standard risk and 31% in high risk patients (P ‫؍‬ 0.44). Patients younger than 40 years (n ‫؍‬ 33) had a similar TRM as patients 40 years and older (n ‫؍‬ 16). The probability of relapse is 41% for the whole group, 29% for standard risk and 55% for high risk pts (P Ͻ 0.05). Our data confirm that UD BMT is an effective treatment for patients with leukemia. TRM is almost similar to related sibling BMT, most probably due to improvements in HLA typing technology, conditioning regimen and supportive patient care.