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Chromosomal transformation in donor cells following allogeneic bone marrow transplantation

Summary:

We report here a monosomy 7 transformation of donor cells following matched-unrelated, same sex, allogeneic bone marrow transplantation in a patient with severe congenital aplastic anemia. A PCR technique was employed to amplify microsatellite markers on chromosome 7 to confirm donor/recipient identity. We found that the transformation of monosomy 7 occurred in previously genetically normal donor cells. This study suggests that the microenvironment of the bone marrow of our patient with severe congenital aplastic anemia may have played a critical role in the development of monosomy 7 of normal donor cells and we conclude that chromosomal microsatellite marker analysis can be a valuable tool for precise donor/recipient differentiation in engraftment monitoring.

Main

The identification of donor or recipient origin of hematopoietic cells after allogeneic bone marrow transplantation (BMT) is important for assessing engraftment, defining any residual population of normal and/or abnormal host cells and for the early recognition of relapse, especially when malignant transformation of normal donor cells is suspected.1 When recipient and donor are of opposite sex, analysis of the sex chromosomes in dividing cells will provide the necessary information.2,3,4,5 When donor and recipient are of the same sex or dividing cells are not available, techniques that employ red cell antigen phenotyping, HLA-typing, immunoglobulin isotypes, leukocyte isoenzymes and restriction fragment length polymorphisms (RFLPs) have been used. However, these techniques are not always reliable and sometimes contradict each other.6,7 Microsatellite DNA markers are dispersed and highly polymorphic tandem-repetitive minisatellite regions in the human genome. These microsatellite markers have the same degree of individual specificity as do fingerprints8 and have been used to distinguish between tissue of donor and recepient origin following BMT.9,10,11,12,13 To determine the origin of monosomy 7 transformation following matched-unrelated same sex donor allogeneic bone marrow transplantation, we used a PCR technique to amplify microsatellite markers on chromosome 7. A comparison was made between donor and recipient, both before and after BMT revealed that this chromosomal transformation occurred in normal donor cells.

Case report

In June 1991, a Caucasian girl presented at 8 days of life with a platelet count of 9000 and easy bruisability. Bone marrow aspiration was consistent with congenital amegakaryocytosis and cytogenetic analysis showed normal female 46,XX[15] (Table 1). Chromosomal breakage test to rule out Fanconi's anemia was normal. At two and half years of age, she progressed to severe aplastic anemia and became transfusion dependent. She underwent her first allogeneic BMT with T-cell depletion by elutriation at 3 years of age from a matched-unrelated 37-year-old female donor. Cytogenetic studies on the patient prior to BMT were normal on multiple occasions. The preparative regimen included total body irradiation (TBI) 1200 cGy (200 cGy b.i.d. for 3 days), Ara-C 1.5 g/kg (250 mg/kg b.i.d. for 3 days) and Cytoxan 100 mg/kg (50 mg/kg/day for 2 days). Peripheral myeloid engraftment was evident on day +56. However, the dose of granulocyte colony stimulating factor (GCSF) was increased to 20 μg/kg/day from day +43 to +74 due to slow engraftment. She was platelet transfusion-dependent until 19 months post-BMT. Her BMT course was complicated by grade I skin GVHD and Herpes Simplex viral infection of her foot. Post-BMT engraftment monitoring RFLP studies at 3, 8 and 13 months all showed 90–100% donor. However, 3 years after her first BMT, she developed pancytopenia again and cytogenetic study at that time revealed monosomy 7, while restriction fragment length polymorphism indicated that the genetic constitution of her hematopoietic cells was still 90–100% donor (Table 1). The decision was made for her to undergo bone marrow transplant again due to the malignant potential of monosomy 7 and her transfusion-dependent thrombocytopenia. No additional unrelated donors were identified. At 43 months after her first BMT, she underwent a matched-unrelated, non-T-depleted allogeneic BMT from the same donor (who had remained clinically well) after a preparative regimen of Cyclophos-phamide 100 mg/kg (50 mg/kg/day for 2 days), Busulfan 160 mg/m2 (40 mg/m2/day for 4 days) and ATG 30 mg/kg (15 mg/kg/day for 2 days). Her second post-BMT course was without any complications. Myeloid engraftment was evident at day +29 and she was platelet transfusion independent from day +23. RFLP studies 3, 6 and 12 months after second BMT all showed 100% donor and with normal female cytogenetics (46,XX)[20]. The donor remains clinically healthy to date. In order to determine the origin of her chromosomal transformation after the first BMT with precision, we employed the new PCR-based chromosomal microsatellite marker analysis technique to ascertain the origin of such an unusual transformation.

Table 1 Blood and marrow findings

Materials and methods

Stored peripheral blood specimens from the recipient before her first BMT and the donor before the second BMT were obtained from National Marrow Donor Program (NMDP) after informed consent was obtained. The recipient's fibroblasts were cultured as previously described14 from her bone marrow aspirate that was obtained just before her second BMT. A peripheral blood sample was obtained with consent from the recipient 2 years after second BMT. Genomic DNA preparations were extracted from recipient's and donor's peripheral blood and bone marrow nucleated cells (both before and after BMT) and recipient's fibroblasts, using Quiagen columns according to the manufacturer's standard protocol. Two sets of fluorescent-labeled primers at loci D7S2465 and D7S669 were used in PCR reaction to amplify microsatellite markers on chromosome 7 by using ABI PRISM™ Fluorescent Genotyping Kit (Perkin Elmer Applied Biosystems). Amplified products were analyzed on ABI PRISM™ 310 Genetic Analyzer (Perkin Elmer Applied Biosystems). Only data from locus D7S2465 are presented.

Results and discussion

Figure 1 shows allelic composition at locus D7S2465 from all the samples. Sample 1 was from patient's peripheral blood prior to her first BMT and it displayed two alleles at nucleotides 332 and 339, respectively. The fibroblasts that were cultured from patient's bone marrow specimen that was obtained prior to her second BMT were represented by sample 2. The patient's bone marrow fibroblasts had an allelic composition identical to that of her peripheral blood (sample 1). (The origin of bone marrow fibroblasts after bone marrow transplantation has been similarly demonstrated to be that of the host).15

Figure 1
figure1

Profiles from ABI PRISM™ 310 Genetic Analyzer of PCR-amplified locus D7S2465 using fluorescence-tagged primers. Sample 1, recipient's peripheral blood specimen prior to first BMT; Sample 2, recipient's bone marrow fibroblasts prior to second BMT; Sample 3, recipient's bone marrow specimen prior to second BMT; Sample 4, donor's peripheral blood specimen prior to second BMT; and Sample 5, recipient's peripheral blood specimen 2 years after second BMT.

Sample 3 represents patient's bone marrow specimen prior to her second BMT, at the time of clinical relapse. This sample has one massive peak at nucleotide 326 and 1 min peak at 340. When compared to sample 4, which is the donor's peripheral blood prior to second BMT, it is clear that the genetic constitution of the patient's bone marrow cells at the time of relapse after her first BMT was of donor origin (the positions of the peaks are identical to the donor's rather than recipient's) and disappearance of the allele at nucleotide 340 led to loss of heterozygosity at locus D7S2465 of chromosome 7. It should be noted that the small peak at nucleotide 340 in sample 3 might suggest that a small number of normal donor cells still remain in recipient's circulation. Table 2 shows the peak height for each allele and peak height ratio was calculated to demonstrate loss of heterozygosity. By general consensus, a peak height ratio greater than 1.5 is considered loss of heterozygosity. Sample 5 represents recipient's peripheral blood 2 years after her second BMT, which is identical to donor's, confirming 100% engraftment as confirmed by RFLP.

Table 2 Assessment of loss of heterozygosity

This study demonstrates chromosomal transformation of normal donor cells in a patient with severe aplastic anemia after allogeneic BMT. The most common types of secondary cancer in allogeneic marrow transplant recipients are non-Hodgkin's lymphoma and solid nonhematopoietic tumors. Occasional cases of leukemia developing in the donor cells have been reported.16 Several mechanisms have been postulated for the development of leukemia in donor cells following allogeneic BMT, including transfer of an oncogenic gene into a new environment, viral infection, transformation of incoming donor cells by irradiation-damaged residual recipient cells and a microenvironmental stromal defect that may support leukemogenesis in donor cells.17 In a report of a patient with severe aplastic anemia that transformed into acute myeloblastic leukemia with monosomy 7, the authors postulated that treatment with GCSF and cyclosporin A might have played an important role.18 Our patient received BMT-associated irradiation and chemotherapy, as well as post-BMT GCSF support. It is not clear exactly which, if any, of these factors played a more important role. The fact that our patient was retransplanted using the same donor and both donor and recipient remain cytogenetically normal and clinically healthy 3 years following transplantation further complicate determination of the etiology. We are continuing vigilant clinical and laboratory monitoring of both the recipient and donor.

Differentiation of donor/recipient origin of hemato-poietic cells post bone marrow transplant is important for assessing engraftment and defining any residual population of normal host cells, especially when secondary malignancies develop. However, some pitfalls exist in defining host or donor origin of secondary malignancies. Cytogenetic studies are limited to a small number of spontaneously dividing cells in culture compared to the results from in situ hybridization of sex chromosome markers in interphase. Restriction fragment length polymorphism (RFLP) studies do not depend upon dividing cells and are generally informative when recipient and donor are of the same sex. However, they require relatively large amount of DNA and have relatively lower sensitivity in comparison to PCR-based microsatellite marker analysis,8 which requires as little as 3 ng of DNA. This is especially important when a retrospective investigation must be conducted with only limited pretransplant specimens available, such as in this study.

In summary, we were able to definitively identify the origin of the monosomy 7 transformation to be in the donor cells. This observation suggests that the microenvi-ronment of the bone marrow of our patient with severe aplastic anemia may have played a role in the development of monosomy 7 of normal donor cells. However, the potential impact of post-BMT immunosuppression, stem cell stimulating growth factor, and local viral infection cannot be assessed. We conclude that PCR-based chromosomal microsatellite marker analysis can be a valuable tool for precise donor/recipient differentiation post stem cell transplantation.

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Correspondence to G Reaman.

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Lang, Z., Dinndorf, P., Ladisch, S. et al. Chromosomal transformation in donor cells following allogeneic bone marrow transplantation. Bone Marrow Transplant 33, 1253–1256 (2004). https://doi.org/10.1038/sj.bmt.1704450

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Keywords

  • donor cell transformation
  • microsatellite analysis

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