A young female patient in a second remission of acute lymphoblastic leukemia underwent bone marrow transplantation after total body irradiation and high-dose cytarabine from her HLA-matched brother. Following successful engraftment, mixed chimerism was seen 75 days post transplant. The karyotype contained numerous abnormalities in residual recipient cells. Chromosomes 1, 7, 13, and X were significantly more affected than other chromosomes. The high-frequency breakpoints identified were 1p22.2, 5q31.2, and 13q14.2. Some karyotypes specific for leukemia, such as t(9;22)(q34.1;q11.2) and t(8;21)(q22.2;q22.2), not seen with the original disease, were also present. As the frequency of aberrant chromosomes increased markedly with time, donor leukocytes were infused 14 months after BMT, which effectively eradicated the abnormal karyotypes.
Allogeneic bone marrow transplantation (BMT) is a potentially curative treatment in patients with hematologic disorders. However, a second leukemia/myelodysplastic syndrome (MDS) occurring after BMT is one of the major obstacles in recipients.1, 2 Both radiation therapy and chemotherapy are thought to cause genomic instability and induce secondary malignancies, similar to the inherent chromosomal instability syndromes associated with ataxia telangiectasia and Fanconi's anemia.3, 4, 5, 6, 7, 8, 9 The relationship between a high frequency of chromosomal aberrations and predisposition to cancer is well established in such syndromes.5, 6 Similarly, post-BMT secondary leukemia may arise from genetically unstable residual recipient cells damaged by radiation or chemotherapy prior to BMT.7 The continuous emergence of abnormal identical clones is known to occur following BMT. Here, we report a case showing numerous, aberrant singular chromosomal changes in recipient cells after allogeneic BMT.
A 2.3-year-old girl was diagnosed with CD10+CD19+ HLA-DR+ precursor B-cell ALL. There were no physical features of a congenital genomic instability syndrome. The bone marrow karyotype showed 27 normal female metaphases and three metaphases with hyperdiploidy (modal chromosome number 53-57). Two of the three hyperdiploid karyotypes were identified as 54,XX,+1,−3,+8,+11,+15,+17,+21,+21,+22,+X, and 57,XX,+1,+1,+2,−3,+4,+6,+6,+8,+11,+11,+12,+13,+14. Philadelphia chromosome was not detected. CSF was involved at presentation. The patient achieved CR but relapsed in the CNS 10 months later. At relapse, no cytogenetic analysis was performed on the leukemic cells in the CSF, but the marrow revealed normal karyotype. Reinduction treatment included systemic chemotherapy as well as intrathecal methotrexate (MTX), cytarabine (Ara-C) and prednisolone. At 7 months after CNS relapse, the patients underwent BMT from her HLA-identical brother in second CR.
The conditioning regimen consisted of total body irradiation (TBI) (1.5 Gy twice a day for 4 days) and high-dose Ara-C (3 g/m2/day × 3 days). GVHD prophylaxis comprised cyclosporine and short-course MTX. G-CSF was given post transplant for 3 weeks. A marrow examination carried out 20 days post transplant showed a normal male karyotype in 20 of 20 cells studied.
Mixed chimerism was found at day 75, consisting of various singular abnormal karyotypes of recipient origin with total chromosome number of 46 (n=11), along with normal 46,XY (n=31) and normal 46,XX (n=1). Thereafter, the fraction of cells of recipient origin with these abnormal chromosomal features gradually increased with time (Figure 1). No leukemic cells were morphologically observed in these specimens. Laboratory findings, including blood cell counts and biochemical data, remained within the normal range. All the abnormal karyotypes were singular, and observed only once. In addition, several specific leukemic karyotypes were noted. However, these were nonclonal, singular, and transient. In view of the potential risk of late graft failure and/or secondary leukemia, we performed a donor leukocyte infusion (DLI) 14 months after BMT. Donor leukocytes were infused twice with a month between treatments. The infused cell numbers were 3.3 × 108 and 5.4 × 108/kg, respectively. GVHD developed 2 to 3 months after DLI. The patient exhibited a mild skin rash and mild liver dysfunction. The fraction of abnormal karyotypes gradually decreased and mixed chimerism disappeared completely by 5 months after DLI (Figure 1). The patient has maintained complete chimerism for 5 years, and neither ALL recurrence nor secondary leukemia/MDS has developed. Chronic GVHD has not been observed.
Mononuclear cells from bone marrow and peripheral blood were cultured for 48 h without stimulation, and karyotypes were analyzed with standard G-banding procedures. Statistical analyses were performed using the χ2 test.10
A total of 661 karyotypes prepared from G-banded metaphases were used to analyze chromosomal breakpoints, and data were mapped on the chromosomes as black dots (Figure 2). The breakpoints disclosed nonrandom distribution. Some chromosomes displayed a significantly higher number of breakpoints than expected, including #1 (P<0.005), #7 (P<0.005), #13 (P<0.005), and X chromosome (P<0.005). Others had a significantly lower number of breaks than predicted, including #5 (P<0.05), #9 (P<0.005), #15 (P<0.01), #16 (P<0.05), #19 (P<0.005), and chromosome Y (P<0.01). Moreover, clusters of breakpoints were observed at regions, such as 1p22.2, 5q31.2, and 13q14.2. Several abnormal karyotypes specific for leukemic cells, such as t(9;22)(q34.1;q11.2), del(5)(q31.2), t(8;21)(q22.2;q22.2), and t(11;14)(p15.4;q11.2), were additionally noted. However, no dominant and consistent chromosome changes were evident.
Genetic abnormalities resulting from specific chromosomal aberrations lead to malignant transformation. Both radiation and chemotherapeutic agents, such as topoisomerase II inhibitors or alkylating agents, cause such genetic instabilities.3, 4, 9 Secondary malignancies have been reported after chemotherapy and/or radiotherapy in patients with cancer and hematologic malignancies.1, 2, 3, 4, 7, 11 Thus, measures should be taken in BMT to reduce the risk of secondary leukemia/MDS as much as possible.
The continuous emergence of identical abnormal clones has been reported as post-BMT cytogenetic abnormalities. However, the numerous and aberrant chromosomal changes in our case had not been described after SCT. Lin et al12 reported the appearance and disappearance of a few clonal chromosomal abnormalities in a patient receiving allogeneic BMT with a TBI-based conditioning regimen. Radiotherapy in the pre-transplant regimen may have been the major cause of chromosomal damage.10, 13, 14, 15, 16 Similar phenomena are observed in atomic bomb survivors,17, 18 who have a higher incidence of chromosomal aberrations in bone marrow than peripheral blood, indicating that more active and malignant clones appear in the bone marrow. Likewise, TBI may have induced the bizarre cytogenetic abnormalities in our patient. The number of abnormal karyotypes was higher in the bone marrow, and 1p22.2 was the site most affected by breakpoints. Interestingly, 1p22 is a specific site of chromosomal rearrangement in irradiated human diploid cells and lymphocytes from patients after radiotherapy.10, 16 In vitro radiation experiments additionally showed that chromosome 1 and band 1p22 are more frequently implicated, consistent with our results.10
The persistence of various types of nonclonal cytogenetic abnormalities in our patient indicates that none of the cells acquire any growth advantages during the post-BMT period. Perot et al19 described 20 patients with various types of chromosomal damage, including 11q23 after autologous BMT with TBI. In addition, transient clonal changes with disappearance and reappearance of abnormal karyotypes were reported in patients with Fanconi's anemia.6 However, such chromosomal aberrations did not immediately affect patient outcomes. Malignant transformation from such an aberrant situation may occur only upon additional genomic alterations as a second hit. Thus, post-BMT genomic instability in our patient may have led to the development of secondary leukemia if preemptive measures such as DLI had not been taken.
In conclusion, numerous nonclonal cytogenetic changes (possibly indicating genetic instability in residual recipient hematopoietic cells) occurring post BMT are described here for the first time. Further studies are necessary to clarify the genetic mechanism(s) of secondary leukemia associated with BMT.
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We thank Dr Hiroshi Nishida for help with statistical analysis.
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Yoshihara, T., Hibi, S., Yamane, Y. et al. Numerous nonclonal chromosomal aberrations arising in residual recipient hematopoietic cells following allogeneic bone marrow transplantation. Bone Marrow Transplant 35, 587–589 (2005) doi:10.1038/sj.bmt.1704860
- chromosomal aberration
- donor leukocyte infusion
- total body irradiation
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