Molecular and cytogenetic analyses can be used to characterize the origin of engrafted cells and the development and evolution of recur rent malignancies after allogeneic HSCT. Hematopoietic cells that emerge after allogeneic HSCT may be of host origin, donor origin, or both. Genetic studies of post-transplant hematopoiesis are termed chimerism analysis. Chimerism should be distinguished from mosaicism, which is characterized by two or more different cell populations originating from one zygote. Monitoring chimerism in recipients of allogeneic HSCT is essential to identify early engraftment, monitor residual disease, predict relapse, and optimize post-transplantation therapy in case of graft failure.
Historically, karyotype analyses were used to evaluate engraftment after sex-mismatched allogeneic HCT. Polymorphism of chromosomes 1, 9, and 16, as well as satellite polymorphism of chromosomes 13, 14, 15, 21, and 22, have been used to differentiate donor from recipient cells in sex-matched allogeneic HCT. Karyotype analyses can identify not only chimerism but also recurrence of the hematologic malignancy. However, this is a time-consuming process and has low sensitivity (5%). In the past two decades, many additional methods for detection of chimerism have been developed. All follow the basic principle of using the differences in polymorphic genetic markers to distinguish donor from recipient hematopoiesis. These methods include restriction fragment length polymorphism, red cell phenotyping, and interphase FISH. The most widely used technique is PCR for a variable number of tandem repeats/short tandem repeats (VNTR/STR-PCR). This technique has a sensitivity of 3% to 5%, but the quantitation of donor and recipient cells may be cumbersome. The method that allows study of chimerism in all patients involves fluorescence labeling of the primers and resolution of PCR products with capillary electrophoresis. It provides a high degree of quantitative accuracy with 1% to 5% sensitivity. Real-time PCR or RQ-PCR for analysis of the SRY gene on the Y chromosome allows identification of male cells in the background of 100,000 female cells, providing high sensitivity for mixed chimerism. However, this approach is limited to the 50% of patients who receive sex-mismatched transplants. Nevertheless, it remains the most sensitive and the fastest method of chimerism analysis, providing reliable quantitative results within 2 hours.
Detection of SNPs by chimerism analysis (SNP-PCR) is a highly sensitive approach. In one study using 11 different SNP loci, SNP PCR analysis identified independent predictors of relapse after HSCT. The two most commonly used methods for detection of chimerism after HSCT are fluorescence-based PCR amplification of short tandem repeats (STR-PCR) and interphase FISH. Both methods are accurate and reproducible. The sensitivity of both methods approaches 1%; however, STR-PCR is sex-chromosome independent and can be applied to all patients. FISH analysis, on the other hand, permits simultaneous evaluation of chimerism and residual disease in the same cell when high sensitivity is not a requirement. FISH analysis for diagnostic genomic abnormalities in conjunction with conventional cytogenetics remains useful and reliable in determining the presence of MRD (Fig.1).
Fig1. DETECTION OF ENGRAFTMENT AND RESIDUAL DISEASE WITH FISH IN SEX-MISMATCHED HEMATOPOIETIC CELL TRANSPLANTATION. Metaphase and non-dividing cell (blue) after DAPI counterstaining, hybridized with X (large red) and Y (large green) for detection of engraftment and with ABL (small red) and BCR (small green) for detection of residual chronic myelogenous leukemia (top panel). Left nucleus (bottom panel) shows a donor male (XY) cell origin and lack of BCR-ABL fusion. In contrast, a host female (XX), BCR-ABL fusion (yellow)–positive cell is shown on the right. Combination of XY FISH probes with diagnostic genomic markers is a powerful and fast FISH method for simultaneous detection of chimerism and minimal residual disease. FISH, fluorescence in situ hybridization.
Donor cell leukemia (DCL) after HSCT is a rare complication occur ring in 0.12% to 5% of cases. Careful genetic analysis of relapse cells is essential. VNTR, restriction fragment length polymorphism, STR analysis, or FISH XY analysis alone may not definitively assign the origin of the leukemic clone because genomic deletions or amplifications of chromosomal segments may occur during transplantation or disease progression. The increased use of unrelated cord blood as a source of stem cells for HSCT raises the concern that HSCs containing preleukemic clonal molecular rearrangements may be inadvertently transplanted. Systematic screening of unselected cord blood samples has revealed putative preleukemic rearrangements such as ETV6-RUNX1 and RUNX1-RUNXT1. Cord blood grafts have been reported to be at the highest risk of leading to DCL. The median duration between the occurrence of DCL following cord blood and bone marrow transplantation has been reported to be 14.5 and 36 months, respectively (P < .0001). Among 30 patients who developed DCL between 2005 and 2018, an abnormal karyotype involving chromosome 7 were observed in 52.4% of cord blood recipients and 17.3% of marrow recipients (P < .003). Patients with DCL generally have a poor prognosis. Proposed mechanisms underlying DCL include: (a) sustained host-origin antigenic stimulation, (b) impaired hematopoietic microenvironment and a defective microenvironmental support system, (c) immune surveillance escape secondary to posttransplant immunosuppressive therapy, (d) similar genetic susceptibility in cases of related donors, (e) viral driven pathogenesis (cytomegalovirus, EBV), (f) delayed effects of conditioning regimens, and (g) transfection of host cell oncogene into donor cells. Most likely, the underlying cause is a combination of these mechanisms operating in individual cases. A very compelling hypothesis underlying DCL is the “2-hit” hypothesis. A donor HSC that has an inherent susceptibility to malignant transformation (hit 1) is placed within a defective microenvironment that elaborates repeated stress signals (hit 2), inducing additional genetic or even epigenetic mutations promoting the appearance of DCL. An examination of every graft prior to transplantation is suggested to determine whether the graft contain abnormal clones (Fig. 2).
Fig2. DONOR-DERIVED MYELODYSPLASTIC SYNDROME FOLLOWING CORD BLOOD TRANSPLANTATION IN ACUTE LYMPHOBLASTIC LEUKEMIA. (A) At diagnosis of acute lymphoblastic leukemia, in January 2006, cytogenetic analysis of bone marrow revealed a 56, XY, +X, inv(2)(p11.2q13), +4, +6, +9, +14, +14, +17, +18, +21, +21 hyperdiploid karyotype (arrows indicate abnormal chromosomes). (B) A bone marrow nucleus from a specimen obtained in January 2007 after FISH showed 5% of cells with tetrasomy 21 (red signals), two copies of X chromosome (green signals), and one copy of Y chromosome (aqua). (C) A partial karyotype of chromosome 2 from the peripheral blood specimen, PHA-stimulated for 72 hours, obtained in May 2006, showing a constitutional inversion (2)(p11.2q13). (D) At the time of the diagnosis of MDS in May 2011, bone marrow cytogenetic analysis revealed 65% of cells with a 44, XY, −3,del(4)(q23q33), der(5;17)(p10;q10), −7,t(8;22)(p21;q13), +mar karyotype (arrows indicate abnormal chromosomes). (E) Five bone marrow nuclei after FISH studies from the June 2011 specimen showing 75% cells with deletion of EGR1 at 5q31 chromosomal location (red) and dele tion of P53 at 17p13.1 chromosomal localization (red) as a result of der(5;17); 71% showing a loss of 7q31 locus as a result of monosomy 7, as well as disomy 21 (red), one X (green), and one Y (aqua) chromosome. (F) A partial bone marrow karyotype of chromosome 2 from May 2011 specimen showing a normal chromosome 2 from the donor cells and absence of inv(2) observed in the bone marrow and PHA-stimulated PB at the time of diagnosis . FISH, Fluorescence in situ hybridization. (Reproduced with permission from Cotter R, Najfeld V, Isola L, et al. An unusual case of donor-derived myelodysplastic syndrome following double-unit umbilical cord blood transplantation in acute lymphoblastic leukemia. Am J Hematol. 2012;87:931.)
