Rapid Telomere Loss in Hematopoietic Cells Associated with Development of t-MDS after Autologous Transplantation for Lymphoma.

Abstract Therapy-related MDS (t-MDS) is a lethal complication of autologous hematopoietic cell transplantation (HCT) for Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Although t-MDS is known to result from damage to hematopoietic stem cells (HSC) from genotoxic cancer treatment, the sequential cellular and molecular changes leading to its development are not known. To better understand t-MDS pathogenesis we are conducting a prospective study of a cohort of patients undergoing autologous HCT for HL and NHL. Patients are followed longitudinally from pre-HCT to 5 yrs post-HCT, with serial collection of marrow and blood samples. We investigated whether development of t-MDS after HCT was associated with altered telomere dynamics in hematopoietic cells. Telomeres are non-coding sequences at ends of chromosomes. Telomere shortening may be associated with genetic instability and myeloid leukemogenesis, and could predispose to t-MDS after HCT. Telomere length was measured in peripheral blood (PB) samples from 8 patients from the cohort who developed t-MDS after HCT and 24 matched controls who did not develop t-MDS (3 controls per case: matched for primary diagnosis, age at HCT, race and length of follow-up). Samples were analyzed pre-HCT and at d100, 6 months, 1 yr and then annually post-HCT till development of t-MDS. PB cells were mixed with equal numbers of 1301 cells, as internal controls, and hybridized with a telomere PNA-FITC probe. Relative telomere length (RTL) values (ratio of telomere signal of PB sample and control 1301 cells) were determined by flow cytometry (FlowFISH). RTL values of lymphoid and myeloid cells, identified by scatter properties, were separately analyzed. The median PBSC CD34+ count (106/kg) for cases was 4.9 (2.6–17.4) and controls was 5.2 (2.1–33.5). Differences in RTL were seen at several time points in t-MDS cases and controls for total cell, lymphoid and myeloid cells, but were most marked in the myeloid cells. Patients developing t-MDS showed reduced RTL pre-HCT (myeloid RTL: cases=18.2±4.8, controls=27.2±2.8); an increase in RTL at d 100 (myeloid RTL for cases=31.4±4.6, controls=19.6±2.5, p=0.04); and progressive reduction in RTL subsequently (myeloid RTL at 3 years: cases= 9.7±4.1, controls=22.0±2.2). An increase in RTL from pre-BMT to d 100 was seen in t-MDS patients (+16.0) but not in controls (−8.2) (p=0.004). A fixed effect growth curve model fitted to the data from d100 to 3 years to examine the rate of change in RTLs over time revealed a sharp decline in RTL for cases (β per 100 days = −1.93 for myeloid cells), but no change in RTL over time for controls (β = 0.07) (p = 0.02). In summary, we observed severely altered telomere dynamics in hematopoietic cells from patients who develop t-MDS post-HCT, with an initial increase in telomere length followed by a very sharp rate of telomere attrition till development of t-MDS. The initial increase in telomere length may reflect a need for increased recruitment of fresh HSC, with longer telomeres, to achieve hematopoietic recovery post-HCT. Subsequent rapid attrition of telomere length may reflect increased hematopoietic cell proliferation associated with ineffective hematopoiesis characteristic of MDS or represent altered telomere regulation in damaged HSC. Altered telomere regulation and reduced telomere length may contribute to leukemic transformation of HSC in t-MDS.