Longitudinal Assessment of Hematopoietic Abnormalities After Autologous Hematopoietic Cell Transplantation for Lymphoma
http://www.100md.com
《临床肿瘤学》
the Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Cancer Center, Duarte, CA
ABSTRACT
PURPOSE: Autologous hematopoietic cell transplantation (HCT) is being increasingly used as an effective treatment strategy for patients with relapsed or refractory Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL) but is associated with therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) as a major cause of nonrelapse mortality. The phenomenon of hematopoietic reconstitution after autologous HCT and the role of proliferative stress in the pathogenesis of t-MDS/AML are poorly understood.
PATIENTS AND METHODS: Using a prospective longitudinal study design, we evaluated the nature and timing of alterations in hematopoietic progenitors and telomere length after HCT in patients undergoing autologous HCT at City of Hope Cancer Center (Duarte, CA).
RESULTS: A significant reduction in primitive and committed progenitors was observed before HCT compared with healthy controls. Further profound and persistent reduction in primitive progenitors but only transient reduction in committed progenitors were seen after HCT. Primitive progenitor frequency in pre-HCT marrow and peripheral-blood stem cells predicted for primitive progenitor recovery after HCT. Shortening of telomere length was observed in marrow cells early after HCT, with subsequent restoration to pre-HCT levels. Patients within this cohort who developed t-MDS/AML had reduced recovery of committed progenitors and poorer telomere recovery, possibly indicating a functional defect in primitive hematopoietic cells.
CONCLUSION: Our studies suggest that hematopoietic regeneration after HCT is associated with increased proliferation and differentiation of primitive progenitors. Increased proliferative stress on stem cells bearing genotoxic damage could contribute to the pathogenesis of t-MDS/AML. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor and telomere recovery predict for increased risk of t-MDS/AML.
INTRODUCTION
Autologous hematopoietic cell transplantation (HCT) is a highly effective treatment approach for patients with refractory or relapsed Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL).1-5 However, autologous HCT is associated with an increased risk of therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML).6-13 The estimated cumulative probability of t-MDS/AML ranges from 4%12 to 20%14 at 5 years after transplantation, and t-MDS/AML is increasingly recognized as a major cause of nonrelapse-related mortality in this population.6,11,13,14
The association of t-MDS/AML with pretransplantation therapeutic exposures,11,12,15-18 its extremely rare occurrence after allogeneic HCT, and the detection of specific cytogenetic abnormalities before transplantation in the marrow or peripheral-blood stem cell (PBSC) grafts19,20 suggest a role for infused stem cells in the pathogenesis of t-MDS/AML. However, the transplantation process itself could potentiate the risk of t-MDS/AML. Potential mechanisms include damage to hematopoietic stem cells (HSCs) during mobilization, collection, and storage; damage to the microenvironment and/or surviving HSCs from the chemotherapy and radiation used for transplantation conditioning; and proliferative stress related to hematopoietic regeneration.21,22
HSCs in the infused PBSC product, which represent a small fraction of the patient's HSCs, must reconstitute hematopoiesis and sustain it long term, imposing an enormous replicative stress on these cells. Superficially, hematopoietic reconstitution appears complete, with normalization of peripheral counts and marrow cellularity within a few months after transplantation. However, studies in allogeneic HCT recipients reveal marked deficits in hematopoiesis persisting for over 10 years after HCT.23-27 There is a paucity of information regarding hematopoietic reconstitution after autologous HCT where HSC number and function may be altered by the underlying malignancy and prior therapy.
Hematopoietic regeneration after allogeneic HCT has been associated with accelerated telomeric shortening.28-30 Telomeres are noncoding regions of DNA that provide a cap at the ends of chromosomes and prevent dicentric fusion and other chromosomal aberrations.31 Each somatic cell division is associated with a loss of telomere length.32 Cumulative telomere shortening can impose a limit on cell divisions and lead to senescence and genetic instability.33 Telomere loss could be of even greater importance in cellular recovery after autologous HCT because telomere length in transplanted cells may already be reduced because of prior chemotherapy and older age of patients.34,35
We are conducting a prospective, longitudinal evaluation of patients with HL or NHL undergoing autologous HCT to understand the pathogenesis of t-MDS/AML. As part of this large comprehensive study, we are investigating the process of hematopoietic regeneration after HCT. An improved understanding of the post-HCT regenerative process may provide clues to the pathogenesis of t-MDS/AML in this setting. Although previous studies evaluating small numbers of patients have indicated that committed and primitive progenitor frequency may be reduced after HCT,25,36-40 a systematic and longitudinal assessment of the nature and kinetics of these abnormalities has not been performed. In this study, we report the prevalence, nature, and timing of occurrence of changes in primitive and committed progenitors and telomere length after HCT; the factors predicting for development of hematopoietic abnormalities; and the relationship between pre-HCT and post-HCT abnormalities.
PATIENTS AND METHODS
Patients
Patients undergoing autologous HCT for NHL or HL at the City of Hope Cancer Center (Duarte, CA), who were older than 18 years at the time of transplantation, were eligible to participate in this study. This study was approved by the Institutional Review Board at City of Hope Cancer Center, in accordance with an assurance filed with and approved by the Department of Health and Human Services, and met all requirements of the Declaration of Helsinki. The study was explained in detail to the patients, including the need for multiple bone marrow aspirations at specified time points, as well as abstraction of pre- and post-HCT data from medical records. All patients provided written informed consent. This report focuses on the first consecutive 103 patients recruited to the study before HCT between March 1999 and July 2003 and observed longitudinally after transplantation. Blood and marrow samples were collected before autologous HCT and at day 100, 6 months, and 1, 2, and 3 years after HCT and were either tested directly or banked for subsequent laboratory analyses. Samples were also obtained from the PBSC products. Marrow samples obtained from eight healthy volunteers served as controls.
Clinical data were abstracted from medical records and included disease characteristics, pre-HCT therapeutic exposures, conditioning regimen, priming with growth factors and/or chemotherapy for PBSC collection, number of PBSC collections, dose of CD34+ cells infused, data on recovery of peripheral WBC counts, vital status and disease status after HCT, and therapy received after HCT for relapse. Hematopathology and classical cytogenetic (karyotyping) data from the marrow specimens were obtained at each predetermined time point. Hematopathologic data included marrow cellularity, evidence of myelodysplasia in any of the cell lineages, blast count, and evidence of acute myeloid leukemia. Bone marrow cellularity, which was defined as the percentage of hematopoietic cells occupying the marrow space, was determined by microscopic examination of the trephine biopsy at low power.41 Biopsies with primarily subcortical bone or abundant aspirate artifact were considered inadequate for evaluation. If no trephine biopsy was available or if the trephine was considered inadequate, the cellularity was determined by scanning the bone marrow aspirate smears.
Sample Collection and Processing
Bone marrow mononuclear cells (BMMC) were isolated by Ficoll-Hypaque (Sigma Diagnostics, St Louis, MO) density gradient centrifugation (specific gravity = 1.077) for 30 minutes at 400 x g. Peripheral-blood mononuclear cells were isolated by RBC sedimentation using hetastarch for 30 minutes. PBSC samples were not subjected to cell separation processing. Cell counts and viability were determined by Trypan blue staining and hemocytometer evaluation. Samples were aliquoted and resuspended in medium containing Iscove’s Modified Dulbecco’s Medium, 20% fetal bovine serum, and 10% dimethylsulfoxide, frozen using rate-controlled freezing, and stored in a liquid nitrogen freezer in the vapor phase.
Progenitor Assays
Progenitor assays were performed using bone marrow or PBSC mononuclear cells, and results were expressed as progenitor frequency per 106 mononuclear cells.
Committed progenitors or colony-forming cells (CFC). Cells were plated in semisolid methylcellulose progenitor culture for 14 to 18 days and assessed for the presence of colony-forming unit granulocyte-macrophage and burst-forming unit erythroid colonies as previously described.42
Primitive progenitors or long-term culture-initiating cells (LTCIC). LTCIC frequency was assayed by evaluating the ability of cells to generate hematopoiesis in long-term culture on M2-10B4 murine fibroblast feeders for 6 weeks. To allow accurate determination of LTCIC frequency, limiting dilution analyses were performed as previously described by plating cells on M2-10B4 feeders subcultured in 96-well plates.42 Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 for 6 weeks. Subsequently, wells were overlaid with CFC growth-supporting medium. Wells were scored as positive or negative after 14 days. The frequency of LTCIC was calculated, using L-CALC software (Stem Cell Technologies, Vancouver, British Columbia, Canada), from the reciprocal of the cell concentration that gave 37% negative wells.
Assessment of telomere shortening. The mean telomere restriction fragment length (TRF) was determined by Southern blot analysis. Genomic DNA purified from cells was digested overnight with HinfI and RsaI enzymes and then separated by agarose gel electrophoresis. DNA was transferred to membrane and hybridized with a biotinylated DNA oligonucleotide consisting of five telomeric repeats (TTAGGG). The blots were developed using the North2South Chemiluminescent Hybridization and Detection Kit (Pierce Biotechnology, Rockford, IL), and densitometry was used to analyze the mean TRF for each sample. Each lane was subdivided into 50 regions, and the signal intensity in each region was determined (SIn). A curve fitted to the migration distances of DNA markers ranging from 0.5 to 23.1 kilobase (kb) was used to determine DNA size at the midpoint of each region (Ln). Mean TRF was calculated using the following formula: mean TRF = (Ln * SIn)/ (SIn).
Statistical Analyses
Statistical analysis was performed using Epilog plus software (Pasadena Epicenter Software, Pasadena, CA).43 Comparisons of means between the various groups were conducted using the analysis of variance techniques. Wilcoxon's paired rank sum test was used for comparing the progenitor frequencies in the same patient at two different time points. Multiple regression analysis was used to investigate predictors of low progenitor frequencies (defined arbitrarily as those less than the 25th percentile for the entire cohort) and t-MDS/AML. Variables in the multivariate model included primary diagnosis, age at HCT, sex, pre-HCT therapeutic exposures (cyclophosphamide, ifosfamide, anthracyclines, and etoposide and pre-HCT radiation), and the number of collections required to achieve the target number of CD34+ cells. Transplantation conditioning regimens and pre-HCT progenitor frequencies were additional variables examined in the t-MDS/AML analysis. Institutional guidelines recommend a target number of CD34+ cells of 5.0 x 106 cells/kg, with a minimum of 2.0 x 106 cells/kg. Patients who collected the target number in two or fewer collections were classified as rapid collectors, whereas those requiring three or more collections were classified as slow collectors. The level of statistical significance was set at P = .05. All P values were two sided.
RESULTS
The clinical characteristics of the study cohort, including primary diagnosis, demographic characteristics, and pretransplantation and transplantation-related therapeutic exposures are listed in Table 1. Sufficient marrow samples for analysis were obtained before HCT in 67 patients (65% of assessable patients), from the PBSC product in 67 patients (65% of assessable patients), at 100 days after transplantation in 44 patients (65% of assessable patients), at 6 months in 32 patients (51% of assessable patients), at 1 year in 45 patients (80% of assessable patients), at 2 years in 25 patients (78% of assessable patients), and at 3 years in nine patients (69% of assessable patients). We were able to capture 50% to 80% of the planned samples as a result of a combination of reasons. In some cases, the sample obtained was inadequate to allow all tests to be performed. In other cases, the patient was either too sick to participate at that particular time point or did not want a sample drawn at a particular time point but agreed to resume from subsequent time points. These situations are commonly observed in prospective, longitudinal studies, and attrition as a result of these reasons was built into the sample size projections. Disease status at last follow-up was as follows: 17 patients were deceased, seven were alive with relapsed or progressive disease, 61 were in first complete remission, 10 were in second or greater complete remission, one had developed a secondary rhabdomyosarcoma, and eight had developed t-MDS/AML.
The median age at HCT was 48.6 years, and 57% of the patients were males. The cohort has been observed for a median of 2.9 years (range, 1.0 to 5.4 years). The median number of PBSC collections was four (range, one to 17 collections), and the median number of CD34+ cells collected was 5.0 x 106 cells/kg. Sixty-seven patients were slow collectors, requiring three or more collections to achieve the target number of cells. Peripheral WBC recovery was observed at a median of 10 days (range, 7 to 21 days) after autologous HCT. Median peripheral-blood counts at 1 year after HCT were within the normal range (median WBC count, 5.0 x 103/μL; range, 2.0 to 11.0 x 103/μL; median absolute neutrophil count, 3.0 x 109/L; range, 1.0 to 7.0 x 109/L; median hemoglobin, 13.0 g/dL; range, 9.0 to 16.0 g/dL; and median platelet count, 156,000/μL; range, 12,000 to 341,000/μL). Median bone marrow cellularity at 1 year after HCT was 40% (range, 10% to 70%).
As shown in Table 2, a comparison of the progenitor frequencies between the study population and the healthy controls revealed a 12.7-fold reduction in primitive progenitor frequency (P < .001) and an eight-fold reduction in committed progenitor frequency (P < .001) in pre-HCT samples compared with the healthy controls, demonstrating a significant perturbation in hematopoiesis in this patient population even before transplantation.
Marrow samples obtained at 100 days and 6 months after HCT revealed further reduction in LTCIC and CFC frequencies when compared with pre-HCT samples (Fig 1 and Table 2). Reduction of LTCIC frequency (three- to six-fold reduction compared with before HCT) was more profound than reduction in CFC frequency (0.3-fold). The initial reduction of CFC frequency was followed by recovery to levels equal to or greater than those seen in the pre-HCT samples at the 1- to 3-year post-HCT time points. However, persistent reduction in LTCIC frequency was seen for up to 3 years after HCT.
We compared progenitor frequency in slow versus rapid collectors. Rapid collectors had a higher LTCIC frequency at day 100 after HCT (P = .006) and at 12 months after HCT (P = .08) compared with slow collectors. Furthermore, rapid collectors had a higher CFC frequency at day 100 after HCT compared with slow collectors (P = .08). PBSC progenitor frequencies were measured, and a comparison between rapid and slow collectors revealed a significantly higher primitive (23.8 in rapid collectors v 7.0 in slow collectors, P = .02) and committed (1,346 in rapid collectors v 479 in slow collectors, P = .004) progenitor frequency among rapid collectors.
The pre-HCT LTCIC and CFC frequency in the marrow correlated with the frequencies of LTCIC (P = .02) and CFC (per kg; P = .003) in the PBSC products (Table 3). The PBSC LTCIC frequency correlated with the LTCIC frequency in the bone marrow at 6 months (P = .0003) and 2 years after HCT (P = .05). There was no correlation between PBSC CFC dose and CFC frequency after HCT. Higher PBSC LTCIC frequency (P = .03), but not CFC frequency (P = .9), was associated with an increased probability of engrafting within 10 days. There was a significant correlation between the bone marrow LTCIC frequency before HCT and the frequency at 6 months (P = .02), 1 year (P = .0009), and 2 years (P = .009) after HCT. Bone marrow CFC frequency before HCT correlated with CFC frequency at 100 days after HCT (P = .04) but not at 6 months, 1 year, and 2 years after HCT.
Multivariate analysis of risk factors associated with low progenitor frequency before autologous HCT showed that prior exposure to ifosfamide (relative risk [RR] = 4.9; 95% CI, 1.4 to 17.9) and a slow collector status (RR = 4.7; 95% CI, 1.0 to 22.8) were associated with low primitive progenitor frequency (Table 4). No specific risk factors for low committed progenitor frequency before HCT could be identified.
We assessed telomere length in BMMC obtained before HCT (n = 15), from PBSC (n = 27), and after HCT at 100 days (n = 18), 6 months (n = 10), 1 year (n = 15), 2 years (n = 8), and 3 years (n = 3). As shown in Figure 2, reduction in telomere length was seen in BMMC obtained 100 days (P = .1) and 6 months (P = .03) after HCT compared with pretransplantation BMMC. However, at 1 year after HCT, the telomere length was similar to that observed in pre-HCT BMMC. Age at transplantation inversely correlated with BMMC telomere length 1 year after HCT (r = –0.62, P = .02) but not before HCT, in the PBSC collections, or at 100 days and 6 months after HCT. We did not observe a significant correlation in the telomere length between pre-HCT and post-HCT BMMC samples.
The telomere length of PBSC (median, 5.2 kb; range, 3.4 to 9.0 kb) was less than the length of BMMC obtained before HCT (P = .01) and 1 year after HCT (P = .06), but it was not significantly different than the telomere length of BMMC obtained on day 100 and 6 months after HCT. We did not observe a significant correlation in the telomere length between PBSC and post-HCT BMMC samples. The telomere length in peripheral-blood mononuclear cells (median, 5.1 kb; range, 3.7 to 9.0 kb) was also significantly less than the length for pre-HCT BMMC (P = .008).
At the time of this analysis, seven patients had developed morphologic and cytogenetic evidence of t-MDS/AML at a median of 24 months after HCT (range, 6 to 44 months; Table 5). The morphologic and cytogenetic characteristics of marrow samples obtained from before HCT to the time of development of t-MDS/AML are listed in Table 6. Table 7 provides a comparison of the clinical characteristics and progenitor frequencies of patients with and without t-MDS/AML. The bone marrow CFC frequency at the 1-year time point was significantly depressed (P = .05) among patients with t-MDS/AML compared with patients without t-MDS/AML. Multivariate analysis of risk factors associated with development of t-MDS/AML after HCT did not reveal a significant association with underlying diagnosis, sex, age, pre-HCT chemotherapeutic exposure, prior radiation exposure, or total-body irradiation. A low CFC frequency at 1 year after HCT was predictive of t-MDS/AML (RR = 10.9; P = .05). Although not clinically significant, slow collectors were found to be at a four-fold higher risk of developing t-MDS/AML (RR = 4.0; 95% CI, 0.5 to 35.1) compared with rapid collectors.
Of the patients in whom telomere length was measured, four have developed t-MDS/AML. In this limited study, there seemed to be a trend towards increased telomere length in PBSC from patients who developed t-MDS/AML compared with patients who did not (P = .06), but a significant difference in telomere lengths in pre-HCT and post-HCT BMMC was not seen. Patients developing t-MDS/AML demonstrated reduced telomere length at 1 year after HCT compared with before HCT (P = .06) and PBSC (P = .03) samples, whereas patients who did not develop t-MDS/AML did not show a significant difference in telomere length 1 year after HCT compared with pre-HCT samples and showed a significant increase in telomere length compared with PBSC samples (P = .04).
DISCUSSION
We have performed a prospective, longitudinal assessment of hematopoietic alterations in patients undergoing autologous HCT for HL or NHL. Our studies have yielded several important and novel observations related to pre- and post-HCT hematopoietic abnormalities, the kinetics of regeneration of primitive and committed progenitors after HCT, factors predisposing to hematopoietic defects before HCT, the relationship between pre-HCT and post-HCT abnormalities, and alterations in telomere length after HCT. These results may provide insights into the pathogenesis of t-MDS/AML after HCT.
Significant defects in hematopoiesis with marked reduction in primitive and committed progenitors are present before transplantation. Further profound reduction in primitive progenitors occurs after HCT. Regeneration of primitive progenitors is not seen until at least 3 years after HCT, indicating that recovery, if it takes place at all, is delayed. Despite the profound defects in primitive progenitors after HCT, hematopoietic defects were less apparent at the level of committed progenitors, which are reduced to a lesser extent than primitive progenitors and recover to or above pretransplantation levels by 1 year after HCT. Furthermore, hematopoietic precursor cells and mature blood cells recovered to normal levels in the majority of patients at 1 year after HCT, as evidenced by the normal range of peripheral-blood counts and bone marrow cellularity. Restoration of normal marrow cellularity and peripheral-blood counts and partial restoration of committed progenitors during hematopoietic regeneration after HCT is best explained by increased proliferation and differentiation of primitive progenitors.
Prior exposure to cytotoxic agents likely contributes to reduction in progenitor frequency in patients before HCT. The variability in reduction in progenitors before HCT may be related to direct or indirect effects of the underlying disease on hematopoiesis and interindividual differences in response to pharmacologic agents. In our analysis, prior exposure to ifosfamide and slow collector status were associated with reduced primitive progenitor frequency before HCT, but an association with other specific exposures, demographic factors, or the underlying diagnosis (HL v NHL) could not be identified.
Primitive progenitor frequency in pre-HCT bone marrow and PBSC was predictive for post-HCT primitive progenitor recovery, whereas committed progenitor frequency in marrow and PBSC before HCT did not correlate with committed progenitor recovery after HCT. Pre-HCT marrow primitive progenitor frequency also correlated with the number of primitive progenitors collected in the PBSC products. Higher PBSC primitive but not committed progenitor frequency was also associated with an increased probability of rapid recovery of peripheral WBC count. These results indicate the importance of primitive progenitors as determinants of progenitor mobilization and hematopoietic regeneration after HCT and are consistent with results of experimental transplantation studies showing that primitive hematopoietic cells, rather than committed progenitors, determine both short- and long-term repopulation after HCT.44-47
Reconstitution of hematopoiesis has also been studied retrospectively in patients after allogeneic transplantation. However, we are not aware of any other studies that have used a similarly rigorous prospective longitudinal study design to assess hematopoietic function after allogeneic HCT. Selleri et al23 investigated bone marrow progenitor cells in normal donors and in patients 1 to 8 years after allogeneic HCT. Patients who had undergone transplantation had normal blood counts and bone marrow cellularity, but CFCs were decreased two-fold, and LTCIC were decreased eight-fold in the marrow of transplantation patients compared with normal donors. Podesta et al24 studied survivors at a median interval of 6 years (range, 2 to 20 years) after allogeneic HCT. Colony-forming unit granulocyte-macrophage and burst-forming unit erythroid numbers did not differ from normal controls; colony-forming unit-granulocyte/erythrocyte/monocyte/megakaryocyte numbers were reduced three-fold; and LTCIC were reduced 17-fold and did not improve with time. These studies suggest a long-term reduction of the stem-cell reservoir after allogeneic HCT. Comparison with the results of the present study suggests that the degree of reduction in primitive progenitors seen after autologous HCT is greater than that seen after allogeneic HCT. Damage to stem cells from prior treatment may contribute to the greater degree of depletion of primitive progenitors that is seen in patients after autologous HCT. Indeed, the degree of reduction seen after allogeneic HCT is similar to that seen in pre-HCT samples from patients undergoing autologous HCT.
Our studies indicate that there is initial shortening of telomere length in bone marrow cells after HCT followed by subsequent restoration to pre-HCT levels. Telomere length in BMMC after HCT likely reflects the telomere length of the HSC from which mature cells are derived, the extent of telomere loss related to mitotic divisions occurring in the course of regeneration of mature HSC, and the counterbalancing of telomere loss by telomerase activity in hematopoietic cells.48 A hierarchy of HSC with short- and long-term repopulating capacity is recognized.45,46,49,50 The initial telomeric shortening after HCT may result from rapid proliferation during post-HCT hematopoietic recovery. The increased telomere length in bone marrow samples obtained 1 year or later after HCT could be explained by long-term recovery via HSC populations without transplantation-associated shortening in telomere length. Because telomerase activity was not assessed in this study, we cannot exclude a role for increased telomerase activity in the recovery of telomere length in 1-year post-HCT samples. Reduction in telomere length has also been reported in allogeneic HCT recipients. Telomere length of cells in the marrow from recipients of allogeneic transplantation is considerably shorter than the telomere length of cells from the donor. Most of this decrease occurs in the first year after transplantation. Rufer et al51 showed that the rate of loss of telomeres in myeloid cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. Thornley et al52 observed no consistent pattern of change in telomere length from 1 to 12 months after HCT, with marked, seemingly random, fluctuations being common. They attributed these fluctuations in telomere length to hematopoietic recovery after HCT being achieved via a limited number of HSC and stochastic determination of HSC behavior.51,52
Comparison of the results of our studies with the studies comparing telomere length in allogeneic transplantation donors and recipients indicate several differences. We did not detect a relationship between patient age and telomere length in pre-HCT samples, suggesting that factors such as recovery from prior cycles of cytotoxic therapy may contribute to alterations in telomere length in pre-HCT cells. Interestingly, an inverse relationship between telomere length and age was apparent at 1 year after HCT, which is a time relatively remote from therapeutic exposure and associated hematopoietic stress. Allogeneic transplantation recipients show persistent telomere shortening at 1 year compared with donors. In contrast, we observed that the average telomere length in autologous HCT recipients returned to pre-HCT levels at 1-year after HCT. Restoration of telomere length at 1 year may reflect derivation of hematopoiesis from stem-cell subsets other than those that were active during the recovery process.
Primitive HSC are usually quiescent, and the increased proliferative drive associated with regeneration may necessitate overcoming the normal physiologic mechanisms maintaining a quiescent state. Our studies and those of others suggest that replicative stress after HCT is borne predominantly by a diminished primitive progenitor population.53 Increased proliferative stress on stem cells and primitive progenitor cells bearing genotoxic damage after HCT could play a role in the establishment and amplification of an abnormal clone in patients developing t-MDS/AML in the setting of autologous HCT (similar to what has been described in other models of neoplastic evolution).54 Increased stem-cell proliferation can cooperate with mechanisms leading to increased adduct formation and persistence and can result in a higher mutational load and increased cancer risk.55 In addition, alteration in mechanisms regulating cell replication can couple with increased proliferative signaling to lead to transformation.56 The numerous replication cycles imposed on HSC after transplantation could contribute to genomic instability through excessive shortening of telomeres in descendent cells.33
Seven patients within this cohort developed t-MDS/AML. Although our study is not well powered for statistical analysis at this time, several interesting observations could be made about hematopoietic regeneration in patients developing t-MDS/AML compared with patients who did not develop t-MDS/AML. Patients who developed t-MDS/AML had reduced recovery of committed progenitors at 1 year after HCT compared with other patients. In fact, those patients with a reduced committed progenitor frequency at 1 year after HCT were 10-fold more likely to develop t-MDS/AML compared with patients without a reduced committed progenitor frequency. Reduced regeneration of committed progenitors may reflect a functional defect in more primitive progenitors. Impairment of primitive progenitor function has been observed in patients with primary MDS.57,58 Reduced expansion capacity could also increase or prolong replicative stress on primitive progenitors associated with hematopoietic regeneration. Patients who developed t-MDS/AML demonstrated reduced telomere length recovery after HCT, which may again be indicative of a defect in primitive hematopoietic cells in patients susceptible to t-MDS/AML. In allogeneic HCT recipients, telomere shortening does not reach levels that may compromise marrow function. However, telomere shortening may be more important after autologous HCT, possibly because of older age and hematopoietic stress from prior therapy. Beauchamp-Nicoud et al59 also observed a reduction in telomere length in the course of evolution of t-MDS/AML in two patients. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor recovery and telomere dynamics as well as emergence of clonal hematopoiesis are associated with increased risk of t-MDS/AML, and these studies are currently ongoing at City of Hope.
Our observations of delayed and poor recovery of primitive hematopoiesis after autologous HCT are of clinical significance because they demonstrate the occurrence of long-term reduction in hematopoietic reserve in autologous HCT survivors. Long-term survivors may be at risk for marrow failure, especially under conditions of hematopoietic stress. The long-term follow-up after HCT should include careful monitoring of hematopoietic function. In addition, our results have possible implications for the pathogenesis of t-MDS/AML and generate testable hypotheses amenable to further testing in the clinical and laboratory settings. For example, they support further studies investigating the interaction between hematopoietic proliferative stress, genotoxic exposures, and the cellular response to DNA damage in the propagation and progression of genetic lesions in hematopoietic cells. Our observations that hematopoietic function 1 year after transplantation was associated with risk of incipient or impending t-MDS/AML after HCT support our plans for extended follow-up of a larger number of patients to confirm and refine the associations between laboratory assessments of hematopoietic function and risk of marrow failure and t-MDS/AML. Early detection of incipient t-MDS/AML after HCT could guide institution of preventive or disease-modulating therapies in the future. Finally, our studies suggest that increasing the transplanted HSC dose could enhance recovery of hematopoietic reserve after autologous HCT. This may be increasingly feasible with the availability of improved mobilizing agents and, in fact, may provide an additional rationale to use such agents.
In conclusion, our results suggest that hematopoietic recovery in patients with HL or NHL after autologous HCT may result from extensive proliferation and differentiation of primitive progenitors, resulting in their profound and persistent depletion. Continuation of these studies and extension to a larger number of patients will allow a more detailed analysis of the factors underlying development of t-MDS/AML after HCT and identification of changes predictive of increased risk of developing t-MDS/AML.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Olga Pabustan for assistance with patient accrual to this study; Liton Francisco for assistance with data management and statistical analysis; Vicki Bedell, Tinisha McDonald, and Dana Miller for help with sample processing; and Melissa Holtz for assistance with establishing the telomere assays. We are indebted to the physicians, nurses, and staff of the Division of Hematology and Hematopoietic Cell Transplantation for assistance with obtaining samples for these studies.
NOTES
Supported by the Leukemia Lymphoma Society of America Translational Research Award Nos. 6165-02, P01 CA 30206 (National Cancer Institute), and General Clinical Research Center Grant No. 5M01 RR00043.
Presented in part at the 43rd Annual Meeting of the American Society of Hematology, Orlando, FL, December 4-7, 2001; the 44th Annual Meeting of the American Society of Hematology, Philadelphia, PA, December 6-10, 2002; and the 45th Annual Meeting of the American Society of Hematology, San Diego, CA, December 6-9, 2003.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Philip T, Armitage JO, Spitzer G, et al: High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate grade or high-grade non-Hodgkin's lymphoma. N Engl J Med 316:1493-1498, 1987
Nademanee A, Schmidt GM, O'Donnell MR, et al: High-dose chemoradiotherapy followed by autologous bone marrow transplantation as consolidation therapy during first complete remission in adult patients with poor-risk aggressive lymphoma: A pilot study. Blood 80:1130-1134, 1992
Freedman AS, Takvorian T, Anderson KC, et al: Autologous bone marrow transplantation in B-cell non-Hodgkin's lymphoma: Very low treatment-related mortality in 100 patients in sensitive relapse. J Clin Oncol 8:784-791, 1990
Laport GF, Williams SF: The role of high-dose chemotherapy in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Semin Oncol 25:503-517, 1998
Fleury J, Legros M, Colombat PH, et al: High-dose chemotherapy and autologous bone marrow transplantation in first complete or partial remission for poor prognosis Hodgkin's disease. Leuk Lymphoma 20:259-266, 1996
Bhatia S, Ramsay NKC, Steinbuch M, et al: Malignant neoplasms following bone marrow transplantation. Blood 87:3633-9639, 1996
Traweek ST, Slovak ML, Nademanee AP, et al: Clonal karyotypic hematopoietic cell abnormalities occurring after autologous bone marrow transplantation for Hodgkin's disease and non-Hodgkin's lymphoma. Blood 84:957-963, 1994
Stone RM, Neuberg D, Saiffer R, et al: Myelodysplastic syndrome as a late complication following autologous bone marrow transplantation for non-Hodgkin's lymphoma. J Clin Oncol 12:2535-2542, 1994
Marolleau JP, Brice P, Morel P, et al: Secondary acute myeloid leukemia after autologous bone marrow transplantation for malignant lymphoma. J Clin Oncol 11:590-591, 1993
Miller JS, Arthur DC, Litz CE, et al: Myelodysplastic syndrome after autologous bone marrow transplantation: An additional late complication of curative cancer therapy. Blood 83:3780-3786, 1994
Krishnan A, Bhatia S, Slovak ML, et al: Predictors of therapy-related leukemia and myelodysplasia following autologous transplantation for lymphoma: An assessment of risk factors. Blood 95:1588-1593, 2000
Darrington DL, Vose JM, Anderson JR, et al: Incidence and characterization of secondary myelodysplastic syndrome and acute myelogenous leukemia following high-dose chemoradiotherapy and autologous stem cell transplantation for lymphoid malignancies. J Clin Oncol 12:2527-2534, 1994
Andre M, Henry-Amar M, Blaise D, et al: Treatment-related deaths and second cancer risk after autologous stem cell transplantation for Hodgkin's disease. Blood 92:1933-1940, 1998
Friedberg JW, Neuberg D, Stone RM, et al: Outcome of patients with myelodysplastic syndrome after autologous bone marrow transplantation for non-Hodgkin's lymphoma. J Clin Oncol 17:3128-3135, 1999
Pedersen-Bjergaard J, Pedersen M, Myhre J, et al: High risk of therapy-related leukemia after BEAM chemotherapy and autologous stem cell transplantation for previously treated lymphomas is mainly related to primary chemotherapy and not to BEAM-transplantation procedure. Leukemia 11:1654-1660, 1997
Gilliland DG, Gribben JG: Evaluation of the risk of therapy-related MDS/AML after autologous stem cell transplantation. Biol Blood Marrow Transplant 8:9-16, 2002
Milligan DW, Ruiz De Elvira MC, Kolb H-J, et al: Secondary leukemia and myelodysplasia after autografting for lymphoma: Results from the EBMT. Br J Haematol 106:1020-1026, 1999
Wheeler C, Khurshid A, Ibrahim J, et al: Incidence of post transplant myelodysplasia/acute leukemia in non-Hodgkin's lymphoma patients compared with Hodgkin's disease patients undergoing autologous transplantation following cyclophosphamide, carmustine, and etoposide (CBV). Leuk Lymphoma 40:499-509, 2001
Lillington DM, Micallef IN, Carpenter E, et al: Detection of chromosome abnormalities pre-high-dose treatment in patients developing therapy-related myelodysplasia and secondary acute myelogenous leukemia after treatment for non-Hodgkin's lymphoma. J Clin Oncol 19:2472-2481, 2001
Abruzzese E, Radford JE, Miller JS, et al: Detection of abnormal pretransplant clones in progenitor cells of patients who developed myelodysplasia after autologous transplantation. Blood 94:1814-1819, 1999
Pedersen-Bjergaard J, Andersen MK, Christiansen DH: Therapy-related acute myeloid leukemia and myelodysplasia after high-dose chemotherapy and autologous stem cell transplantation. Blood 95:3273-3279, 2000
Relling MV, Boyett JM, Blanco JG, et al: Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment. Blood 101:3862-3867, 2003
Selleri C, Maciejewski JP, De Rosa G, et al: Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant. Bone Marrow Transplantation 23:1029-1037, 1999
Podesta M, Piaggio G, Frassoni F, et al: Deficient reconstitution of early progenitors after allogeneic bone marrow transplantation. Exp Hematol 19:1011-1017, 1997
Novitzky N, Mohammed R: Alterations in the progenitor cell population follow recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol 25:471-477, 1997
Ma DD, Varga DE, Biggs JC: Hematopoietic reconstitution after allogeneic bone marrow transplantation in man: Recovery of hematopoietic progenitors (CFU-Mix, BFU-E and CFU-GM). Br J Haematol 65:5-10, 1987
Messner H, Curtis JE, Minden MD, et al: Clonogenic hematopoietic precursors in bone marrow transplantation. Blood 70:1425-1432, 1987
Wynn RF, Cross MA, Hatton C, et al: Accelerated telomere shortening in young recipients of allogeneic bone marrow transplants. Lancet 351:178-181, 1998
Seligman SJ: Telomere shortening in recipients of bone-marrow transplants. Lancet 351:1287-1288, 1998
Shay JW: Accelerated telomere shortening in bone marrow recipients. Lancet 351:153-154, 1998
Blackburn EH: Structure and function of telomeres. Nature 350:569-573, 1991
Harley CB, Futcher AB, Greider CW: Telomeres shorten during aging of human fibroblasts. Nature 345:458-460, 1990
Sharpless NE, DePinho RA: Telomeres, stem cells, senescence and cancer. J Clin Invest 113:160-168, 2004
Akiyama M, Asai O, Kuraishi Y, et al: Shortening of telomeres in recipients of both autologous and allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplantation 25:441-447, 2000
Schroder CP, Wisman GB, de Jong S, et al: Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation. Br J Cancer 84:1348-1351, 2001
Domenech J, Gihana E, Dayan A, et al: Haemopoiesis of transplanted patients with autologous marrows assessed by long-term marrow culture. Br J Haematol 88:488-496, 1994
Domenech J, Linassier C, Gihana E, et al: Prolonged impairment of hematopoiesis after high-dose therapy followed by autologous bone marrow transplantation. Blood 85:3320-3327, 1995
Novitzky N, Mohamed R: Alterations in both the hematopoietic microenvironment and the progenitor cell population follow the recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol 23:1661-1666, 1995
Soligo DA, Lambertenghi Deliliers G, Sevida F, et al: Hematopoietic abnormalities after autologous stem cell transplantation for leukemia. Bone Marrow Transplant 21:15-22, 1998
del Canizo C, Lopez N, Cabalerro D, et al: Haematopoietic damage persists 1 year after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 23:901-905, 1999
Brown DC, Gatter KC: The bone marrow trephine biopsy: A review of normal histology. Histopathology 22:411-422, 1993
Bhatia R, Munthe HA, Williams AD, et al: Increased sensitivity of chronic myelogenous leukemia primitive hematopoietic progenitors to growth factor induced cell division and maturation. Exp Hematol 28:1401-1412, 2000
Buckley JD: Epilog Windows Procedure. Pasadena, CA, Pasadena Epicenter Software, 1993
Uchida A, Tsukamoto A, He D, et al: High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 101:961-966, 1998
Glimm H, Eisterer W, Lee K, et al: Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 107:199-206, 2001
Christensen JL, Weissman IL: Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 98:14541-14546, 2001
Sitnicka E, Buza-Vidas N, Larsson S, et al: Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. Blood 102:881-886, 2003
Greenwood MJ, Lansdorp PM: Telomeres, telomerase, and hematopoietic stem cell biology. Arch Med Res 34:489-495, 2003
Guenechea G, Gan OI, Dorrell C, et al: Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2:75-82, 2001
Mazurier F, Gan OI, McKenzie JL, et al: Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103:545-552, 2004
Rufer N, Brummendorf TH, Chapuis B, et al: Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation. Blood 97:575-577, 2001
Thornley I, Sutherland R, Wynn RF, et al: Early hematopoietic reconstitution after clinical stem cell transplantation: Evidence for scholastic stem cell behavior and limited acceleration in telomere loss. Blood 99:2387-2396, 2002
Thornley I, Sutherland R, Nayar R, et al: Replicative stress after allogeneic bone marrow transplantation: Changes in cycling of CD34+CD90+ and CD34+CD90- hematopoietic progenitors. Blood 97:1876-1878, 2001
Chan KS, Sano S, Kiguchi K, et al: Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis. J Clin Invest 114:720-728, 2004
Jackson PE, O'Connor PJ, Cooper DP, et al: Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumor yield in mice treated with 1,2-dimethylhydrazine. Carcinogenesis 24:527-533, 2003
Peeper DS, Dannenberg JH, Douma S, et al: Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nat Cell Biol 3:198-203, 2001
Sato T, Kim S, Selleri C, et al: Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome. Leukemia 12:1187-1194, 1998
Thanapoulou E, Cashman J, Kakagianne T, et al: Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome. Blood 103:4285-4293, 2004
Beauchamp-Nicoud A, Feneux D, Bayle C, et al: Therapy-related myelodysplasia and/or acute myeloid leukemia after autologous haematopoietic progenitor cell transplantation in a prospective single center cohort of 221 patients. Br J Haematol 122:109-117, 2003(Ravi Bhatia, Khristine Va)
ABSTRACT
PURPOSE: Autologous hematopoietic cell transplantation (HCT) is being increasingly used as an effective treatment strategy for patients with relapsed or refractory Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL) but is associated with therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML) as a major cause of nonrelapse mortality. The phenomenon of hematopoietic reconstitution after autologous HCT and the role of proliferative stress in the pathogenesis of t-MDS/AML are poorly understood.
PATIENTS AND METHODS: Using a prospective longitudinal study design, we evaluated the nature and timing of alterations in hematopoietic progenitors and telomere length after HCT in patients undergoing autologous HCT at City of Hope Cancer Center (Duarte, CA).
RESULTS: A significant reduction in primitive and committed progenitors was observed before HCT compared with healthy controls. Further profound and persistent reduction in primitive progenitors but only transient reduction in committed progenitors were seen after HCT. Primitive progenitor frequency in pre-HCT marrow and peripheral-blood stem cells predicted for primitive progenitor recovery after HCT. Shortening of telomere length was observed in marrow cells early after HCT, with subsequent restoration to pre-HCT levels. Patients within this cohort who developed t-MDS/AML had reduced recovery of committed progenitors and poorer telomere recovery, possibly indicating a functional defect in primitive hematopoietic cells.
CONCLUSION: Our studies suggest that hematopoietic regeneration after HCT is associated with increased proliferation and differentiation of primitive progenitors. Increased proliferative stress on stem cells bearing genotoxic damage could contribute to the pathogenesis of t-MDS/AML. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor and telomere recovery predict for increased risk of t-MDS/AML.
INTRODUCTION
Autologous hematopoietic cell transplantation (HCT) is a highly effective treatment approach for patients with refractory or relapsed Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL).1-5 However, autologous HCT is associated with an increased risk of therapy-related myelodysplasia and acute myeloid leukemia (t-MDS/AML).6-13 The estimated cumulative probability of t-MDS/AML ranges from 4%12 to 20%14 at 5 years after transplantation, and t-MDS/AML is increasingly recognized as a major cause of nonrelapse-related mortality in this population.6,11,13,14
The association of t-MDS/AML with pretransplantation therapeutic exposures,11,12,15-18 its extremely rare occurrence after allogeneic HCT, and the detection of specific cytogenetic abnormalities before transplantation in the marrow or peripheral-blood stem cell (PBSC) grafts19,20 suggest a role for infused stem cells in the pathogenesis of t-MDS/AML. However, the transplantation process itself could potentiate the risk of t-MDS/AML. Potential mechanisms include damage to hematopoietic stem cells (HSCs) during mobilization, collection, and storage; damage to the microenvironment and/or surviving HSCs from the chemotherapy and radiation used for transplantation conditioning; and proliferative stress related to hematopoietic regeneration.21,22
HSCs in the infused PBSC product, which represent a small fraction of the patient's HSCs, must reconstitute hematopoiesis and sustain it long term, imposing an enormous replicative stress on these cells. Superficially, hematopoietic reconstitution appears complete, with normalization of peripheral counts and marrow cellularity within a few months after transplantation. However, studies in allogeneic HCT recipients reveal marked deficits in hematopoiesis persisting for over 10 years after HCT.23-27 There is a paucity of information regarding hematopoietic reconstitution after autologous HCT where HSC number and function may be altered by the underlying malignancy and prior therapy.
Hematopoietic regeneration after allogeneic HCT has been associated with accelerated telomeric shortening.28-30 Telomeres are noncoding regions of DNA that provide a cap at the ends of chromosomes and prevent dicentric fusion and other chromosomal aberrations.31 Each somatic cell division is associated with a loss of telomere length.32 Cumulative telomere shortening can impose a limit on cell divisions and lead to senescence and genetic instability.33 Telomere loss could be of even greater importance in cellular recovery after autologous HCT because telomere length in transplanted cells may already be reduced because of prior chemotherapy and older age of patients.34,35
We are conducting a prospective, longitudinal evaluation of patients with HL or NHL undergoing autologous HCT to understand the pathogenesis of t-MDS/AML. As part of this large comprehensive study, we are investigating the process of hematopoietic regeneration after HCT. An improved understanding of the post-HCT regenerative process may provide clues to the pathogenesis of t-MDS/AML in this setting. Although previous studies evaluating small numbers of patients have indicated that committed and primitive progenitor frequency may be reduced after HCT,25,36-40 a systematic and longitudinal assessment of the nature and kinetics of these abnormalities has not been performed. In this study, we report the prevalence, nature, and timing of occurrence of changes in primitive and committed progenitors and telomere length after HCT; the factors predicting for development of hematopoietic abnormalities; and the relationship between pre-HCT and post-HCT abnormalities.
PATIENTS AND METHODS
Patients
Patients undergoing autologous HCT for NHL or HL at the City of Hope Cancer Center (Duarte, CA), who were older than 18 years at the time of transplantation, were eligible to participate in this study. This study was approved by the Institutional Review Board at City of Hope Cancer Center, in accordance with an assurance filed with and approved by the Department of Health and Human Services, and met all requirements of the Declaration of Helsinki. The study was explained in detail to the patients, including the need for multiple bone marrow aspirations at specified time points, as well as abstraction of pre- and post-HCT data from medical records. All patients provided written informed consent. This report focuses on the first consecutive 103 patients recruited to the study before HCT between March 1999 and July 2003 and observed longitudinally after transplantation. Blood and marrow samples were collected before autologous HCT and at day 100, 6 months, and 1, 2, and 3 years after HCT and were either tested directly or banked for subsequent laboratory analyses. Samples were also obtained from the PBSC products. Marrow samples obtained from eight healthy volunteers served as controls.
Clinical data were abstracted from medical records and included disease characteristics, pre-HCT therapeutic exposures, conditioning regimen, priming with growth factors and/or chemotherapy for PBSC collection, number of PBSC collections, dose of CD34+ cells infused, data on recovery of peripheral WBC counts, vital status and disease status after HCT, and therapy received after HCT for relapse. Hematopathology and classical cytogenetic (karyotyping) data from the marrow specimens were obtained at each predetermined time point. Hematopathologic data included marrow cellularity, evidence of myelodysplasia in any of the cell lineages, blast count, and evidence of acute myeloid leukemia. Bone marrow cellularity, which was defined as the percentage of hematopoietic cells occupying the marrow space, was determined by microscopic examination of the trephine biopsy at low power.41 Biopsies with primarily subcortical bone or abundant aspirate artifact were considered inadequate for evaluation. If no trephine biopsy was available or if the trephine was considered inadequate, the cellularity was determined by scanning the bone marrow aspirate smears.
Sample Collection and Processing
Bone marrow mononuclear cells (BMMC) were isolated by Ficoll-Hypaque (Sigma Diagnostics, St Louis, MO) density gradient centrifugation (specific gravity = 1.077) for 30 minutes at 400 x g. Peripheral-blood mononuclear cells were isolated by RBC sedimentation using hetastarch for 30 minutes. PBSC samples were not subjected to cell separation processing. Cell counts and viability were determined by Trypan blue staining and hemocytometer evaluation. Samples were aliquoted and resuspended in medium containing Iscove’s Modified Dulbecco’s Medium, 20% fetal bovine serum, and 10% dimethylsulfoxide, frozen using rate-controlled freezing, and stored in a liquid nitrogen freezer in the vapor phase.
Progenitor Assays
Progenitor assays were performed using bone marrow or PBSC mononuclear cells, and results were expressed as progenitor frequency per 106 mononuclear cells.
Committed progenitors or colony-forming cells (CFC). Cells were plated in semisolid methylcellulose progenitor culture for 14 to 18 days and assessed for the presence of colony-forming unit granulocyte-macrophage and burst-forming unit erythroid colonies as previously described.42
Primitive progenitors or long-term culture-initiating cells (LTCIC). LTCIC frequency was assayed by evaluating the ability of cells to generate hematopoiesis in long-term culture on M2-10B4 murine fibroblast feeders for 6 weeks. To allow accurate determination of LTCIC frequency, limiting dilution analyses were performed as previously described by plating cells on M2-10B4 feeders subcultured in 96-well plates.42 Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 for 6 weeks. Subsequently, wells were overlaid with CFC growth-supporting medium. Wells were scored as positive or negative after 14 days. The frequency of LTCIC was calculated, using L-CALC software (Stem Cell Technologies, Vancouver, British Columbia, Canada), from the reciprocal of the cell concentration that gave 37% negative wells.
Assessment of telomere shortening. The mean telomere restriction fragment length (TRF) was determined by Southern blot analysis. Genomic DNA purified from cells was digested overnight with HinfI and RsaI enzymes and then separated by agarose gel electrophoresis. DNA was transferred to membrane and hybridized with a biotinylated DNA oligonucleotide consisting of five telomeric repeats (TTAGGG). The blots were developed using the North2South Chemiluminescent Hybridization and Detection Kit (Pierce Biotechnology, Rockford, IL), and densitometry was used to analyze the mean TRF for each sample. Each lane was subdivided into 50 regions, and the signal intensity in each region was determined (SIn). A curve fitted to the migration distances of DNA markers ranging from 0.5 to 23.1 kilobase (kb) was used to determine DNA size at the midpoint of each region (Ln). Mean TRF was calculated using the following formula: mean TRF = (Ln * SIn)/ (SIn).
Statistical Analyses
Statistical analysis was performed using Epilog plus software (Pasadena Epicenter Software, Pasadena, CA).43 Comparisons of means between the various groups were conducted using the analysis of variance techniques. Wilcoxon's paired rank sum test was used for comparing the progenitor frequencies in the same patient at two different time points. Multiple regression analysis was used to investigate predictors of low progenitor frequencies (defined arbitrarily as those less than the 25th percentile for the entire cohort) and t-MDS/AML. Variables in the multivariate model included primary diagnosis, age at HCT, sex, pre-HCT therapeutic exposures (cyclophosphamide, ifosfamide, anthracyclines, and etoposide and pre-HCT radiation), and the number of collections required to achieve the target number of CD34+ cells. Transplantation conditioning regimens and pre-HCT progenitor frequencies were additional variables examined in the t-MDS/AML analysis. Institutional guidelines recommend a target number of CD34+ cells of 5.0 x 106 cells/kg, with a minimum of 2.0 x 106 cells/kg. Patients who collected the target number in two or fewer collections were classified as rapid collectors, whereas those requiring three or more collections were classified as slow collectors. The level of statistical significance was set at P = .05. All P values were two sided.
RESULTS
The clinical characteristics of the study cohort, including primary diagnosis, demographic characteristics, and pretransplantation and transplantation-related therapeutic exposures are listed in Table 1. Sufficient marrow samples for analysis were obtained before HCT in 67 patients (65% of assessable patients), from the PBSC product in 67 patients (65% of assessable patients), at 100 days after transplantation in 44 patients (65% of assessable patients), at 6 months in 32 patients (51% of assessable patients), at 1 year in 45 patients (80% of assessable patients), at 2 years in 25 patients (78% of assessable patients), and at 3 years in nine patients (69% of assessable patients). We were able to capture 50% to 80% of the planned samples as a result of a combination of reasons. In some cases, the sample obtained was inadequate to allow all tests to be performed. In other cases, the patient was either too sick to participate at that particular time point or did not want a sample drawn at a particular time point but agreed to resume from subsequent time points. These situations are commonly observed in prospective, longitudinal studies, and attrition as a result of these reasons was built into the sample size projections. Disease status at last follow-up was as follows: 17 patients were deceased, seven were alive with relapsed or progressive disease, 61 were in first complete remission, 10 were in second or greater complete remission, one had developed a secondary rhabdomyosarcoma, and eight had developed t-MDS/AML.
The median age at HCT was 48.6 years, and 57% of the patients were males. The cohort has been observed for a median of 2.9 years (range, 1.0 to 5.4 years). The median number of PBSC collections was four (range, one to 17 collections), and the median number of CD34+ cells collected was 5.0 x 106 cells/kg. Sixty-seven patients were slow collectors, requiring three or more collections to achieve the target number of cells. Peripheral WBC recovery was observed at a median of 10 days (range, 7 to 21 days) after autologous HCT. Median peripheral-blood counts at 1 year after HCT were within the normal range (median WBC count, 5.0 x 103/μL; range, 2.0 to 11.0 x 103/μL; median absolute neutrophil count, 3.0 x 109/L; range, 1.0 to 7.0 x 109/L; median hemoglobin, 13.0 g/dL; range, 9.0 to 16.0 g/dL; and median platelet count, 156,000/μL; range, 12,000 to 341,000/μL). Median bone marrow cellularity at 1 year after HCT was 40% (range, 10% to 70%).
As shown in Table 2, a comparison of the progenitor frequencies between the study population and the healthy controls revealed a 12.7-fold reduction in primitive progenitor frequency (P < .001) and an eight-fold reduction in committed progenitor frequency (P < .001) in pre-HCT samples compared with the healthy controls, demonstrating a significant perturbation in hematopoiesis in this patient population even before transplantation.
Marrow samples obtained at 100 days and 6 months after HCT revealed further reduction in LTCIC and CFC frequencies when compared with pre-HCT samples (Fig 1 and Table 2). Reduction of LTCIC frequency (three- to six-fold reduction compared with before HCT) was more profound than reduction in CFC frequency (0.3-fold). The initial reduction of CFC frequency was followed by recovery to levels equal to or greater than those seen in the pre-HCT samples at the 1- to 3-year post-HCT time points. However, persistent reduction in LTCIC frequency was seen for up to 3 years after HCT.
We compared progenitor frequency in slow versus rapid collectors. Rapid collectors had a higher LTCIC frequency at day 100 after HCT (P = .006) and at 12 months after HCT (P = .08) compared with slow collectors. Furthermore, rapid collectors had a higher CFC frequency at day 100 after HCT compared with slow collectors (P = .08). PBSC progenitor frequencies were measured, and a comparison between rapid and slow collectors revealed a significantly higher primitive (23.8 in rapid collectors v 7.0 in slow collectors, P = .02) and committed (1,346 in rapid collectors v 479 in slow collectors, P = .004) progenitor frequency among rapid collectors.
The pre-HCT LTCIC and CFC frequency in the marrow correlated with the frequencies of LTCIC (P = .02) and CFC (per kg; P = .003) in the PBSC products (Table 3). The PBSC LTCIC frequency correlated with the LTCIC frequency in the bone marrow at 6 months (P = .0003) and 2 years after HCT (P = .05). There was no correlation between PBSC CFC dose and CFC frequency after HCT. Higher PBSC LTCIC frequency (P = .03), but not CFC frequency (P = .9), was associated with an increased probability of engrafting within 10 days. There was a significant correlation between the bone marrow LTCIC frequency before HCT and the frequency at 6 months (P = .02), 1 year (P = .0009), and 2 years (P = .009) after HCT. Bone marrow CFC frequency before HCT correlated with CFC frequency at 100 days after HCT (P = .04) but not at 6 months, 1 year, and 2 years after HCT.
Multivariate analysis of risk factors associated with low progenitor frequency before autologous HCT showed that prior exposure to ifosfamide (relative risk [RR] = 4.9; 95% CI, 1.4 to 17.9) and a slow collector status (RR = 4.7; 95% CI, 1.0 to 22.8) were associated with low primitive progenitor frequency (Table 4). No specific risk factors for low committed progenitor frequency before HCT could be identified.
We assessed telomere length in BMMC obtained before HCT (n = 15), from PBSC (n = 27), and after HCT at 100 days (n = 18), 6 months (n = 10), 1 year (n = 15), 2 years (n = 8), and 3 years (n = 3). As shown in Figure 2, reduction in telomere length was seen in BMMC obtained 100 days (P = .1) and 6 months (P = .03) after HCT compared with pretransplantation BMMC. However, at 1 year after HCT, the telomere length was similar to that observed in pre-HCT BMMC. Age at transplantation inversely correlated with BMMC telomere length 1 year after HCT (r = –0.62, P = .02) but not before HCT, in the PBSC collections, or at 100 days and 6 months after HCT. We did not observe a significant correlation in the telomere length between pre-HCT and post-HCT BMMC samples.
The telomere length of PBSC (median, 5.2 kb; range, 3.4 to 9.0 kb) was less than the length of BMMC obtained before HCT (P = .01) and 1 year after HCT (P = .06), but it was not significantly different than the telomere length of BMMC obtained on day 100 and 6 months after HCT. We did not observe a significant correlation in the telomere length between PBSC and post-HCT BMMC samples. The telomere length in peripheral-blood mononuclear cells (median, 5.1 kb; range, 3.7 to 9.0 kb) was also significantly less than the length for pre-HCT BMMC (P = .008).
At the time of this analysis, seven patients had developed morphologic and cytogenetic evidence of t-MDS/AML at a median of 24 months after HCT (range, 6 to 44 months; Table 5). The morphologic and cytogenetic characteristics of marrow samples obtained from before HCT to the time of development of t-MDS/AML are listed in Table 6. Table 7 provides a comparison of the clinical characteristics and progenitor frequencies of patients with and without t-MDS/AML. The bone marrow CFC frequency at the 1-year time point was significantly depressed (P = .05) among patients with t-MDS/AML compared with patients without t-MDS/AML. Multivariate analysis of risk factors associated with development of t-MDS/AML after HCT did not reveal a significant association with underlying diagnosis, sex, age, pre-HCT chemotherapeutic exposure, prior radiation exposure, or total-body irradiation. A low CFC frequency at 1 year after HCT was predictive of t-MDS/AML (RR = 10.9; P = .05). Although not clinically significant, slow collectors were found to be at a four-fold higher risk of developing t-MDS/AML (RR = 4.0; 95% CI, 0.5 to 35.1) compared with rapid collectors.
Of the patients in whom telomere length was measured, four have developed t-MDS/AML. In this limited study, there seemed to be a trend towards increased telomere length in PBSC from patients who developed t-MDS/AML compared with patients who did not (P = .06), but a significant difference in telomere lengths in pre-HCT and post-HCT BMMC was not seen. Patients developing t-MDS/AML demonstrated reduced telomere length at 1 year after HCT compared with before HCT (P = .06) and PBSC (P = .03) samples, whereas patients who did not develop t-MDS/AML did not show a significant difference in telomere length 1 year after HCT compared with pre-HCT samples and showed a significant increase in telomere length compared with PBSC samples (P = .04).
DISCUSSION
We have performed a prospective, longitudinal assessment of hematopoietic alterations in patients undergoing autologous HCT for HL or NHL. Our studies have yielded several important and novel observations related to pre- and post-HCT hematopoietic abnormalities, the kinetics of regeneration of primitive and committed progenitors after HCT, factors predisposing to hematopoietic defects before HCT, the relationship between pre-HCT and post-HCT abnormalities, and alterations in telomere length after HCT. These results may provide insights into the pathogenesis of t-MDS/AML after HCT.
Significant defects in hematopoiesis with marked reduction in primitive and committed progenitors are present before transplantation. Further profound reduction in primitive progenitors occurs after HCT. Regeneration of primitive progenitors is not seen until at least 3 years after HCT, indicating that recovery, if it takes place at all, is delayed. Despite the profound defects in primitive progenitors after HCT, hematopoietic defects were less apparent at the level of committed progenitors, which are reduced to a lesser extent than primitive progenitors and recover to or above pretransplantation levels by 1 year after HCT. Furthermore, hematopoietic precursor cells and mature blood cells recovered to normal levels in the majority of patients at 1 year after HCT, as evidenced by the normal range of peripheral-blood counts and bone marrow cellularity. Restoration of normal marrow cellularity and peripheral-blood counts and partial restoration of committed progenitors during hematopoietic regeneration after HCT is best explained by increased proliferation and differentiation of primitive progenitors.
Prior exposure to cytotoxic agents likely contributes to reduction in progenitor frequency in patients before HCT. The variability in reduction in progenitors before HCT may be related to direct or indirect effects of the underlying disease on hematopoiesis and interindividual differences in response to pharmacologic agents. In our analysis, prior exposure to ifosfamide and slow collector status were associated with reduced primitive progenitor frequency before HCT, but an association with other specific exposures, demographic factors, or the underlying diagnosis (HL v NHL) could not be identified.
Primitive progenitor frequency in pre-HCT bone marrow and PBSC was predictive for post-HCT primitive progenitor recovery, whereas committed progenitor frequency in marrow and PBSC before HCT did not correlate with committed progenitor recovery after HCT. Pre-HCT marrow primitive progenitor frequency also correlated with the number of primitive progenitors collected in the PBSC products. Higher PBSC primitive but not committed progenitor frequency was also associated with an increased probability of rapid recovery of peripheral WBC count. These results indicate the importance of primitive progenitors as determinants of progenitor mobilization and hematopoietic regeneration after HCT and are consistent with results of experimental transplantation studies showing that primitive hematopoietic cells, rather than committed progenitors, determine both short- and long-term repopulation after HCT.44-47
Reconstitution of hematopoiesis has also been studied retrospectively in patients after allogeneic transplantation. However, we are not aware of any other studies that have used a similarly rigorous prospective longitudinal study design to assess hematopoietic function after allogeneic HCT. Selleri et al23 investigated bone marrow progenitor cells in normal donors and in patients 1 to 8 years after allogeneic HCT. Patients who had undergone transplantation had normal blood counts and bone marrow cellularity, but CFCs were decreased two-fold, and LTCIC were decreased eight-fold in the marrow of transplantation patients compared with normal donors. Podesta et al24 studied survivors at a median interval of 6 years (range, 2 to 20 years) after allogeneic HCT. Colony-forming unit granulocyte-macrophage and burst-forming unit erythroid numbers did not differ from normal controls; colony-forming unit-granulocyte/erythrocyte/monocyte/megakaryocyte numbers were reduced three-fold; and LTCIC were reduced 17-fold and did not improve with time. These studies suggest a long-term reduction of the stem-cell reservoir after allogeneic HCT. Comparison with the results of the present study suggests that the degree of reduction in primitive progenitors seen after autologous HCT is greater than that seen after allogeneic HCT. Damage to stem cells from prior treatment may contribute to the greater degree of depletion of primitive progenitors that is seen in patients after autologous HCT. Indeed, the degree of reduction seen after allogeneic HCT is similar to that seen in pre-HCT samples from patients undergoing autologous HCT.
Our studies indicate that there is initial shortening of telomere length in bone marrow cells after HCT followed by subsequent restoration to pre-HCT levels. Telomere length in BMMC after HCT likely reflects the telomere length of the HSC from which mature cells are derived, the extent of telomere loss related to mitotic divisions occurring in the course of regeneration of mature HSC, and the counterbalancing of telomere loss by telomerase activity in hematopoietic cells.48 A hierarchy of HSC with short- and long-term repopulating capacity is recognized.45,46,49,50 The initial telomeric shortening after HCT may result from rapid proliferation during post-HCT hematopoietic recovery. The increased telomere length in bone marrow samples obtained 1 year or later after HCT could be explained by long-term recovery via HSC populations without transplantation-associated shortening in telomere length. Because telomerase activity was not assessed in this study, we cannot exclude a role for increased telomerase activity in the recovery of telomere length in 1-year post-HCT samples. Reduction in telomere length has also been reported in allogeneic HCT recipients. Telomere length of cells in the marrow from recipients of allogeneic transplantation is considerably shorter than the telomere length of cells from the donor. Most of this decrease occurs in the first year after transplantation. Rufer et al51 showed that the rate of loss of telomeres in myeloid cells, although accelerated initially, became comparable to the loss of telomeres in healthy controls from the second year after transplantation. Thornley et al52 observed no consistent pattern of change in telomere length from 1 to 12 months after HCT, with marked, seemingly random, fluctuations being common. They attributed these fluctuations in telomere length to hematopoietic recovery after HCT being achieved via a limited number of HSC and stochastic determination of HSC behavior.51,52
Comparison of the results of our studies with the studies comparing telomere length in allogeneic transplantation donors and recipients indicate several differences. We did not detect a relationship between patient age and telomere length in pre-HCT samples, suggesting that factors such as recovery from prior cycles of cytotoxic therapy may contribute to alterations in telomere length in pre-HCT cells. Interestingly, an inverse relationship between telomere length and age was apparent at 1 year after HCT, which is a time relatively remote from therapeutic exposure and associated hematopoietic stress. Allogeneic transplantation recipients show persistent telomere shortening at 1 year compared with donors. In contrast, we observed that the average telomere length in autologous HCT recipients returned to pre-HCT levels at 1-year after HCT. Restoration of telomere length at 1 year may reflect derivation of hematopoiesis from stem-cell subsets other than those that were active during the recovery process.
Primitive HSC are usually quiescent, and the increased proliferative drive associated with regeneration may necessitate overcoming the normal physiologic mechanisms maintaining a quiescent state. Our studies and those of others suggest that replicative stress after HCT is borne predominantly by a diminished primitive progenitor population.53 Increased proliferative stress on stem cells and primitive progenitor cells bearing genotoxic damage after HCT could play a role in the establishment and amplification of an abnormal clone in patients developing t-MDS/AML in the setting of autologous HCT (similar to what has been described in other models of neoplastic evolution).54 Increased stem-cell proliferation can cooperate with mechanisms leading to increased adduct formation and persistence and can result in a higher mutational load and increased cancer risk.55 In addition, alteration in mechanisms regulating cell replication can couple with increased proliferative signaling to lead to transformation.56 The numerous replication cycles imposed on HSC after transplantation could contribute to genomic instability through excessive shortening of telomeres in descendent cells.33
Seven patients within this cohort developed t-MDS/AML. Although our study is not well powered for statistical analysis at this time, several interesting observations could be made about hematopoietic regeneration in patients developing t-MDS/AML compared with patients who did not develop t-MDS/AML. Patients who developed t-MDS/AML had reduced recovery of committed progenitors at 1 year after HCT compared with other patients. In fact, those patients with a reduced committed progenitor frequency at 1 year after HCT were 10-fold more likely to develop t-MDS/AML compared with patients without a reduced committed progenitor frequency. Reduced regeneration of committed progenitors may reflect a functional defect in more primitive progenitors. Impairment of primitive progenitor function has been observed in patients with primary MDS.57,58 Reduced expansion capacity could also increase or prolong replicative stress on primitive progenitors associated with hematopoietic regeneration. Patients who developed t-MDS/AML demonstrated reduced telomere length recovery after HCT, which may again be indicative of a defect in primitive hematopoietic cells in patients susceptible to t-MDS/AML. In allogeneic HCT recipients, telomere shortening does not reach levels that may compromise marrow function. However, telomere shortening may be more important after autologous HCT, possibly because of older age and hematopoietic stress from prior therapy. Beauchamp-Nicoud et al59 also observed a reduction in telomere length in the course of evolution of t-MDS/AML in two patients. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor recovery and telomere dynamics as well as emergence of clonal hematopoiesis are associated with increased risk of t-MDS/AML, and these studies are currently ongoing at City of Hope.
Our observations of delayed and poor recovery of primitive hematopoiesis after autologous HCT are of clinical significance because they demonstrate the occurrence of long-term reduction in hematopoietic reserve in autologous HCT survivors. Long-term survivors may be at risk for marrow failure, especially under conditions of hematopoietic stress. The long-term follow-up after HCT should include careful monitoring of hematopoietic function. In addition, our results have possible implications for the pathogenesis of t-MDS/AML and generate testable hypotheses amenable to further testing in the clinical and laboratory settings. For example, they support further studies investigating the interaction between hematopoietic proliferative stress, genotoxic exposures, and the cellular response to DNA damage in the propagation and progression of genetic lesions in hematopoietic cells. Our observations that hematopoietic function 1 year after transplantation was associated with risk of incipient or impending t-MDS/AML after HCT support our plans for extended follow-up of a larger number of patients to confirm and refine the associations between laboratory assessments of hematopoietic function and risk of marrow failure and t-MDS/AML. Early detection of incipient t-MDS/AML after HCT could guide institution of preventive or disease-modulating therapies in the future. Finally, our studies suggest that increasing the transplanted HSC dose could enhance recovery of hematopoietic reserve after autologous HCT. This may be increasingly feasible with the availability of improved mobilizing agents and, in fact, may provide an additional rationale to use such agents.
In conclusion, our results suggest that hematopoietic recovery in patients with HL or NHL after autologous HCT may result from extensive proliferation and differentiation of primitive progenitors, resulting in their profound and persistent depletion. Continuation of these studies and extension to a larger number of patients will allow a more detailed analysis of the factors underlying development of t-MDS/AML after HCT and identification of changes predictive of increased risk of developing t-MDS/AML.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank Olga Pabustan for assistance with patient accrual to this study; Liton Francisco for assistance with data management and statistical analysis; Vicki Bedell, Tinisha McDonald, and Dana Miller for help with sample processing; and Melissa Holtz for assistance with establishing the telomere assays. We are indebted to the physicians, nurses, and staff of the Division of Hematology and Hematopoietic Cell Transplantation for assistance with obtaining samples for these studies.
NOTES
Supported by the Leukemia Lymphoma Society of America Translational Research Award Nos. 6165-02, P01 CA 30206 (National Cancer Institute), and General Clinical Research Center Grant No. 5M01 RR00043.
Presented in part at the 43rd Annual Meeting of the American Society of Hematology, Orlando, FL, December 4-7, 2001; the 44th Annual Meeting of the American Society of Hematology, Philadelphia, PA, December 6-10, 2002; and the 45th Annual Meeting of the American Society of Hematology, San Diego, CA, December 6-9, 2003.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Philip T, Armitage JO, Spitzer G, et al: High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate grade or high-grade non-Hodgkin's lymphoma. N Engl J Med 316:1493-1498, 1987
Nademanee A, Schmidt GM, O'Donnell MR, et al: High-dose chemoradiotherapy followed by autologous bone marrow transplantation as consolidation therapy during first complete remission in adult patients with poor-risk aggressive lymphoma: A pilot study. Blood 80:1130-1134, 1992
Freedman AS, Takvorian T, Anderson KC, et al: Autologous bone marrow transplantation in B-cell non-Hodgkin's lymphoma: Very low treatment-related mortality in 100 patients in sensitive relapse. J Clin Oncol 8:784-791, 1990
Laport GF, Williams SF: The role of high-dose chemotherapy in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Semin Oncol 25:503-517, 1998
Fleury J, Legros M, Colombat PH, et al: High-dose chemotherapy and autologous bone marrow transplantation in first complete or partial remission for poor prognosis Hodgkin's disease. Leuk Lymphoma 20:259-266, 1996
Bhatia S, Ramsay NKC, Steinbuch M, et al: Malignant neoplasms following bone marrow transplantation. Blood 87:3633-9639, 1996
Traweek ST, Slovak ML, Nademanee AP, et al: Clonal karyotypic hematopoietic cell abnormalities occurring after autologous bone marrow transplantation for Hodgkin's disease and non-Hodgkin's lymphoma. Blood 84:957-963, 1994
Stone RM, Neuberg D, Saiffer R, et al: Myelodysplastic syndrome as a late complication following autologous bone marrow transplantation for non-Hodgkin's lymphoma. J Clin Oncol 12:2535-2542, 1994
Marolleau JP, Brice P, Morel P, et al: Secondary acute myeloid leukemia after autologous bone marrow transplantation for malignant lymphoma. J Clin Oncol 11:590-591, 1993
Miller JS, Arthur DC, Litz CE, et al: Myelodysplastic syndrome after autologous bone marrow transplantation: An additional late complication of curative cancer therapy. Blood 83:3780-3786, 1994
Krishnan A, Bhatia S, Slovak ML, et al: Predictors of therapy-related leukemia and myelodysplasia following autologous transplantation for lymphoma: An assessment of risk factors. Blood 95:1588-1593, 2000
Darrington DL, Vose JM, Anderson JR, et al: Incidence and characterization of secondary myelodysplastic syndrome and acute myelogenous leukemia following high-dose chemoradiotherapy and autologous stem cell transplantation for lymphoid malignancies. J Clin Oncol 12:2527-2534, 1994
Andre M, Henry-Amar M, Blaise D, et al: Treatment-related deaths and second cancer risk after autologous stem cell transplantation for Hodgkin's disease. Blood 92:1933-1940, 1998
Friedberg JW, Neuberg D, Stone RM, et al: Outcome of patients with myelodysplastic syndrome after autologous bone marrow transplantation for non-Hodgkin's lymphoma. J Clin Oncol 17:3128-3135, 1999
Pedersen-Bjergaard J, Pedersen M, Myhre J, et al: High risk of therapy-related leukemia after BEAM chemotherapy and autologous stem cell transplantation for previously treated lymphomas is mainly related to primary chemotherapy and not to BEAM-transplantation procedure. Leukemia 11:1654-1660, 1997
Gilliland DG, Gribben JG: Evaluation of the risk of therapy-related MDS/AML after autologous stem cell transplantation. Biol Blood Marrow Transplant 8:9-16, 2002
Milligan DW, Ruiz De Elvira MC, Kolb H-J, et al: Secondary leukemia and myelodysplasia after autografting for lymphoma: Results from the EBMT. Br J Haematol 106:1020-1026, 1999
Wheeler C, Khurshid A, Ibrahim J, et al: Incidence of post transplant myelodysplasia/acute leukemia in non-Hodgkin's lymphoma patients compared with Hodgkin's disease patients undergoing autologous transplantation following cyclophosphamide, carmustine, and etoposide (CBV). Leuk Lymphoma 40:499-509, 2001
Lillington DM, Micallef IN, Carpenter E, et al: Detection of chromosome abnormalities pre-high-dose treatment in patients developing therapy-related myelodysplasia and secondary acute myelogenous leukemia after treatment for non-Hodgkin's lymphoma. J Clin Oncol 19:2472-2481, 2001
Abruzzese E, Radford JE, Miller JS, et al: Detection of abnormal pretransplant clones in progenitor cells of patients who developed myelodysplasia after autologous transplantation. Blood 94:1814-1819, 1999
Pedersen-Bjergaard J, Andersen MK, Christiansen DH: Therapy-related acute myeloid leukemia and myelodysplasia after high-dose chemotherapy and autologous stem cell transplantation. Blood 95:3273-3279, 2000
Relling MV, Boyett JM, Blanco JG, et al: Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment. Blood 101:3862-3867, 2003
Selleri C, Maciejewski JP, De Rosa G, et al: Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant. Bone Marrow Transplantation 23:1029-1037, 1999
Podesta M, Piaggio G, Frassoni F, et al: Deficient reconstitution of early progenitors after allogeneic bone marrow transplantation. Exp Hematol 19:1011-1017, 1997
Novitzky N, Mohammed R: Alterations in the progenitor cell population follow recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol 25:471-477, 1997
Ma DD, Varga DE, Biggs JC: Hematopoietic reconstitution after allogeneic bone marrow transplantation in man: Recovery of hematopoietic progenitors (CFU-Mix, BFU-E and CFU-GM). Br J Haematol 65:5-10, 1987
Messner H, Curtis JE, Minden MD, et al: Clonogenic hematopoietic precursors in bone marrow transplantation. Blood 70:1425-1432, 1987
Wynn RF, Cross MA, Hatton C, et al: Accelerated telomere shortening in young recipients of allogeneic bone marrow transplants. Lancet 351:178-181, 1998
Seligman SJ: Telomere shortening in recipients of bone-marrow transplants. Lancet 351:1287-1288, 1998
Shay JW: Accelerated telomere shortening in bone marrow recipients. Lancet 351:153-154, 1998
Blackburn EH: Structure and function of telomeres. Nature 350:569-573, 1991
Harley CB, Futcher AB, Greider CW: Telomeres shorten during aging of human fibroblasts. Nature 345:458-460, 1990
Sharpless NE, DePinho RA: Telomeres, stem cells, senescence and cancer. J Clin Invest 113:160-168, 2004
Akiyama M, Asai O, Kuraishi Y, et al: Shortening of telomeres in recipients of both autologous and allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplantation 25:441-447, 2000
Schroder CP, Wisman GB, de Jong S, et al: Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation. Br J Cancer 84:1348-1351, 2001
Domenech J, Gihana E, Dayan A, et al: Haemopoiesis of transplanted patients with autologous marrows assessed by long-term marrow culture. Br J Haematol 88:488-496, 1994
Domenech J, Linassier C, Gihana E, et al: Prolonged impairment of hematopoiesis after high-dose therapy followed by autologous bone marrow transplantation. Blood 85:3320-3327, 1995
Novitzky N, Mohamed R: Alterations in both the hematopoietic microenvironment and the progenitor cell population follow the recovery from myeloablative therapy and bone marrow transplantation. Exp Hematol 23:1661-1666, 1995
Soligo DA, Lambertenghi Deliliers G, Sevida F, et al: Hematopoietic abnormalities after autologous stem cell transplantation for leukemia. Bone Marrow Transplant 21:15-22, 1998
del Canizo C, Lopez N, Cabalerro D, et al: Haematopoietic damage persists 1 year after autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 23:901-905, 1999
Brown DC, Gatter KC: The bone marrow trephine biopsy: A review of normal histology. Histopathology 22:411-422, 1993
Bhatia R, Munthe HA, Williams AD, et al: Increased sensitivity of chronic myelogenous leukemia primitive hematopoietic progenitors to growth factor induced cell division and maturation. Exp Hematol 28:1401-1412, 2000
Buckley JD: Epilog Windows Procedure. Pasadena, CA, Pasadena Epicenter Software, 1993
Uchida A, Tsukamoto A, He D, et al: High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 101:961-966, 1998
Glimm H, Eisterer W, Lee K, et al: Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 107:199-206, 2001
Christensen JL, Weissman IL: Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 98:14541-14546, 2001
Sitnicka E, Buza-Vidas N, Larsson S, et al: Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells. Blood 102:881-886, 2003
Greenwood MJ, Lansdorp PM: Telomeres, telomerase, and hematopoietic stem cell biology. Arch Med Res 34:489-495, 2003
Guenechea G, Gan OI, Dorrell C, et al: Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2:75-82, 2001
Mazurier F, Gan OI, McKenzie JL, et al: Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103:545-552, 2004
Rufer N, Brummendorf TH, Chapuis B, et al: Accelerated telomere shortening in hematological lineages is limited to the first year following stem cell transplantation. Blood 97:575-577, 2001
Thornley I, Sutherland R, Wynn RF, et al: Early hematopoietic reconstitution after clinical stem cell transplantation: Evidence for scholastic stem cell behavior and limited acceleration in telomere loss. Blood 99:2387-2396, 2002
Thornley I, Sutherland R, Nayar R, et al: Replicative stress after allogeneic bone marrow transplantation: Changes in cycling of CD34+CD90+ and CD34+CD90- hematopoietic progenitors. Blood 97:1876-1878, 2001
Chan KS, Sano S, Kiguchi K, et al: Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis. J Clin Invest 114:720-728, 2004
Jackson PE, O'Connor PJ, Cooper DP, et al: Associations between tissue-specific DNA alkylation, DNA repair and cell proliferation in the colon and colon tumor yield in mice treated with 1,2-dimethylhydrazine. Carcinogenesis 24:527-533, 2003
Peeper DS, Dannenberg JH, Douma S, et al: Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nat Cell Biol 3:198-203, 2001
Sato T, Kim S, Selleri C, et al: Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome. Leukemia 12:1187-1194, 1998
Thanapoulou E, Cashman J, Kakagianne T, et al: Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome. Blood 103:4285-4293, 2004
Beauchamp-Nicoud A, Feneux D, Bayle C, et al: Therapy-related myelodysplasia and/or acute myeloid leukemia after autologous haematopoietic progenitor cell transplantation in a prospective single center cohort of 221 patients. Br J Haematol 122:109-117, 2003(Ravi Bhatia, Khristine Va)