Polyclonal hematopoiesis with variable telomere shortening in human long-term allogeneic marrow graft recipients

Hematopoietic reconstitution after allogeneic marrow transplantation relies on a relatively small number of hematopoietic stem cells (HSCs) compared with the estimated stem cell pool in the donor.1-4 However, given the suggested proliferative potential of HSCs, it has been assumed that even this significantly reduced number of HSCs could provide adequate hematopoiesis throughout the life of the patient.5Preclinical small animal studies have shown that small numbers of donor HSCs may give rise to polyclonal, oligoclonal, and even monoclonal hematopoiesis.1,2,6,7 In human transplantation patients, where both blood volume and life span are greater, polyclonal hematopoiesis is most commonly observed, but monoclonal and oligoclonal hematopoiesis has also been reported.3,4,8,9 According to the intrinsic-timetable model, HSCs have a finite number of divisions; therefore, repeated challenges to a reduced stem cell pool could exhaust the system, resulting in marrow failure.5 It is reasonable to speculate that extreme proliferative demand on a limited number of stem cells would result in significant telomere shortening. Significant differences might then exist between telomere length in granulocytes from the donor and those concurrently isolated from the transplant recipient. Only a few cases of patients more than 15 years after allogeneic hematopoietic stem cell transplantation (HSCT) have been described in the literature, and potentially conflicting observations have been reported.3,4 10-13 In the present study, we tested this hypothesis by evaluating blood samples from 17 long-term marrow transplantation survivors and their donors.

Seventeen patients who received transplants between 1971 and 198014-16 and their HLA-identical donors agreed to participate in the study (Table 1). Approval for the study was obtained from the Fred Hutchinson Cancer Research Center Institutional Review Board.

Granulocyte and mononuclear cell fractions were obtained from peripheral blood by Ficoll-Hypaque density gradient separation. Buccal samples were obtained from patients after mouthwash with 20 mL normal saline. High molecular weight (MW) DNA was extracted after lysis of cell pellets by means of DNazol (Molecular Research Center, Cincinnati, OH).

Chimerism was examined in granulocyte, mononuclear, and T-cell fractions of sex-mismatched recipients by dual-color XY-chromosome fluorescence in situ hybridization (FISH) analysis.17Variable number tandem repeat (VNTR) analyses were performed in sex-matched recipients with the use of polymorphisms for the ApoB, SE33, and 33.6 VNTR loci by a polymerase chain reaction (PCR) method as described.18 19 Pretransplant samples were unavailable; therefore, constitutional DNA was extracted from patient buccal mucosal samples.

Clonal analysis of tissues used X-linked polymorphisms of theM27β (DXS255) and the phosphoglycorate kinase (PGK) genes as described previously.3 20 

Terminal restriction fragment length (TRF) analysis on peripheral blood granulocytes and flow-FISH were performed on granulocytes and mononuclear cells as previously described.21-23 

The Wilcoxon signed rank test was used to determine statistical significance of differences in recipient and donor complete blood cell count, mean corpuscular volume, and difference in TRF (dTRF; ie, the donor's TRF minus the patient's TRF).

The absolute neutrophil counts (ANCs) of all the recipients except one were in the normal range. Patient 1 was pancytopenic with an ANC of 960/μL when contacted for the study. However, the average difference between recipients' and donors' ANCs was statistically significant, with recipients' ANCs lower than those of their donors (P = .03; Wilcoxon signed rank test). The significant difference in neutrophil counts may be related to posttransplant factors. All marrow recipients except one had normal hemoglobin (Hgb) levels (patient 1, Hgb = 12.1). The average difference between recipients' and donors' Hgb, was not statistically significant (P = .11; Wilcoxon signed rank test). In 15 evaluable recipients, the average difference between recipients' and donors' platelet counts was not statistically significant (P = .10; Wilcoxon signed rank test). However, 3 recipients had platelet counts lower than the normal range: 45 000/μL (patient 1), 116 000/μL (patient 9), and 131 000/μL (patient 12). Arnold et al24 and Li et al25found that the majority of recipients 2 months to 8.5 years after transplantation had normal leukocyte counts and normal hemoglobin levels. This further supports previous observations that long-term survivors of uncomplicated marrow transplants have normal hematopoiesis many years after bone marrow transpant (BMT).

Of the 17 patients, 15 had full (greater than 97%) donor-derived hematopoiesis. One patient had indeterminate chimerism owing to lack of informative markers (Table 1), and one patient/donor pair consists of monozygous twins.

Twelve evaluable recipients with female donors had polyclonal donor-derived hematopoiesis (Table 1). Furthermore, there was not a significant shift in the recipients' clonal ratios of the X-linked alleles compared with those from the donors. Our current findings further support our initial observations3 and demonstrate that hematopoietic reconstitution remains polyclonal many years after uncomplicated marrow transplantation.

Results from a representative TRF experiment are shown in Figure1A, which depicts the analysis of 3 patient/donor pairs. We tested the null hypothesis that the average dTRF was equal to zero, where the variance of the estimated mean was adjusted to account for the fact that multiple experiments were done in individual patients. The average dTRF across all 50 experiments was estimated to be 0.94 kilobases (kb) (95% confidence interval, 0.69-1.20; P < .0001) (Figure 1B). When the null hypothesis that the average dTRF was equal to 0.369 kb was tested instead, the estimated average dTRF was still significantly different than 0.365 kb (P < .0001). Telomere length was also calculated by flow-FISH independently in recipient and donor pairs 1 and 9, and similar differences between donor and recipient telomere lengths were observed. Statistical analysis revealed no correlation between dTRF and total number of nucleated marrow cells infused, donor age, or time after transplant (data not shown).

Our findings indicate that, although telomere shortening was consistently seen in long-term marrow recipients, the degree of telomere shortening was variable among recipients and, on average, was not greater than what has been previously reported early after transplant.10-13,26,27 If we accept that telomere shortening reflects an increased number of stem cell divisions after transplantation, then the observed variability among patients suggests that either a variable number of HSCs contributed to engraftment and hematopoietic reconstitution or secondary demands in hematopoiesis vary among these recipients. Furthermore, assuming that telomeres lose 100 bp per cell division and given an average dTRF of 0.94 kb, transplanted HSCs would theoretically undergo an average of 9 to 10 extra divisions after transplant. In granulocytes, telomere length is estimated to decrease by 30 bp per year23; thus a decrease of 0.94 kb would correspond to 30 years of normal hematopoiesis, consistent with previous theoretical estimates.28 The similar degree of telomere shortening in both short- and long-term recipients is consistent with a model in which demand for HSC replication stabilizes following an initial accelerated period. Late after transplant, the demand for stem cell replication appears to be no greater than in the normal donor. A more rapid loss of telomere length over time relative to the donor may occur in a setting in which donor-derived hematopoietic reconstitution is monoclonal or oligoclonal.

In summary, our findings suggest that many years after BMT, despite increased demands early after transplantation, donor-derived hematopoiesis can sustain normal counts and remains polyclonal. These observations emphasize the extensive replicative reserve of HSCs.