Telomere shortening has been documented in the blood cells of recipients of allogeneic bone marrow transplants compared with their donors. Allogeneic ...
Bone Marrow Transplantation (2001) 27, 1283–1286 2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt
Telomere length Accelerated telomere shortening following allogeneic transplantation is independent of the cell source and occurs within the first year post transplant JD Robertson1, NG Testa1, NH Russell2, G Jackson3, AN Parker4, DW Milligan5, C Stainer2, S Chakrabarti5, M Dougal1 and R Chopra1 1
CRC Laboratory of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester; Nottingham City Hospital, Nottingham; 3Royal Victoria Hospital, Newcastle upon Tyne; 4Glasgow Royal Infirmary, Glasgow; and 5 Birmingham Heartlands Hospital, Birmingham, UK 2
Summary: Telomere shortening has been documented in the blood cells of recipients of allogeneic bone marrow transplants compared with their donors. Allogeneic peripheral blood progenitor cells (PBPCs) have been increasingly used as an alternative to bone marrow. Their advantages include earlier engraftment and immune reconstitution following transplantation. We have measured telomere length of neutrophils and T cells in fully engrafted recipients of allogeneic bone marrow (n = 19) and allogeneic PBPC (n = 17) and also measured sequential telomere length in four patients after transplantation. Overall, significant telomere shortening occurred in recipients in neutrophils (0.3 kb, P ⬍ 0.001) and T cells (0.2 kb, P = 0.045). The data demonstrate that first, the degree of shortening was the same for BM and PBPC transplants and was not related to the time taken to engraft neutrophils and platelets and second, telomere shortening occurs in the first year post transplant without further shortening during the period of observation. These data suggest that the superiority of engraftment seen in PBPC transplants is independent of telomere shortening and other mechanisms such as homing or seeding may be more important. Bone Marrow Transplantation (2001) 27, 1283–1286. Keywords: telomere; allogeneic; transplantation
Accelerated telomere shortening has been described following allogeneic BM transplantation.1–5 This reflects the replicative stress in the progenitor cell compartment, and may lead to an increased risk of developing secondary clonal blood disorders. There are limited data regarding the kinetics of telomere shortening and no data directly comparing bone marrow-derived progenitor cells with G-CSF mobil-
ised allogeneic PBPC transplantation. PBPC transplants result in earlier neutrophil and platelet recovery6,7 and more rapid immune reconstitution8 compared to BM transplants. This may be attributed to the higher numbers of progenitor cells that are present in PBPC harvests9,10 or the biological differences that exist between progenitor cells derived from these two sources. PBPCs have a higher proportion of circulating progenitors in the G0/G1 phase of the cell cycle,11 a high content of clonogenic progenitor cells12 and an altered adhesion molecule profile compared to BM. This may result in improved homing and seeding into the bone marrow.13–15 The increased availability of progenitor cells for engraftment may, at least in part, abrogate the initial replicative stress following transplantation. We aimed to define whether patients receiving PBPC transplants as compared to BM transplants have a different degree of telomere shortening, which may be a reflection of haemopoietic stress post transplant. This may have a bearing on the choice of stem cells for allogeneic transplantation. Patients and methods Local Ethical Committee approval was obtained prior to commencement of this study. Samples of peripheral blood were obtained from 36 patients who had previously undergone either allogeneic BM transplantation (n = 19) or allogeneic PBPC transplantation (n = 17). All recipients were demonstrated to have 100% donor chimerism using variable tandem nucleotide repeat (VNTR) analysis or cytogenetic (X:Y) analysis. Concurrent control samples were obtained from their healthy donors in the majority of cases. In seven cases, donor blood was obtained some months prior to recipient blood. In these cases, the measured telomere length of the donor blood was adjusted to take into account normal telomere shortening with ageing.16–18 Patient characteristics
Correspondence: Dr R Chopra, Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester M20 4BX, UK Received 14 November 2000; accepted 25 March 2001
Nineteen BM donor/recipient pairs were compared with 17 PBPC pairs. The results are summarised in Table 1. BM transplant patients received a median of 3.05 × 108 mono-
Telomere shortening post allogeneic PBPCT and BMT JD Robertson et al
1284
Table 1 Characteristics of donors and recipients of allogeneic bone marrow transplants (19 pairs) and PBPC transplants (17 pairs)
Donor age (median; range) Recipient age (median; range) Diagnosis AML ALL CML Other Conditioning TBI-containinga Chemotherapy aloneb Cell dose (median; range) MNC × 108/kg CD34+ × 106/kg Days to engraftment (median; range) neutrophils ⬎0.5 × 109/l neutrophils ⬎1.0 × 109/l platelets ⬎20 × 109/l platelets ⬎50 × 109/l Years since transplant (median; range)
Bone marrow (n = 19)
PBSC (n = 17)
32 (18–55) 30 (20–53)
43.5 (16–65) 42.5 (18–55)
4 4 9 2
7 2 4 4
16 3
16 1
3.05 (1.44–3.9) 4. 6 (1.58–7.04) 17 (11–29) 20 (12–33) 23 (8–47) 30 (18–67) 2.5 (0.9–13.3)
15 (8–31) 16 (10–35) 17 (11–31) 21 (9–44) 0.95 (0.25–3.25)
a
Cyclophosphamide/total body irradiation or melphalan/total body irradiation. b Busulphan/cyclophosphamide or BEAM.
nuclear cells (MNCs)/kg at transplant. In PBPC recipients the number of cells infused was quantified by CD34+ estimation. As CD34+ cells account for approximately 1% of the cells in a marrow harvest19,20 it is apparent that the recipients of BM and PBPC transplants received similar CD34+ doses. Neutrophil and T cell preparations Neutrophils and T cells were obtained from whole blood as previously described.18 Neutrophils were obtained from all 36 donor/recipient pairs but sufficient T cells for telomere analysis were available for only 50 individuals. Where comparative analyses were undertaken, only samples with paired neutrophil and T cell data were included. Terminal restriction fragment (TRF) analysis This was carried out using in-gel hybridisation as previously described.18 Statistical analysis The data were parametric. Student’s t-test and Levine’s test for the equality of variances were used to assess the significance of observed differences. Regression analysis and Spearman’s correlation coefficient were used to define any association between the extent of TRF shortening post transplant and clinical variables. Bone Marrow Transplantation
Results and discussion This study is the first to compare telomere (TRF) shortening following allogeneic BMT and PBPC transplantation and the first to prospectively document changes in TRF length over a period of time in the same patient. When all transplants were analysed together, significant TRF shortening was observed in neutrophils (0.3 kb, P ⬍ 0.001) and T cells (0.2 kb, P = 0.045). Figure 1 shows the TRF shortening in neutrophils and T cells following both types of transplant by comparison of the mean donor TRF length with recipient TRF length. In addition, a direct analysis of individual paired samples was carried out. By this method, TRF shortening was observed in neutrophils (321 ± 90 base pairs (bp) for BMT recipients (n = 19, P = 0.002) and 296 ± 127 bp for PBPCT recipients (n = 17, P = 0.03)). Although there was telomere shortening in T cells of recipients compared to their donors this was statistically significant following BMT but not following PBSCT (289 ± 133 bp for bone marrow recipients (n = 15, P = 0.03) and 113 ± 379 bp (n = 9, P = NS) for PBPCT recipients) (Figure 1). The nonsignificance of the TRF shortening in T cells post PBPCT may be due to the small T cell sample number in the PBPC group (n = 9); alternatively it may suggest that the larger T cell dose associated with PBPC transplantation8 results in a reduction in T cell proliferative stress. Insufficient T cell numbers precluded further analysis of telomere shortening in T cell sub-populations following transplantation, but it is possible that the observed T cell telomere shortening overall may partly reflect differences in these. It has been shown, for example, that transplant recipients have low percentages of naive CD4+ and CD8+ T cells compared with normal adults.21 Such cells, with the phenotype CD45RA+RO−, have been observed to have longer telomeres and to undergo telomere shortening during proliferation at a slower rate than memory T cells.22 There was no statistically significant difference between BMT and PBPCT in terms of the amount of observed TRF shortening. Assuming a rate of shortening of 20–40 base pairs per year in the healthy general population,17,18 the post-transplant shortening is the equivalent of approximately 7.5–15 years ageing for neutrophils and 5–10 years ageing for T cells. In order to compare TRF shortening between neutrophils and T cells, a direct analysis of individual paired samples was carried out only in those pairs where both neutrophil and T cell results were available for donor and recipient. The degree of shortening was equivalent for neutrophils and T cells. The extent of TRF shortening was not associated with donor age, recipient age or donor telomere length. It was observed that T cells had significantly longer TRFs than neutrophils in both donors (n = 25, mean difference 311 ± 82 bp, P = 0.001) and transplant recipients (n = 25, mean difference 447 ± 99 bp, P ⬍ 0.001) (Figure 1), in agreement with previous studies.5,17,18 PBPC donors, who had received G-CSF, had similar TRF lengths to BM donors both in neutrophils (8.06 kb vs 8.36 kb; 95% CI −0.32, 0.92; P = NS) and T cells (8.74 kb vs 8.71 kb; 95% CI −0.42, 1.23; P = NS) in measurements undertaken at different time intervals, 3–39 months post donation. These results suggest that, within the timeframe
Telomere shortening post allogeneic PBPCT and BMT JD Robertson et al
BMT T cells
1285
BMT neutrophils
10.5 10.5
9.5
TRF (kb)
TRF (kb)
10 9 8.5 8
8.73
8.45
Donor
8.5 8.36
7.5
7.5 7
9.5
6.5
Recipient
8.04
Donor
PBSCT T cells
Recipient
PBSCT neutrophils
10.5 10.5
TRF (kb)
TRF (kb)
10 9.5 9 8.5 8
8.45
8.33
Donor
8.5 8.06
7.5
7.5 7
9.5
6.5
Recipient
7.77
Donor
Recipient
12 11.5 11 10.5 10 9.5 9 8.5 8
0
3
6
11
18
21
24
Months post transplant
TRF (kb)
studied, G-CSF mobilisation has limited impact on telomere length, which has important implications for the counseling of potential PBPC cell donors. Accelerated telomere shortening occurred in the first year following transplantation. In 10 patients whose TRF length was assessed in the first year post transplant, the mean TRF shortening from the time of the transplant was 537 ± 155 bp compared to a mean TRF shortening of 221 ± 81 bp in 26 patients assessed between 1 and 13.3 years after the procedure. In four patients, an initial TRF measurement was made from the donor at the time of apheresis or bone marrow harvest, followed by sequential TRF assessments in the recipient over a 2 year period (Figure 2). There was evidence of significant telomere shortening within the first year of the transplant (P = 0.01) but thereafter there was no additional telomere loss. These observations are in keeping with studies in Safari cats undergoing autologous transplantation using glucose-6-phosphate dehydrogenase isoenzymes to track haemopoiesis.23 Two phases of repopulating stem cell kinetics were described: initially there were significant fluctuations in the contributions of stem cell clones followed by more stable haemopoiesis. These may be mirrored by the two phases of telomere shortening, the more rapid early shortening suggesting extensive proliferation of engrafted stem and progenitor cells. Subsequent stabilisation in telomere shortening is likely to be the result of the establishment of steady-state haemopoiesis. In agreement with previous studies,6,7,24 transplantation of allogeneic peripheral blood progenitor cells resulted in more rapid platelet and neutrophil recovery although the difference was only statistically significant for platelet recovery to ⬎20 × 109/l (P = 0.05) and ⬎50 × 109/l (P = 0.04) (Table 1). In our study, the numbers of CD34+ cells transplanted were similar for both groups. Therefore,
TRF (kb)
Figure 1 Telomere length in T cells and neutrophils of donors and recipients of BM and PBPC transplants. TRF shortening was detected in recipients as compared to donors, although this was not significant in T cells of PBPC transplant recipients. T cells were observed to have significantly longer telomeres than neutrophils in both donors and recipients. Mean TRF values are given.
12 11.5 11 10.5 10 9.5 9 8.5 8
10.84 10.04
0
311
10.01
1824
Months post transplant Figure 2 Sequential neutrophil TRF measurements in BMT (왕,왖) and PBPCT (䊊,䊉) recipients over a 2 year period suggest that telomere shortening occurs predominantly in the early post-transplant period. Mean TRF values are shown at the baseline time of harvest (t = 0), 3–11 months and 18–24 months following transplantation.
qualitative differences in the progenitor cells transplanted are more likely to explain differences in engraftment. These qualitative differences do not, however abrogate the rapid telomere shortening described here for PBPC and BMT recipients. There was no correlation between the time taken to engraftment of neutrophils or platelets and the extent Bone Marrow Transplantation
Telomere shortening post allogeneic PBPCT and BMT JD Robertson et al
1286
of TRF shortening. Furthermore, we found no correlation between the dose of MNCs and TRF shortening in BMT recipients (within the range 1.44–3.9 × 109/kg) or between the CD34+ cell dose and TRF shortening in PBPCT recipients (within the range 1.58–7.04 × 106/kg). This latter observation has been controversial with one study1 demonstrating a correlation between stem cell dose and telomere shortening and others showing no such relationship. Our data suggest that stem cell usage after transplantation of adequate numbers of cells occurs independently of cell dose and is similar for both G-CSF-mobilised PBPCs and unmanipulated bone marrow cells. This implies that engraftment post transplant must initially occur from a limited stem cell pool which accounts for the rapid fall in telomere length described within the first year of transplantation. An understanding of the mechanisms by which stem cells contribute to stable haemopoiesis post transplant is important for the development of gene therapy protocols. Future studies must concentrate on defining more accurately the stem and progenitor cell populations bearing this stress and the processes that influence stem cell usage post transplant.
8
9
10
11
12 13 14 15
Acknowledgements We would like to thank Srs T Howe, D Sheekey, K Bowyer and C Richardson for their help with this study. NG Testa is supported by the Cancer Research Campaign, R Chopra is supported by the Christie Hospital Leukaemia Research Endowment funds and Chugai Pharmaceuticals. This work was supported by a grant from the Leukaemia Research Fund.
References 1 Notaro R, Cimmino A, Tabarini D et al. In vivo telomere dynamics of human hematopoietic stem cells. Proc Natl Acad Sci USA 1997; 94: 13782–13785. 2 Wynn RF, Cross MA, Hatton C et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet 1998; 351: 178–181. 3 Akiyama M, Hoshi Y, Sakurai S et al. Changes of telomere length in children after hematopoietic stem cell transplantation. Bone Marrow Transplant 1998; 21: 167–171. 4 Lee J, Kook H, Chung I et al. Telomere length changes in patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 1999; 24: 411–415. 5 Wynn R, Thornley I, Freedman M, Saunders EF. Telomere shortening in leucocyte subsets of long-term survivors of allogeneic bone marrow transplantation. Br J Haematol 1999; 105: 997–1001. 6 Mahmoud H, Fahmy O, Kamel A et al. Peripheral blood vs bone marrow as a source for allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 1999; 24: 355–358. 7 Schmitz N, Bacigalupo A, Hasenclever D et al. Allogeneic bone marrow transplantation vs filgrastim-mobilised peripheral blood progenitor cell transplantation in patients with early leukaemia: first results of a randomised multicentre trial of the
Bone Marrow Transplantation
16
17
18
19
20 21
22 23 24
European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 1998; 21: 995–1003. Ottinger HD, Beelen DW, Scheulen B et al. Improved immune reconstitution after allotransplantation of peripheral blood stem cells instead of bone marrow. Blood 1996; 88: 2775– 2779. Tjonnfjord GE, Steen R, Evensen SA et al. Characterization of CD34+ peripheral blood cells from healthy adults mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1994; 84: 2795–2801. Mavroudis D, Read E, Cottler Fox M et al. CD34+ cell dose predicts survival, post transplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies. Blood 1996; 88: 3223–3229. Lemoli RM, Tafuri A, Fortuna A et al. Cycling status of CD34+ cells mobilized into peripheral blood of healthy donors by recombinant human granulocyte colony-stimulating factor. Blood 1997; 89: 1189–1196. Molineux G, Pojda Z, Hampson IN et al. Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor. Blood 1990; 76: 2153–2158. Simmons P, Leavesley DI, Levesque JP et al. The mobilization of primitive hemopoietic progenitors into the peripheral blood. Stem Cells 1994; 12 (Suppl. 1) 187–201. Habibian HK, Peters SO, Hsieh CC et al. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med 1998; 188: 393–398. Quesenberry PJ, Stewart FM, Zhong S et al. Lymphohematopoietic stem cell engraftment. Ann NY Acad Sci 1999; 872: 40–45. Vaziri H, Dragowska W, Allsopp RC et al. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA 1994; 91: 9857–9860. Rufer N, Brummendorf TH, Kolvraa S et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med 1999; 190: 157–167. Robertson JD, Gale RE, Wynn RF et al. Dynamics of telomere shortening in neutrophils and T cells during ageing and the relationship to skewed X chromosome inactivation patterns. Br J Haematol 2000; 109: 272–279. Korbling YO, Huh A, Durett N et al. Allogeneic blood stem cell transplantation: peripheralization and yield of donorderived primitive hematopoietic progenitor cells (CD34+Thy1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease. Blood 1995; 86: 2842–2848. Lemoli RM, Tafuri A, Fortuna A et al. Biological characterization of CD34+ cells mobilized into peripheral blood. Bone Marrow Transplant 1998; 22 (Suppl. 5) 47–50. Storek J, Witherspoon RP, Storb R. T cell reconstitution after bone marrow transplantation into adult patients does not resemble T cell development in early life. Bone Marrow Transplant 1995; 16: 413–425. Weng NP, Levine BL, June CH et al. Human naive and memory T lymphocytes differ in telomeric length and replicative potential. Proc Natl Acad Sci USA 1995; 92: 11091–11094. Abkowitz JL, Persik MT, Shelton GH et al. Behavior of haemopoietic stem cells in a large animal. Proc Natl Acad Sci USA 1995; 92: 2031–2035. Champlin RE, Horowitz MM, Chapius B et al. Blood stem cells versus bone marrow as a source of haematopoietic cells for allogeneic transplantation. Blood 2000; 95: 3702–3709.
