Sustained telomere erosion due to increased stem cell turnover during triple autologous hematopoietic stem cell transplantation

Experimental Hematology 36 (2008) 104–110

Sustained telomere erosion due to increased stem cell turnover during triple autologous hematopoietic stem cell transplantation Thomas Widmanna, Harald Kneera, Jochem Ko¨nigc, Markus Herrmannb, and Michael Pfreundschuha a

Klinik und Poliklinik fu¨r Innere Medizin I and bKlinische Chemie und Laboratoriumsmedizin, Universita¨tsklinikum Homburg, Homburg, Germany; cInstitut fu¨r Medizinische Biometrie, Epidemiologie und Informatik, Johannes Gutenberg-Universita¨t Mainz, Mainz, Germany (Received 19 July 2007; revised 20 August 2007; accepted 20 August 2007)

Telomeres cap chromosomal ends and are shortened throughout a lifetime. Additional telomere erosion has been documented during conventional chemotherapy or hematopoietic stem cell transplantation. Previous studies of stem cell transplantation reported variable amounts of telomere shortening with inconsistent results regarding the persistence of telomere shortening. Here we have prospectively studied telomere length and proliferation kinetics of hematopoietic cells in aggressive non-Hodgkin lymphoma patients who underwent a fourcourse high-dose chemotherapy protocol combined with triple autologous stem cell transplantation. We observed sustained telomere shortening in hematopoietic cells after triple stem cell transplantation with prolonged stem cell replication during the first year after stem cell transplantation. Ó 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Telomeres cap chromosomal ends and are a marker of somatic cell age in vitro [1] and in vivo [2]. In the human hematopoietic system, increased telomere erosion has been demonstrated in 1) states of increased cellular turnover (rheumatoid arthritis [3], paroxysmal nocturnal hemoglobinuria [4], chronic myeloid leukemia [5], Wegener’s granulomatosis [6]; 2) defects in telomere repair (Werner syndrome [7], ataxia telangiectasia [8], subgroups of aplastic anemia [9], dyskeratosis congenita [10]), 3) telomere DNA damage (oxidative stress [11,12]); and 4) diseases with so far unidentified mechanisms (e.g., aggressive non-Hodgkin lymphoma [13], coronary artery disease [14], vascular dementia [15]). Also, application of conventional [16] or myeloablative chemotherapy combined with stem cell transplantation (SCT) [17] has been associated with increased telomere shortening in leukocytes from patients. Long-term assessment of telomere length (TL) after SCT has shown inconsistent results regarding persistence of telomere shortening in patients [18–21]. Experimental approaches on poor-prognosis patients favor dose and dense intensification protocols with repetitive cycles of high-dose chemotherapy and the need for repetitive autografting [22–24].

Offprint requests to: Thomas Widmann, M.D., Klinik und Poliklinik fu¨r Innere Medizin I, Universita¨tsklinikum Homburg/Saar, 66421 Homburg/ Saar, Germany; E-mail: [email protected]

Especially for long-term cancer survivors, premature telomere erosion may be relevant, as shortened telomeres have been linked to development of secondary myelodysplastic syndrome (MDS), acute myeloid leukemia [18], and, rarely, graft failure after stem cell transplantation [25]. So far, the degree of telomere shortening in humans undergoing repetitive cycles of high-dose chemotherapy followed by autologous SCT has not been examined. This prompted us to study TL and proliferation kinetics of hematopoietic cells in aggressive non-Hodgkin lymphoma (aNHL) patients undergoing a four-course high-dose chemotherapy regimen combined with autologous SCT following courses 2, 3, and 4.

Material and methods This study was approved by the local ethics committee and all study participants gave written informed consent. Patients were recruited from an ongoing phase III clinical trial (MEGA-Choep Phase III) of the Deutsche Studiengruppe fu¨r Hochmaligne NonHodgkin Lymphome study group, comparing a four-course highdose chemotherapy (MEGA-Choep; Fig. 1) vs a conventional, dose-dense chemotherapy (eight cycles of Rituximab þ Choep þ granulocyte colony-stimulating factor [G-CSF] every 14 days) protocol for poor-prognosis aNHL (age adjusted International Prognostic Index [26] 2 or 3). Histopathology from six of six patients’ lymph node biopsies (recruited from 2003 to 2005) was performed by a local

0301-472X/08 $–see front matter. Copyright Ó 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2007.08.028

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

Course 1 CY DOXO VIN ETO PRED R

1500 70 2 600 500 375

R

105

PBSC

PBSC

PBSC

Course 2

Course 3

Course 4

CY DOXO VIN ETO PRED

4500 70 2 960 500

R

CY DOXO VIN ETO PRED

4500 70 2 960 500

R

CY DOXO VIN ETO PRED

6000 70 2 1480 500

Rx R R

Figure 1. Sequential high-dose treatment protocol of aggressive non-Hodgkin lymphoma patients. Cumulative doses per course were given in mg/m2. CY 5 cyclophosphamide; DOXO 5 doxorubicin; ETO 5 etoposide; PBSC 5 peripheral blood stem cells; PRED 5 prednisone; R 5 Rituximab; Rx 5 radiotherapy; VIN 5 vincristine. : indicates blood sampling for telomere-length analysis and/or proliferation studies.

pathologist and reviewed by an expert hematopathologist. aNHL patients received one course of induction chemotherapy (course 1) combined with filgrastim (G-CSF) mobilization at a dose of 480 mg/day, started on day 6 until at least 2  106 CD34 progenitor cells/kg body weight (BW) were harvested for transplantation after course 2. Using the same mobilization regimen, peripheral hematopoietic stem cells were harvested again after the second and third course of chemotherapy for subsequent reinfusion after courses 3 and 4 (Fig. 1). Furthermore, aNHL patients were treated with G-CSF during course 4 to ensure rapid engraftment. In case of a CD20-positive aNHL, patients received applications of the anti-CD20 antibody Rituximab in addition to treatment courses 1 to 4 and twice after completion of course 4 (Fig. 1). In addition to the six aNHL patients (patients A–F), data on hematopoietic stem cell (HSC) mobilization and hematopoietic recovery were available from 11 additional patients treated in the protocol. If hematopoietic reconstitution had not reached a level of 1000 leukocytes or 50,000 platelets per mL peripheral blood at the beginning of the next treatment course, data for hematopoietic reconstitution were censored (22 days). Cell separation and phenotyping Heparinized peripheral blood samples or apheresis product were collected from study participants and peripheral blood mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation. Granulocytes were separated by ammonium chloride lysis of the red cell pellet. Peripheral blood mononuclear cells were stained with anti-human monoclonal antibodies (CD3-allophycocyanin; CD34-phycoerythrin from Becton Dickinson, Heidelberg, Germany; Ki67-fluorescein isothiocyanate [FITC]; and isotype controls from Dako, Hamburg, Germany) and analyzed on a FACSCalibur (Becton Dickinson). Negative controls were run in each experiment. Telomere length analysis by FlowFish TL in leukocyte subsets was determined by the FlowFish method as described elsewhere [13]. In brief, 600,000 cells were washed once (5% dextrose, 0.1% bovine serum albumin [BSA], 10 mM N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid) and resuspended in hybridization buffer (75% deionized formamide; Sigma,

St Louis, MO, USA, 20 mM Tris [pH 7.1], 20mM NaCl, 1% BSA) for 10 minutes. After denaturing cells, 90 minutes incubation with either no probe (unstained control) or 0.18 mg FITC-labeled, telomere-specific (C3TA2)3 PNA probe (Applied Biosystems, Langen, Germany) was performed. After three rounds of washing (75% deionized formamide, 0.1% BSA, 10 mM Tris, 0.1% Tween 20; Sigma), cells were resuspended in phosphate-buffered saline, 0.1% BSA, RNAse A at 10 mg/mL and 0.1 mg/mL LDS 751 (Exciton, Mu¨nchen, Germany) for DNA counterstain. Flow cytometric analysis was carried out on a FACSCalibur (Becton Dickinson, Heidelberg, Germany). TL was expressed as mean fluorescent signal intensity. To control for inter-day variation, FITC-labeled beads (Quantum 24; Caltag, Hamburg, Germany) with defined amounts of fluorescence (molecules of equivalent soluble fluorochrome) were run in parallel each day. Furthermore, fluorescence values from donor samples were calculated relative to a specific donor sample that was run in parallel for each experiment. Statistics Paired test was used for leukocyte proliferation, analysis of CD34 content and for comparison of CD34 doses in stem cell transplants. Trends for telomere lengths were analyzed with mixed model analysis of variance using empirical variance estimation or the paired test for comparison of telomere lengths after course 4 and at follow-up dates. Wilcoxon signed rank test was used for analysis of leukocyte and platelet reconstitution after autografting. All p values reported are two-sided. p Values ! 0.05 are referred to as statistically significant.

Results We prospectively studied six poor-prognosis aNHL patients (average age: 51.0 years, Table 1), undergoing a dose-escalated four-course high-dose therapy regimen. All patients underwent stem cell collections before each myeloablative therapy combined with subsequent autologous SCT of the freshly harvested HSCs (Fig. 1). Four of six patients additionally underwent involved field radiotherapy for bulky or extranodal disease, beginning 3 to 6 weeks after completion

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

106 Table 1. Patient characteristics Patient A B C D E F

Age (y) 56.2 54.3 48.1 47.3 52.9 47.0

Gender

Histological subtype

Stagea

aaIPI

M M M F M M

DLCL, CD20þ Peripheral T-NHL DLCL, CD20þ DLCL, CD20þ DLCL, CD20þ DLCL, CD20þ

IV B III BE IV AE IV AE IV B III B

2 2 2 2 2 2

Radiotherapy

Remission status

No Yes Yes Yes No Yes

CR1 CR1 CR1 CR1 CR1

aaIPI 5 age adjusted International Prognostic Index (including lactate dehydrogenase level above normal, stage and Eastern Cooperative Oncology Group Performance status); CR1 5 first complete remission; DLCL 5 diffuse large cell lymphoma; F 5 female; M 5 male; T-NHL 5 T-cell–derived non-Hodgkin lymphoma. a Staging according to the Ann Arbor classification.

Stem cell mobilization and proliferation kinetics Parallel to increasing courses of HSC mobilization and autologous SCT, aNHL patients (n 5 17) mobilized less

Granulocyte telomere length (MESF)

TL dynamics TL determined as molecules of equivalent soluble fluorochrome was converted into kilobase pairs (kbp) according to a previously established standard curve [27]. Granulocytes (Fig. 2A) and lymphocytes (Fig. 2B) displayed a steadily decreasing TL along with treatment courses and, furthermore, telomere erosion was sustained even at FU1 and FU2. All patients experienced telomere shortening in their granulocytes (range, 0.1–3.2 kbp): TL decreased by 17.1% on average from 7.5 kbp to 6.3 kbp after completion of chemotherapy (p 5 0.016) and was not regained at follow-up dates (p 5 0.541 for TL at R4 vs FU1 and p 5 0.394 for R4 vs FU2). Loss of TL in granulocytes during the first year post-SCT was 164 bp/year, indicating increased turnover in myeloid progenitors. During the extended 2-year follow-up period (FU1 to FU2), telomere loss slowed down to an average rate of 55 bp/year. Telomere erosion in lymphocytes during repetitive autografting was less prominent and statistically not significant (6.6 kbp to 6.0 kbp; p 5 0.075), with no evidence for recovery (p 5 0.825 for comparison of TL after R4 vs FU1 and p 5 0.570 for R4 vs FU2). Four of six patients’ lymphocytes shortened during the treatment protocol (range of all patients, 0.2 to 1.9 kbp). Lymphocytes eroded their telomeres at an average rate of 100 bp/year in the first year and 72 bp/year in the second and third year after SCT.

CD34 HSC into their peripheral blood pool upon stem cell apheresis. Average percentages of CD34 HSC in apheresis product declined from 6.18% (63.16% SD) during the first HSC harvest (following course 1) to an average of 3.20% (62.5% SD) during the second harvest (following

Lymphocyte telomere length (MESF)

of chemotherapy. The MEGA-Choep protocol allowed studying telomere dynamics in granulocytes and lymphocytes after three consecutive rounds of myeloablative treatment and repopulation of serially transplanted HSCs in the same patient. TL in granulocytes and lymphocytes was analyzed before course 1 (C1), after hematopoietic recovery from each autologous SCT procedure (R2–4) and at two follow-up dates (average 12 months [FU1], average 36 months [FU2]). Patient C was lost to follow-up 6 months after completion of the protocol and died shortly thereafter.

30000

A

*

25000 20000 15000 10000 5000 0 C1

R2

R3

R4

FU1

FU2

C1

R2

R3

R4

FU1

FU2

25000

B 20000

15000

10000

5000

0

Figure 2. Telomere erosion in granulocytes and lymphocytes. Average telomere length of granulocytes (A) and lymphocytes (B) is displayed in molecules of equivalent soluble fluorochrome at different time points during the treatment regimen: before course 1 (C1), after hematopoietic recovery following courses 2, 3, 4 (R2, R3, R4, respectively) and at a follow-up dates (FU1 and FU2). Results are expressed as box plots, displaying the median as horizontal lines and boxes as 25th and 75th percentile as well as whiskers indicating 10th and 90th percentile. *p ! 0.05. MESF 5 molecules of equivalent soluble fluorochrome.

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

course 2, p 5 0.006) and finally to a minimum of 1.49% (61.27% SD) HSC during the third harvest following course 3 (p 5 0.077 for comparison second vs third mobilization and p 5 0.003 for comparison first vs third mobilization; Fig. 3A). Therefore, fewer HSC/kg BW could be harvested per apheresis procedure and were transplanted after myeloablative therapy. During the chronologically first SCT procedure, an average of 6.2  106 CD34/kg BW HSCs (SD 6 4.0  106 CD34/kg BW) were reinfused. After declining to 5.4  106 CD34/kg BW (SD 6 2.8  106 CD34/kg BW) for the second SCT procedure, the third stem cell transplantation was performed with 4.7  106 CD34/kg BW HSCs (SD 6 2.6  106 CD34/kg BW; p 5 0.045 for first vs third SCT; Fig. 3B). Because a decreasing content of HSCs in stem cell transplants may require increased self-replication after myeloablative treatment, we analyzed the proliferation status of HSC by staining for the proliferation marker Ki67 (Fig. 3C). Baseline proliferation before treatment C1 of HSCs increased to from 0.4% CD34þKi67þ cells (SD 6 0.2%) to 2.8% after R3 (SD 6 1.3%, p 5 0.005) and remained high at FU1 (mean: 4.3%, SD 6 2.2%, p 5 0.009 for FU1 vs baseline proliferation and p 5 0.173 for FU1 vs R3). Thirty-six months after the last SCT (FU2),

the proliferation of HSCs decreased (mean: 0.7%, SD 6 0.7%, p 5 0.008 for FU2 vs FU1) and was comparable to baseline characteristics (p 5 0.416 for FU2 vs C1). As documented with HSCs, CD3 lymphocytes showed a low baseline proliferation activity (Fig. 3D, mean CD3þKi67þ: 1.2%, SD 6 0.4%). Proliferation of CD3 lymphocytes increased during serial SCT (R3, mean: 7.1%, SD 6 2.0%, p 5 0.011), but in contrast to HSCs, showed a decrease in proliferation rates at FU1 (mean: 1.9%, SD 6 2.3%, p 5 0.013 for R3 vs FU1). Proliferating CD3 lymphocytes at FU2 were even lower than the proliferation rate at baseline (mean: 0.4%, SD 6 0.2%, p 5 0.019 for FU2 vs C1). Hematopoietic recovery Data on hematopoietic recovery after autografting were available in 17 aNHL patients. Leukocyte recovery to at least 1000/mL was documented on average after 9.1 days (SD 6 1.1), 9.8 days (SD 6 1.1), and 12.3 days (SD 6 4.3) following course 2, 3, and 4, respectively (p 5 0.049 for 2nd vs 3rd course, p 5 0.006 for 3rd vs 4th course, p 5 0.003 for 2nd vs 4th course; Figure 4A). Recovery of platelets O50,000/mL was completed after 13.5 days (SD 6 5.5), 15.6 days (SD 6 5.3), and 19.1 days (SD 6 4.5) following course 2, 3, and 4, respectively (p 5 0.023

18

12

A

** **

14

Proliferating CD34 (%)

% CD34/ nucleated cells

16

12 10 8 6 4 2

harvest 1

C

**

**

*

8 6 4 2

harvest 2

harvest 3

C1 14

B

*

12

Proliferating CD3 (%)

14

CD34x106/kgBW

10

0

0

16

107

12 10 8 6 4 2

D

R3

*

Fu1

Fu2

Fu1

Fu2

*

10 8 6 4 2 0

0 Tx 1

Tx 2

Tx 3

C1

R3

Figure 3. Hematopoietic stem cell (HSC) mobilization and leukocyte proliferation kinetics. (A) HSC content in % of total nucleated cells during HSC harvests (harvest 1, harvest 2, and harvest 3) after course 1, 2, 3, respectively (n 5 17 aggressive non-Hodgkin lymphoma [aNHL] patients). (B) Transplanted CD34  106/kg body weight (BW) of 17 aNHL patients in chronologic autologous stem cell transplantation (Tx 1–3). (C) Cellular proliferation of HSC (% of total HSC) from n 5 5 aNHL patients displayed as CD34Ki-67–positive cells before therapy (C1), after hematopoietic recovery following course 3 (R3) and at a follow-up date (FU). (D) Proliferation of CD3 lymphocytes displayed as CD3Ki67-positive cells (% of total CD3 lymphocytes) before therapy (C1), following course 3 (R3) and at FU1 and FU2. *p ! 0.05; **p ! 0.01.

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

108

for 2nd vs 3rd course, p 5 0.049 for 3rd vs 4th course, p 5 0.010 for 2nd vs 4th course; Figure 4B).

Discussion To the best of our knowledge, this is the first report of TL analysis during triple myeloablative treatment and autologous SCT in humans. The safety and efficacy of repetitive high-dose chemotherapy followed by autografting has been proven in the phase I/II MEGAChoep clinical trial [22] with documented favorable outcome parameters compared to historic, conventionally treated aNHL patients [26]. However, the price these patients pay for maximum dose escalation is a sustained and possible permanent telomere length reduction in peripheral leukocytes. The telomere erosion per round of myeloablative therapy and autologous SCT of z400 bp in granulocytes (1.2 kbp after three rounds), is comparable to previously reported telomere erosion shortly after single autologous [28] or allogeneic SCT [20]. Telomere loss as high as 3.4 kbp in T lymphocytes [19] was reported in a study of four patients receiving Tcell–depleted allografts. Previous studies determining TL

Leucocyte recovery >1,000/µl (days)

25

A

**

20

*

**

15

10

5

0 R2

R3

R4

Platelet recovery >50.000/µl (days)

40

B

* *

30

*

20

10

0 R2

R3

R4

Figure 4. Prolonged hematopoietic recovery after consecutive courses of autografting. Days to recovery of leukocytes O1000/mL (A) or platelets O50,000/mL (B) after courses 2 to 4 (R2–R4, respectively) were displayed as mean (bars) and standard deviations (whiskers). *p ! 0.05; **p ! 0.01.

during single treatment courses of autografting cannot be directly compared to this study, however, we observed a sustained telomere erosion rather than telomere homeostasis [18,20,29], in line with reports from other groups [19,21]. Lower CD34 content in transplants may require more extensive HSC replication during hematopoietic recovery and thus entail shortening in HSCs [30]. In accordance with this model, transplantation of high CD34 doses lead to telomere conservation in a study of allogeneic stem cell transplantation [20]. Because granulocytes differentiate after a limited number of divisions from HSCs, and because we have previously shown that TL in granulocytes and CD34 HSCs highly correlate [27], we suggest that accelerated telomere loss in granulocytes reflects telomere loss in HSCs. This is supported by the documented increased proliferation of HSCs during the first year post-SCT with increased telomere shortening in granulocytes. The phenomenon of increased proliferative activity of HSCs after SCT has been documented in humans up to 12 months post-SCT [28] and in murine studies up to 4 months post-Tx [31]. During the extended follow-up period (12–36 months post-SCT), we were, furthermore, able to show a return to baseline proliferation kinetics in HSCs, with a corresponding decrease in telomere loss, comparable to telomere loss per year in healthy individuals [2,27]. Regeneration of different progenitor compartments seems a likely explanation for prolonged HSC replication, because we have documented decreasing HSC content with consecutive autologous SCT, and a deficiency of marrow progenitors over several years after autologous SCT was reported in lymphoma patients by Benboubker et al. [32]. The model of increased, stochastically determined replication of HSCs for a limited time after SCT is supported by our study and may explain why telomeres shorten at a physiological rate following the first year of transplantation [19,33,28]. Recent molecular studies on murine HSCs only begin to uncover the mechanisms that regulate the balance between quiescence and proliferation [34,35]. However, the exact mechanisms by which ‘‘true’’ HSC and more differentiated progenitors repopulate a myeloablatively treated patient still need to be established. Unlike granulocytes, T lymphocytes primarily expand through peripheral self-replication shortly after SCT [36], and their maturation through the thymus is enabled for several months [37]. Here, we document massively increased proliferation of lymphocytes during three tightly spaced courses of myeloablative treatment, indicating a stimulus for self-replication in the myeloablatively treated host. Larger quantities of transplanted peripheral lymphocytes compared to HSCs during autologous SCT and the influx of naı¨ve T cells via the thymus beginning 6 months postSCT [37], may account for the limited demand of increased proliferation of lymphocytes seen in our study at follow-up dates. In line with these results is a limited telomere loss

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

(72 bp/year) in aNHL patients during years 2 and 3 after SCT, again, comparable to healthy individuals (59 bp/year [2]). Serially transplanted murine bone marrow cells experience excessive telomere shortening with, ultimately, failure to engraft after four rounds of transplantation. Interestingly, failure to engraft after four rounds of transplantation was also documented among mice with mTERT overexpression and conserved telomeres, pointing to telomere-independent barriers in this system [38]. Nevertheless, the important role of telomerase to conserve telomeres during several rounds of autologous SCT was outlined in these murine studies [38,39]. Human HSCs seem to tolerate triple myeloablative treatment and autologous SCT, as none of the studied patients here developed graft failure or MDS. Nevertheless, caution and close patient monitoring is warranted, as aNHL patients already harbor shorter telomeres in their leukocytes at diagnosis [13], equivalent to 20 years of age, and undergo approximately 30 to 36 years of somatic cell aging (granulocytes) during the MEGA-Choep protocol, based on calculations from our previous study in granulocytes and HSC [27] and from Rufer et al. [2]. In a previous study, our group found no correlation between absolute telomere length in granulocytes from allogeneic stem cell donors and time to hematopoietic recovery in their hosts [27]. The current study allows intra-patient comparison of hematopoietic recovery and TL and documented a prolonged hematopoietic recovery after consecutive cycles of high-dose therapy, paralleled by decreasing granulocyte telomere length. This result may be confounded by the declining pool of transplanted HSCs with consecutive rounds of autografting, although quantities of transplanted HSCs were never !2.0 CD34/kg BW and, thus, within the range of practical guidelines for autologous SCT [40]. Furthermore, chemotherapy induced marrow toxicity in both the HSC and stromal compartments [41] may have prolonged hematopoietic recovery. Alternatively, TL itself may be part of the phenomenon ‘‘marrow toxicity’’ as an animal model showed increased sensitivity to irradiation [42] or chemotherapeutic agents [43] upon shortened telomeres in hematopoietic cells. In conclusion, we show extensive and sustained telomere erosion upon triple autografting in aNHL patients due to increased stem cell proliferation during antineoplastic therapy and in the first year thereafter. So far, only one patient treated in the phase I/II MEGA-Choep trial developed a secondary MDS [22]. Nevertheless, close patient monitoring (including TL) of this heavily treated cohort is indicated. Upon detection of an increased incidence of secondary MDS or therapy-associated acute leukemia (as reported by Bhatia et al. [18]), a prospective evaluation of TL and consecutive telomere shortening in patients undergoing high-dose chemotherapy could identify patients at risk for these conditions and possibly lead to modified treatment strategies in the future.

109

Acknowledgment We thank the Deutsche Studiengruppe Hochmaligne Non-Hodgkin Lymphome for providing patient material and clinical data and Klaus Roemer for critical reading the manuscript. T.W. was supported by a grant from HOMFOR.

References 1. Allsopp RC, Vaziri H, Patterson C, et al. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci U S A. 1992;89:10114–10118. 2. 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. 3. Schonland SO, Widmann T, Lopez-Benitez CM, et al. Premature telomeric loss in rheumatoid arthritis is genetically determined and involves both myeloid and lymphoid cell lineages. Proc Natl Acad Sci U S A. 2003;100:13471–13476. 4. Beier F, Balabanov S, Buckley T, et al. Accelerated telomere shortening in glycosylphosphatidylinositol (GPI)-negative compared with GPI-positive granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH. detected by proaerolysin flow-FISH. Blood. 2005;106:531–533. 5. Brummendorf TH, Holyoake TL, Rufer N, et al. Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry. Blood. 2000;95:1883–1890. 6. Vogt S, Iking-Konert C, Hug F, Andrassy K, Hansch GM. Shortening of telomeres: Evidence for replicative senescence of T cells derived from patients with Wegener’s granulomatosis. Kidney Int. 2003;63: 2144–2151. 7. Crabbe L, Verdun RE, Haggblom CI, Karlseder J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science. 2004;306:1951–1953. 8. Metcalfe JA, Parkhill J, Campbell L, et al. Accelerated telomere shortening in ataxia telangiectasia. Nat Genet. 1996;13:350–353. 9. Vulliamy T, Marrone A, Dokal I, Mason PJ. Association between aplastic anaemia and mutations in telomerase RNA. Lancet. 2002; 359:2168–2170. 10. Vulliamy T, Marrone A, Goldman F, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature. 2001;413:432–435. 11. Epel ES, Blackburn EH, Lin J, et al. Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci U S A. 2004;101:17312– 17315. 12. Von Zglinicki T, Petrie J, Kirkwood TB. Telomere-driven replicative senescence is a stress response. Nat Biotechnol. 2003;21:229–230. 13. Widmann TA, Herrmann M, Taha N, Konig J, Pfreundschuh M. Short telomeres in aggressive non-Hodgkin’s lymphoma as a risk factor in lymphomagenesis. Exp Hematol. 2007;35:939–946. 14. Fitzpatrick AL, Kronmal RA, Gardner JP, et al. Leukocyte telomere length and cardiovascular disease in the cardiovascular health study. Am J Epidemiol. 2007;165:14–21. 15. Von Zglinicki T, Serra V, Lorenz M, et al. Short telomeres in patients with vascular dementia: an indicator of low antioxidative capacity and a possible risk factor? Lab Invest. 2000;80:1739–1747. 16. Lee JJ, Nam CE, Cho SH, et al. Telomere length shortening in non-Hodgkin’s lymphoma patients undergoing chemotherapy. Ann Hematol. 2003;82:492–495. 17. Akiyama M, Asai O, Kuraishi Y, et al. Shortening of telomeres in recipients of both autologous and allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2000;25:441–447.

110

T. Widmann et al./ Experimental Hematology 36 (2008) 104–110

18. Bhatia R, Van Heijzen K, Palmer A, et al. Longitudinal assessment of hematopoietic abnormalities after autologous hematopoietic cell transplantation for lymphoma. J Clin Oncol. 2005;23:6699–6711. 19. 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. 2001;97:575–577. 20. Roelofs H, de Pauw ES, Zwinderman AH, et al. Homeostasis of telomere length rather than telomere shortening after allogeneic peripheral blood stem cell transplantation. Blood. 2003;101:358–362. 21. Wynn RF, Cross MA, Hatton C, et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet. 1998;351:178–181. 22. Glass B, Kloess M, Bentz M, et al. Dose-escalated CHOP plus etoposide (MegaCHOEP. followed by repeated stem cell transplantation for primary treatment of aggressive high-risk non-Hodgkin lymphoma. Blood. 2006;107:3058–3064. 23. Lotz JP, Bui B, Gomez F, et al. Sequential high-dose chemotherapy protocol for relapsed poor prognosis germ cell tumors combining two mobilization and cytoreductive treatments followed by three high-dose chemotherapy regimens supported by autologous stem cell transplantation. Results of the phase II multicentric TAXIF trial. Ann Oncol. 2005;16:411–418. 24. Elias AD, Ibrahim J, Richardson P, et al. The impact of induction duration and the number of high-dose cycles on the long-term survival of women with metastatic breast cancer treated with high-dose chemotherapy with stem cell rescue: an analysis of sequential phase I/II trials from the Dana-Farber/Beth Israel STAMP program. Biol Blood Marrow Transplant. 2002;8:198–205. 25. Awaya N, Baerlocher GM, Manley TJ, et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol Blood Marrow Transplant. 2002;8:597– 600. 26. Shipp MA. A predictive model for aggressive non-Hodgkin’s lymphoma. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med. 1993;329:987–994. 27. Widmann TA, Willmann B, Pfreundschuh M, Beelen DW. Influence of telomere length on short-term recovery after allogeneic stem cell transplantation. Exp Hematol. 2005;33:1257–1261. 28. Thornley I, Sutherland R, Wynn R, et al. Early hematopoietic reconstitution after clinical stem cell transplantation: evidence for stochastic stem cell behavior and limited acceleration in telomere loss. Blood. 2002;99:2387–2396. 29. Ricca I, Compagno M, Ladetto M, et al. Marked telomere shortening in mobilized peripheral blood progenitor cells (PBPC) following two tightly spaced high-dose chemotherapy courses with G-CSF. Leukemia. 2005;19:644–651.

30. Notaro R, Cimmino A, Tabarini D, Rotoli B, Luzzatto L. In vivo telomere dynamics of human hematopoietic stem cells. Proc Natl Acad Sci U S A. 1997;94:13782–13785. 31. Allsopp RC, Cheshier S, Weissman IL. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J Exp Med. 2001;193:917–924. 32. Benboubker L, Cartron G, Roingeard F, et al. Long-term marrow reconstitutive ability of autologous grafts in lymphoma patients using peripheral blood mobilized with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor compared to bone marrow. Exp Hematol. 2003;31:89–97. 33. Robertson JD, Testa NG, Russell NH, et al. Accelerated telomere shortening following allogeneic transplantation is independent of the cell source and occurs within the first year post transplant. Bone Marrow Transplant. 2001;27:1283–1286. 34. Passegue E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med. 2005;202:1599–1611. 35. Lacorazza HD, Yamada T, Liu Y, et al. The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells. Cancer Cell. 2006;9:175–187. 36. Roux E, Dumont-Girard F, Starobinski M, et al. Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity. Blood. 2000;96:2299–2303. 37. Dumont-Girard F, Roux E, van Lier RA, et al. Reconstitution of the Tcell compartment after bone marrow transplantation: restoration of the repertoire by thymic emigrants. Blood. 1998;92:4464–4471. 38. Allsopp RC, Morin GB, Horner JW, et al. Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells. Nat Med. 2003;9:369–371. 39. Allsopp RC, Morin GB, DePinho R, Harley CB, Weissman IL. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. Blood. 2003;102:517–520. 40. Serke S, Johnsen HE. A European reference protocol for quality assessment and clinical validation of autologous haematopoietic blood progenitor and stem cell grafts. Bone Marrow Transplant. 2001;27: 463–470. 41. Domenech J, Roingeard F, Herault O, et al. Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation. Leuk Lymphoma. 1998;29:533–546. 42. Goytisolo FA, Samper E, Martin-Caballero J, et al. Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med. 2000;192:1625–1636. 43. Rudolph KL, Chang S, Lee HW, et al. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell. 1999;96:701–712.