Marked telomere shortening in mobilized peripheral blood progenitor cells (PBPC) following two tightly spaced high-dose chemotherapy courses with G-CSF I Ricca 1 , M Compagno 1 , M Ladetto 1 , A Rocci 1 , M Dell’Aquila 1 , P Omede ` 1 , F De Marco 1 , S D’Antico 2 , D Caracciolo 1 , D Ferrero 1 , C Carlo-Stella 3 and C Tarella 1 1 Divisione di Ematologia, Dipartimento di Medicina ed Oncologia Sperimentale, Universita’ di Torino and A. O. S. Giovanni Battista, Torino, Italy; 2 Banca del Sangue di Torino – A. O. S. Giovanni Battista, Torino, Italy; and 3 Medical Oncology Unit, Istituto Nazionale Tumori, University of Milano, Milano, Italy The purpose of the study was to compare telomere length (TL) in peripheral blood progenitor cells (PBPC) collected after two tightly spaced high-dose (hd) chemotherapy courses. We assessed 37 previously untreated lymphoma patients under- going a hd-chemotherapy program with autografting. They sequentially received hd-cyclophosphamide (CY) and hd-Ara-C, both followed by PBPC harvesting. Both post-CY and post-Ara-C harvests were assessed for TL by Southern blot analysis. In 12 patients, the assay was also performed on purified CD34 þ cells. All patients displayed high PBPC mobilization following both hd- CY and hd-Ara-C. In all but one patient, TL was shorter in PBPC collected after Ara-C compared to CY: 7226bp (range: 4135– 9852) vs 8282 bp (range 4895–14860) (Po0.0001). This result was confirmed on CD34 þ cells. Platelet recovery in patients receiving post-Ara-C PBPC was significantly slower compared to those receiving post-CY PBPC. In conclusion, (i) administra- tion of tightly spaced hd-chemotherapy courses induces marked telomere shortening on harvested PBPC; (ii) engraft- ment kinetics seem slower, with delayed platelet recovery, in patients autografted with PBPC suffering marked TL erosion; (iii) long-term follow-up is required to verify whether PBPC with shortened telomeres display defective engraftment stability and/ or risk of secondary leukemia; (iv) TL evaluation is advisable whenever new mobilization procedures are developed. Leukemia (2005) 19, 644–651. doi:10.1038/sj.leu.2403652 Published online 17 February 2005 Keywords: telomere; non-Hodgkin’s lymphoma; PBPC mobilization; autograft Introduction High-dose (hd) chemotherapy with autologous stem cell transplantation (ASCT) is an effective treatment option for most chemosensitive tumors, particularly Hodgkin’s and non-Hodg- kin’s lymphoma. 1 Its toxicity has been markedly lowered by the transplantation of peripheral blood progenitor cells (PBPC). 2–4 PBPC collection and administration are simple and feasible, and result in fast postgraft recovery and consequently low treatment- related mortality. Extensive use of a highly tolerable intensive approach has improved the survival expectancy of many high- risk lymphoma patients. 5,6 However, the prolongation of survival has been accompanied by an increased risk of developing secondary hematological neoplasms, compared with age-matched controls. 7–11 DNA damage induced by mutagenic agents and the proliferative stress associated with bone marrow repopulation are considered to play major roles in the pathogenesis of these complications. 12,13 Recent observations have focused on the role of telomeres in the pathogenesis of post-transplant hematological neoplasms. Telomeres are linear DNA structures situated at the ends of chromosomes that help to maintain genome stability by preventing degradation, recombination and fusion of double- stranded DNA ends. 14–16 In the absence of effective telomere- protecting mechanisms, chromosomes lose 50–200 base pairs (bp) of their telomere sequences at each mitotic division. Progressive telomere shortening leads to senescence, a peculiar phase of cell life, chiefly characterized by growth inhibition. 17–20 Senescence may ultimately progress to crisis, entailing genetic instability, chromosomal fusions and a high propensity to apoptotic death or malignant transformation. 21,22 In germ line and in some somatic cells, telomere length (TL) is maintained by various DNA-elongating mechanisms, in particular the RNA-dependent telomerase. 17,20,23,24 Despite the ability of immature hemopoietic cells to activate telomerase, the TL of both early and committed progenitors progressively shortens with age, as in other highly replicating tissues. 25–28 This suggests either that the mechanisms for telomere maintenance become less effective with age in stem and progenitor cells, or that replicative stresses increase in intensity throughout cell life and induce extensive telomere shortening that cannot be sufficiently offset by DNA elongation. 29 Considerable and potentially stressful proliferative effort is required from hematopoietic cells following chemotherapy, particularly if delivered at high doses. This abnormal proliferation is even more pronounced during marrow regeneration after autologous or allogeneic transplantation, and several reports have shown marked though variable telomere shortening in transplanted patients. 30–33 Increased susceptibility to genetic damage associated with reduced TL could be crucial in the development of secondary myelodysplastic syndromes and leukemia after transplantation. Less is known about the telomere structure of the BM or PBPC harvests employed in transplant procedures. This information would be of particular value in the autograft setting, since harvesting is frequently scheduled after a variable number of chemotherapy courses. We evaluated the TL of PBPC collected in a hd-sequential chemotherapy program (HDS) designed for high- risk lymphoma. The program includes two consecutive courses of PBPC mobilization, after hd-cyclophosphamide (CY) and hd-Ara- C, delivered at approximately 1 month interval. 34 The aims of the study were (i) to see whether the tightly spaced administration of hd drugs results in detectable telomere shortening; (ii) to see whether in vitro clonogenic assays predict changes in the TL of hemopoietic progenitors; (iii) to define the value of telomere evaluation on PBPC employed in ASCT programs. Patients and methods Patients and treatment schedule TL was assessed in PBPC from 37 previously untreated patients aged o60 years, undergoing an HDS program for advanced- Received 20 May 2004; accepted 6 December 2004; Published online 17 February 2005 Correspondence: Professor C Tarella, Divisione di Ematologia, Dipartimento di Medicina ed Oncologia Sperimentale, Universita’ di Torino, via Genova 3, Torino, Italy; Fax: þ 39 011 6963737; E-mail: corrado.tarella@unito.it Leukemia (2005) 19, 644–651 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu