Significantly shorter telomeres in T-cells of patients with ZAP-70+/CD38+ chronic lymphocytic leukaemia

Authors


Alexander Röth, Department of Haematology, University Hospital, Hufelandstr. 55, D-45122 Essen, Germany. E-mail: alexander.roeth@uni-due.de Gabriela M. Baerlocher, Department of Haematology, University Hospital Bern, Freiburgstrasse 4, CH-3010 Bern, Switzerland. E-mail: Gabriela.Baerlocher@insel.ch

Summary

In contrast to other B-cell neoplasias, chronic lymphocytic leukaemia (CLL) is characterized by increased non-leukaemic T-cells. In order to assess their replicative history, telomere length was analyzed in subsets of leucocytes from CLL patients. Naive and memory T-cells from ZAP-70+/CD38+ patients exhibited significantly shorter average telomere lengths than ZAP-70/CD38 patients. Compared to the age-related percentiles of telomere length values from healthy individuals practically all values of the naive and memory T-cells from the ZAP-70+/CD38+ CLL patients fell below the 50th percentile. This indicated an extensive expansion and a role for T-cells in ZAP-70+/CD38+ CLL patients.

B-cell chronic lymphocytic leukaemia (CLL) is characterized by a clonal expansion of CD5-positive cells expressing CD19, CD23, and dim surface IgM. Interestingly, an increase in the peripheral blood T-cell number and a change in the function of T-cells have been described for patients with CLL. The latter observations seem to be characteristic for CLL because they are not found in other human cancers, and in particular, not in other B-cell neoplasias [reviewed in (Mellstedt & Choudhury, 2006)]. T-cell abnormalities have frequently been described for patients with CLL and those abnormalities are often associated with pathological conditions, such as an increased susceptibility to infections or autoimmune phenomena (e.g., autoimmune hemolytic anemia or thrombocytopenia). Based on those findings, speculation has been raised that T-cells may actually play an important role in contributing to the onset, sustenance, and exacerbation of the disease by creating and supporting the microenvironment to sustain the malignant B-cell clone (Ghia & Caligaris-Cappio, 2000).

Telomeres, specialized chromosome protective structures at the end of eukaryotic chromosomes, shorten with each round of cell division as a result of the inability of DNA polymerase to completely replicate the 3′ end of chromosomes, as well as being due to other causes (Verfaillie et al, 2002). As a consequence, the average telomere length in cells from most human tissues decreases with age in vivo and with culture in vitro. Analysis of telomere length can therefore be used to estimate the replicative history of individual cells. The present study aimed to assess the replicative history of T- and B-cells in samples from CLL patients and to determine whether differences exist between patients with a good (ZAP-70/CD38) and a poor prognosis (ZAP-70+/CD38+).

Design and methods

Patients and samples

Seventy-three patients with CLL (ZAP-70+/CD38+, n = 30; ZAP-70/CD38, n = 29; and ZAP-70/CD38 discordant, n = 14) were included in this retrospective study. Leucocytes were obtained from peripheral venous blood samples after informed consent and according to institutional guidelines. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Freiburg, Germany) and cryopreserved until further analysis. Comprehensive clinical information including treatment histories was available for all patients.

Laboratory analysis

Cytoplasmatic ZAP-70 expression and CD38 expression were determined by flow cytometry, as described previously (Schroers et al, 2005). The average length of telomere repeats at the chromosome ends in individual subsets of leucocytes (B-cells, and naive and memory T-cells) was measured by automated fluorescence in situ hybridization (FISH) and flow cytometry (flow-FISH), as described previously (Baerlocher et al, 2006).

Statistical analysis

Comparison of clinical or laboratory parameters between patient subgroups was performed using the non-parametric Mann–Whitney U-test for independent variables. Differences were regarded as statistically significant when P < 0·05.

Discussion and results

Elevated absolute numbers of circulating T-cells are frequently found in patients with CLL. This increase has been reported to be up to 4-fold when blood samples from CLL patients are compared to blood samples from healthy individuals (Garcia et al, 1989). It is, however, not clear whether this increase in the number of circulating T-cells reflects a shift of T-cells from the lymphoid organs to the circulation or if this is the result of an increase in the absolute number of T-cells, or even both. Using automated multicolour flow-FISH, we measured the telomere length in subsets of leucocytes from patients with CLL and from healthy individuals. Table I summarizes the clinical and laboratory characteristics of the CLL patients.

Table I.   Clinical and laboratory data of the CLL patients.
VariableAll patientsZAP-70+/CD38+ZAP-70/CD38Discordant
  1. WBC, white blood cell count; LDH, lactat dehydrogenase; n.a., not available.

Number of patients73302914
Median age at sample date, years (range)67 (39–82)63 (39–86)71 (39–81)67 (48–82)
Male, n (%)47 (64)21 (70)18 (62)8 (57)
Binet stage at diagnosis, n (%)
 A54 (74)18 (60)24 (83)12 (86)
 B12 (16)7 (23)4 (14)1 (7)
 C7 (10)5 (17)1 (3)1 (7)
Median WBC at sample date, ×109/l (range)59 (13–547)61 (13–276)38 (13–547)60 (12–400)
Median LDH at sample date, U/l (range)223 (138–1038)264 (138–1038)216 (156–340)207 (170–695)
Median time (primary diagnosis – sample), months (range)56 (0–297)33 (0–178)104 (0–297)36 (2–149)
Binet stage at sample date, n (%)
 A36 (49)11 (37)18 (62)7 (50)
 B15 (21)6 (20)5 (17)4 (29)
 C22 (30)13 (43)6 (21)3 (21)
Prior therapy, n (%)27 (37)12 (40)7 (24)8 (57)
 Fludarabine-containing regimens, n (%)9/27 (33)6/12 (50)0/7 (0)3/8 (37)
Median T-cell count (CD3+), ×109/l (range) 2·2 (0·4–11·5)1·5 (0·9–3·1)n.a.

As previous reports (Damle et al, 2004; Grabowski et al, 2005) have demonstrated, the present study confirmed significantly shorter telomere length values for the clonal B-cells of CLL patients. The telomere length was, however, significantly shorter for the ZAP-70+/CD38+ patient samples (mean ± STD, 2·46 ± 1·08 kb) than for the ZAP-70/CD38 patient samples (5·06 ± 1·76 kb, P < 6·7 × 10−9; Fig 1), whereas the telomere length of discordant cases fell between these values (3·1 ± 1·7 kb). Surprisingly, both naive and memory T-cells from ZAP-70+/CD38+ CLL patients also exhibited a significantly shorter average telomere length (4·85 ± 1·58 kb and 4·39 ± 1·09 kb, respectively) than naïve and memory T-cells from ZAP-70/CD38 CLL patients (6·64 ± 1·72 kb, < 2·2 × 10−4; and 6·22 ± 1·5 kb, P < 7·4 × 10−6, respectively). The average telomere length of naïve and memory T-cells from discordant CLL cases fell between those of the ZAP-70+/CD38+ and ZAP-70/CD38 patients (5·6 ± 1·9 kb and 4·8 ± 1·7 kb, respectively). Treatment had no effect on those results.

Figure 1.

 Telomere length analysis of T- and B-cells in CLL patients. The age-dependent telomere length percentiles (1%, 25%, 50%, 75%, and 99%) of naive and memory T-cells and B-cells are shown, based on telomere length measurements in subsets of leucocytes (naive and memory T-cells and B-cells) from 400 healthy individuals (Yamaguchi et al, 2005). The age-related telomere length values of naive and memory T-cells and B-cells of 59 CLL patients from two different prognostic groups (ZAP-70+/CD38+ vs. ZAP-70/CD38) are shown as red and blue symbols.

Given that telomeres shorten approximately 50–100 bp per cell division, the observed differences in telomere length of the analyzed T-cells for ZAP70+/CD38+ samples compared to normal T-cells or ZAP70/CD38 samples equals approximately 18–36 population doublings (PDs). If we consider only one T-cell undergoing 36 cell divisions, an impressive 1 × 1011 T-cells would arise; moreover, if we assume that the T-cells in B-CLL patients are not clonal, but polyclonal (resulting from multiple T-cells), this projected number of T-cells would even be much higher, suggesting an immense expansion of cells within the T-cell compartment. Furthermore, when we compared the telomere length to age-related percentiles calculated from over 400 healthy individuals, 0–102 years of age (Yamaguchi et al, 2005), practically all of the telomere length values of the naive and memory T-cells from the ZAP-70+/CD38+ CLL patients fell below the 50th percentile, whereas the values of naive and memory T-cells from the ZAP-70/CD38 CLL patients fell within the normal distribution, but rather at the higher end with even a few values above the 99th percentile.

In relation to the time from primary diagnosis to the date the sample was obtained, the average telomere loss was higher for naive T-cells in the ZAP-70+/CD38+ group compared to the ZAP-70/CD38 group (7·87 bp/month vs. 5·78 bp/month, respectively). Both findings indicate again that there must be an extensive expansion within the T-cell compartment, especially for the patients with ZAP-70+/CD38+ CLL (Garcia et al, 1989). In addition, T-cells from patients with CLL demonstrate a low responsiveness to mitogens and a decreased cell growth ability, as well as a poor expression of CD28, which is typically observed for senescing T-cells with short telomeres after expansion (Foa et al, 1980; Rossi et al, 1996).

In summary, these results indicate extensive T-cell proliferation and suggest that T-cells play an important role in the pathobiology of CLL, which might have been underestimated to date (Ghia et al, 2002). On the one hand, cytokines produced by T-cells, such as interleukin-2, interleukin-4, and interferon-γ, were shown to support the survival of CLL cells in vitro (Buschle et al, 1993; Huang et al, 1993; Sandoval-Montes & Santos-Argumedo, 2005), which could generate a supporting microenvironment sustaining the growth of the leukaemic B-cells in vivo. On the other hand, CLL cells might facilitate the polyclonal expansion of T-cells and prevent their apoptotic death from normal immunoregulatory mechanisms. Further studies addressing T-cells in CLL are needed and will help to elucidate the role and function of T-cells in this disease.

Acknowledgements

We thank Anja Führer, Barbara Friedmann, and Ute Schmücker for their expert technical assistance. This work was supported by a grant from the Bernese Cancer League (to G.M.B.).

Author contributions

A.R., J.D., and G.M.B. designed the research; A.R., D.B., H.N., L.S., J.D., and G.M.B. performed the experiments and collected the data; A.R., D.B., U.D., J.D., and G.M.B. analyzed and interpreted the data; A.R., U.D., J.D., and G.M.B. wrote the paper.

Conflicts of Interest

The authors declare no competing financial interests.

Ancillary