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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1883-1890
PLENARY PAPER
From the Terry Fox Laboratory and Division of Hematology, British
Columbia Cancer Agency, Vancouver, British Columbia, Canada;
Departments of Medicine, Statistics, and Medical Genetics, University
of British Columbia, Vancouver, British Columbia, Canada.
Chronic myeloid leukemia (CML) is a clonal, multilineage
myeloproliferative disorder characterized by the Philadelphia
chromosome (Ph) and a marked expansion of myeloid cells. Previous
studies have indicated that the telomere length in blood cells may
indicate their replicative history. However, the large variation in
telomere length between individuals complicates the use of this
parameter in CML and other hematologic disorders. To circumvent this
problem, we compared the telomere length in peripheral blood or bone
marrow cells with purified normal (Ph
Chronic myeloid leukemia (CML) is a malignant disorder
that originates in a pluripotent hematopoietic stem cell1
(HSC) that acquires a Philadelphia (Ph) chromosome2,3
encoding the BCR-ABL oncogenic fusion protein.4,5 In
chronic phase (CP), increased numbers of Ph+ myeloid,
erythroid, and megakaryocytic progenitor cells are found both in the
hyperplastic bone marrow and in the peripheral blood of patients with
highly elevated white blood cell counts. Despite convincing evidence
that CML can originate in a lymphohematopoietic precursor cell,
Ph+ cells typically outnumber the progeny of normal cells
only in the myeloid lineages. Some Ph+ B-lineage cells are
found in a proportion of patients and circulating T cells are
predominantly Ph In human somatic cells, chromosomes terminate in several kilobases of
repetitive TTAGGG sequences and associated proteins. Telomere repeats
in such cells are successively lost with repeated cell division; hence,
with age, this process leads to genetic instability and cell
senescence.21-28 Telomerase is required to maintain the
length of telomere repeats in the germ line,29,30 and
variable levels of telomerase activity can also be detected in normal
hematopoietic progenitor cells, activated T cells, germinal center B
lymphocytes, as well as in the majority of human cancers (for reviews,
see Shay and Bacchetti,31 Weng et al,32 and de
Lange33). Recently, studies of telomerase knock-out mice have highlighted the functional importance of telomeres and telomerase in the biology of multicellular organisms.29,34,35 The
finding that overexpression of hTERT in telomerase-negative human
somatic cells results in the extension of the in vitro life span of
otherwise normal epithelial cells and fibroblasts further supports a
linkage between telomere shortening and replicative
senescence.36-38
The level of telomerase found in normal HSCs appears to be too low to
prevent telomere shortening with replication and age.39-42 In addition, we have shown the proliferative capacity of HSCs at
different stages of ontogeny to be correlated with the overall telomere
length of the corresponding hematopoietic tissue.39,43-45 Telomere shortening appears to be accelerated in donor-derived HSCs
regenerated in recipients of allogeneic bone marrow transplants (BMT).46,47 Taken together, these studies suggest that
telomere length measurements may be useful indications of stem cell
turnover in vitro and in vivo.
Recent studies in CML have suggested that telomere length shortens with
progression from CP to BP and that telomerase is up-regulated in
association with the acquisition of additional cytogenetic aberrations.40,48-50 It has also been reported that longer
telomeres may be a feature of patients with a more favorable prognosis
and likelihood to respond to treatment with
interferon- CML patient characteristics
Cells
FACS analysis Cell populations were incubated for 10 minutes at 4°C with Hanks' balanced salt solution (HBSS, StemCell Technologies) containing 5% human serum prior to addition of antihuman CD3-fluorescein isothiocyante (FITC) 1:50 (Becton Dickinson, San Jose, Calif). Cells were incubated at 4°C for another 30 minutes and finally washed twice with HBSS/2% FCS; 1 µg/mL propidium iodide (PI, Sigma) was added to the second wash to allow discrimination of dead (PI+) cells before analysis. Acquisition and analysis of the cells were performed on a FACSort using LYSIS II software (Becton Dickinson).Isolation of CD3+ cells The CD3+ cells were either first separated from myeloid cells by immunomagnetic negative selection on a column (StemSep, StemCell Technologies) or were directly expanded from either low-density or whole blood leukocyte preparations without their pre-enrichment. Monoclonal antibodies against CD36, CD34, IgE, CD66b, and CD66e were added to the standard StemSep T-cell enrichment cocktail to improve the purity of negatively selected CD3+ cells. After the depletion step, the CD3+-enriched cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% human serum and 1.0 µg/mL phytohemagglutinin (PHA, Murex Diagnostics, Schaffausen, Switzerland) and 100 U/mL recombinant human interleukin-2 (rhIL-2; Roche, Nutley, NJ). In some cases, 106/mL irradiated allogeneic low-density normal blood cells were added as feeders as well. After 10 to 15 days of incubation, these cultures typically yielded sufficient numbers of T cells for analysis of telomere length.Flow cytometric detection of telomere FISH (flow-FISH) Telomere length measurements of PBL and selectively expanded T lymphocytes were performed by flow-FISH as previously described.52 Analysis of PBL (Figure 1A-C) and T lymphocytes (Figure 1D-F) of 1 individual CML patient is shown. Daily shifts in the linearity of the flow cytometer and fluctuations in the laser intensity and alignment were compensated using FITC-labeled fluorescent beads (Quantum-24 Premixed; Flow Cytometry Standards, San Juan, Puerto Rico). Green fluorescence (FL1) was measured on a linear scale and results were expressed in molecular equivalents of soluble fluorochrome units (kMESF).53 After gating on single diploid cells (R1, see Figure 1A and D), specific telomere fluorescence was calculated by subtracting the mean fluorescence of the background control (no probe, see Figure 1C and F, light gray) from the mean fluorescence obtained from cells hybridized with the telomere probe (see Figure 1C and F, dark gray, difference indicated by horizontal bars). In previous studies, we have shown that telomere fluorescence of PBL and T lymphocytes is directly proportional to the mean size of terminal restriction fragments measured by Southern blot analysis.54
LTC-IC assay Cells were seeded onto pre-established irradiated murine fibroblasts genetically engineered to produce human IL-3 (10 ng/mL), human granulocyte colony-stimulating factor (G-CSF; 130 ng/mL), and human Steel factor (10 ng/mL) in long-term culture medium (Myelocult, StemCell Technologies) supplemented with freshly dissolved 10 6 mol/L hydrocortisone sodium hemisuccinate
(Sigma). These cultures were then maintained in a humidified atmosphere
of 37°C, 5% CO2 in air for 6 weeks with weekly
exchanges of half the medium and removal of half the nonadherent cells
as described.55 At the end of 6 weeks, the nonadherent
cells were combined with the trypsinized adherent cells and assayed for
their content of granulopoietic, erythroid, and multilineage
colony-forming cells in methylcellulose medium (H4330, StemCell
Technologies) supplemented with 50 ng/mL Steel factor, 20 ng/mL IL-3
(Novartis, Basel, Switzerland), 20 ng/mL IL-6 (Cangene, Mississauga,
ON, Canada), 20 ng/mL granulocyte-macrophage colony-stimulating factor
(Novartis), 20 ng/mL G-CSF (StemCell Technologies) and 3 U/mL
erythropoietin (StemCell Technologies). These cultures were then
incubated for 12 to 16 days at 37°C at the end of which colonies
were scored in situ using standard criteria.55
Cytogenetic analysis Colonies generated from 6-week-old LTC-IC assays and in vitro expanded CD3+ T cells were genotyped as Ph+ or Ph by cytogenetic analysis of Giemsa-banded
metaphases.56
Statistical analysis Fluorescence values in kMESF units for different cell types, donor age, disease stage (CP, AP/BP), Ph status of the patients' LTC-IC, disease duration since diagnosis, and the remaining duration of CP from the time point the sample was taken were the parameters used for statistical analysis of the data. Controls were taken from a previous study performed in this center on 301 normal individuals.52 Age adjustments were made based on the linear regression analysis for total PBL values obtained from a subpopulation of this cohort, aged 16 to 80 years (n = 147). Telomere data of different patient groups consisted of generally mixed cross-sectional and serial measurements. To counteract any weighting effect of using multiple specimens from single patients in the analyses and to identify within-patient, across-patient, and between-group levels of variation, multilevel maximum likelihood analyses were carried out using the MLn program of the University of London (MLn, Multilevel Models Project, University of London, 1998). Differences of paired data within patients were also analyzed by the MLn method.
Initial comparison of telomere fluorescence measurements in blood or marrow cells from patients with CML and healthy controls Flow-FISH measurements of telomere fluorescence were obtained on 123 unseparated or low-density blood or marrow samples from 59 patients (solid circles, Figure 2). Considerable variation between values even when plotted as a function of the patient's age is seen. Also shown in Figure 2 for comparison are results for total fresh blood leukocytes taken from a study on a large series of normal individuals assessed concurrently in our center using the same methodology.52 Much of the variation seen in telomere values at any given age in normal individuals appears to be genetically determined as indicated by an analysis of twins in this previous study. Nevertheless, as summarized in Table 1, the average age-adjusted telomere length determined for CML cells (11.0 ± 0.3 kMESF) proved to be significantly (P < .001) lower than the corresponding value for normal cells from age-adjusted donors (13.2 ± 0.3 kMESF). In CML, a number of factors are expected to exacerbate the normal variation in telomere length. These include effects due to the stage of the disease as well as other factors including the proportion of normal cells in the sample. Indeed (normal) T lymphocytes may have been slightly enriched in some of the CML samples due to a greater loss of (Ph+) myeloid cells on thawing. Accordingly, a comparison was made between paired telomere length measurements for CML cells and purified normal T cells from the same patient.
Comparison of paired telomere fluorescence in CML cells and normal T lymphocytes from the same patients In healthy individuals, granulocytes can be readily separated from lymphocytes on the basis of their different light scattering properties.However, in CML samples, such a separation is hampered due to an abundance of immature myeloid cells (see Figure 1B). Therefore, to obtain a suitably enriched population of normal cells for use as an internal control, we selectively enriched CD3+ T lymphocytes and expanded these in vitro using rhIL-2 and PHA as described in "Materials and Methods." Using this approach, we obtained CD3+ populations of > 90% purity (after 8-10 days) that were cytogenetically Ph in all 28 cases
analyzed. These T lymphocytes were analyzed by flow-FISH when
> 106 cells had been generated in culture. Examples of
flow-FISH analysis of paired unseparated (mostly myeloid) cells and
expanded normal T cells from the peripheral blood or bone marrow of
representative CML patients examined at diagnosis (in CP), several
years after diagnosis but still in CP, and in BP are shown in Figure
3. As indicated in Table 1, telomere
fluorescence in CD3+ cells (15.0 ± 0.4 kMESF,
n = 51 from 42 patients) was significantly (P < .001)
higher than in total leukocytes from the same individual patients
(10.3 ± 0.4 kMESF). No significant difference in telomere length
between lymphocytes and granulocytes from the blood of age-matched
healthy controls was observed in this particular age range (data not
shown).
Telomere fluorescence of CML cells from patients at different stages of disease We next investigated whether any significant changes in telomere length of CML cells could be correlated with the clinical stage of the disease at the time the sample was obtained. Telomere fluorescence in cells obtained from patients in AP/BP was significantly lower than in cells from patients in CP (P = .02) and also significantly lower than in cells from patients in CR (P = .03). Compared to samples obtained in AP (8.6 ± 0.8 kMESF, n = 8), telomere fluorescence tended to be higher in samples obtained in BP (10.5 ± 1.1 kMESF, n = 7); however, this difference did not reach statistical significance.
Correlation between telomere shortening and predominance of
Ph+ LTC-IC
Telomere length differences indicate enhanced stem cell turnover of Ph+ HSC in patients with CML According to prevalent models of hematopoiesis, mature peripheral blood cells are derived from rapidly dividing progenitor cells that, in turn, are derived from HSCs. In adults, the number of cell divisions that separate the bulk of the mature blood cells from the HSC population is assumed to be roughly constant unless the system is perturbed. Thus, considering the relatively short half-life of mature cells, the average length of their telomeres should reflect that of the HSC a limited number of cell generations earlier.52 The present investigations of telomere length in leukocytes from a large series of patients with CML have shown that in CP CML cells, telomere length was approximately 1 kb shorter than in age-matched controls. Assuming that roughly 100 bp (50-200 bp) are lost per cell division in somatic cells,28,39 the reduced telomere length in CP CML cells would indicate that their leukemic stem cells would have undergone on average 10 more divisions than normal HSC during the development of the clone before diagnosis. With disease progression telomere length progressively shortens leading to a significantly increased difference of 2 kb between cells from samples acquired in AP or BP compared to age-adjusted controls. Further evidence of an increased rate of turnover of the Ph+ stem cell population is provided by the finding that the mature Ph+ cells showed significantly shorter telomeres than ex vivo expanded Ph T lymphocytes. In a previous study, we showed
that the average telomere length in (naive) T lymphocytes and
granulocytes of healthy individuals of approximately the same age range
(16-80 years) is similar.52
Telomere length in CML cells correlates with disease evolution A major purpose of this study was to investigate whether telomere length measurements of the neoplastic cells from patients with CML using the recently developed flow-FISH method might have prognostic importance. Three different approaches to address this question were used in this study. First, we compared the telomere length of cells taken from patients at different stages in their disease. Average telomere fluorescence was highest in patients in Ph
CR and in CP followed by significantly lower telomere fluorescence in
AP/BP samples. This finding is in line with recent data showing telomere shortening in all 12 patients who were serially studied in CP
and BP48 as well as in another cross-sectional study
comparing telomere length in CP versus BP,49 both of which
used Southern blot analysis. Secondly, we compared the telomere length
in CP samples with a remaining duration of CP of less than 2 years with those in samples from patients with a remaining duration of CP of more
than 2 years. A significant difference between the 2 groups was found
(P < .05). Thirdly, we compared telomere length in samples from patients grouped according to the prevalence of leukemic or normal
elements in their LTC-IC population. At the time of diagnosis most CML
patients have predominantly normal LTC-IC.8,13,62 However,
in a proportion of CP patients, Ph+ LTC-IC predominate.
This analysis showed that the samples with predominantly
Ph+ LTC-IC tended to have shorter telomeres than
samples with Ph LTC-IC. However, the sample size
in this particular subanalysis was too small to draw
definitive conclusions. In summary, these findings support the
hypothesis that progressive telomere shortening is correlated with
disease progression in CPCML.
Model of telomere biology in CML Based on the data reported so far by other groups and the results presented here, we propose the following model of telomere biology in CML (see also Figure 5). An increased rate of turnover in the leukemic HSC clone will result in a shorter telomere length of Ph+ compared to polyclonal Ph
stem cells at diagnosis. Similar to normal HSCs, the level of telomerase expression in Ph+ stem cells is apparently
unable to prevent replication-dependent telomere shortening at this
stage of the disease. During CP and with continued proliferation of the
Ph+ stem cells, the differences in telomere length as
compared to normal cells are expected to become more pronounced.
Eventually, the telomeres in the cells of the neoplastic clone become
critically short and this may precipitate progression of the disease to
AP/BP. Genetic instability is suspected to be of major importance in the progression of many tumors,63 and the acquisition of
additional cytogenetic alterations is a hallmark of AP/BP CML.
Mechanisms underlying genetic instability, like gene amplification,
aneuploidy, and loss of heterozygosity, may directly result from
telomeric associations arising from cells with short
telomeres,29,64,65 thus linking telomere shortening with
genetic instability.33 One mechanism by which late CP CML
cells might escape replicative senescence is through activation or
up-regulation of telomerase. Because telomerase is expressed at low
levels in normal hematopoietic cells,42 a separate genetic
event may not be required for selection of cells expressing high
levels. Telomerase-positive subclones might either re-elongate their
telomeres or continue to grow without a net lengthening of their
telomeres.66 The selective growth advantage of
Ph+ cells with increased levels of telomerase following
selection of cells mediated by telomere-mediated genetic instability
would thus be expected to result in CML BP.
Gloria Shaw and Elizabeth Chavez are thanked for
cytogenetic analysis, Edwin Mak for help with the statistical
analysis of the data, Jennifer Mak and Pamela Austin for technical
assistance, Karen Lambie for help with retrieval of patient
samples, Daphne Brockington for collection of patient information, and
Colleen MacKinnon for editorial work on the
manuscript.
Submitted August 9, 1999; accepted November 23, 1999.
Supported by grants AI29524 and GM56162 from the National Institutes of Health and by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run. T.H.B. is funded by a grant from the Deutsche Forschungsgemeinschaft. T.L.H. holds a Senior Lecturership from the United Kingdom Leukemia Research Foundation. N.R. is a recipient of a fellowship from the Fonds National Suisse. C.J.E. is a Terry Fox Cancer Research Scientist of the NCIC.
Reprints: Peter M. Lansdorp, Terry Fox Laboratory, BC Cancer Agency, 601 West 10th Ave, Vancouver, British Columbia, V5Z 1L3, Canada; e-mail: plansdor{at}bccancer.bc.ca.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Abstract
Introduction
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