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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 358-361
BRIEF REPORT
Telomere length shortening in chronic myelogenous leukemia is
associated with reduced time to accelerated phase
Jackie Boultwood,
Andrew Peniket,
Fiona Watkins,
Patricia Shepherd,
Paul McGale,
Susan Richards,
Carrie Fidler,
Timothy J. Littlewood, and
James S. Wainscoat
From the Leukaemia Research Fund Molecular Haematology Unit,
Department of Cellular Science, John Radcliffe Hospital, Oxford;
MRC CML Trials Unit, Western General Hospital, Edinburgh; CTSU,
Radcliffe Infirmary, Oxford, UK.
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Abstract |
Telomere shortening is associated with disease evolution in chronic
myelogenous leukemia (CML). We have examined the relationship between
diagnostic telomere length and outcome in 59 patients with CML who
entered into the MRC CMLIII Trial by Southern blot hybridization using
the (TTAGGG)4 probe. Age-adjusted telomere repeat array
(TRA) reduction was found to significantly correlate with time from
diagnosis to acceleration, such that patients with a larger TRA
reduction entered the accelerated phase more rapidly (r =  0.50; P = .008). Cox-regression
analysis for this group was suggestive of a relationship between a
greater TRA-reduction and a shorter time to acceleration
(P = .054). Age-adjusted TRA reduction did not
significantly affect either the time to blast crisis or overall
survival. Our results show that telomere shortening observed at the
time of diagnosis in CML significantly influences the time to progress
to the accelerated phase. The measurement of diagnostic TRA may prove
to be clinically important in the selection of patients at high risk of
disease transformation in CML.
(Blood. 2000;96:358-361)
© 2000 by The American Society of Hematology.
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Introduction |
Telomeres are the termini of eukaryotic chromosomes and
are composed of TTAGGG repeats.1 A reduction in telomere
length has been described in a wide range of human cancers, including both solid tumors and leukemias.2,3 Indeed, progressive
telomere shortening has been shown to contribute to genomic
instability.4 Most late-stage human tumors express
telomerase and this may contribute to the relative immortality of these
cells.5,6 Reduced telomere length has been shown to be
associated with the evolution of certain hematologic malignancies,
including chronic myelogenous leukemia (CML).2,7-13 We have
shown that telomere length in the accelerated and/or blast phase was
reduced compared with serial samples taken at the chronic phase in
patients with CML.11 Similarly, Ohyashiki et
al8 have also shown that telomere length was significantly shortened in the blast phase compared with the chronic phase in CML. We
have previously shown that leukocytes from patients with CML have
shorter telomere length values than healthy controls and that patients
display heterogeneity of telomere length in the chronic
phase,11 which may reflect the relative variation in
time-to-disease progression in CML. These data lead to the hypothesis
that the mean telomere repeat array (TRA) at the time of diagnosis may
be of prognostic importance.
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Materials and methods |
Patients and controls
Diagnostic DNA samples were available from 59 patients with CML who
had entered the UK Medical Research Council CMLIII Trial between 1989 and 1994.14 Patients in this study were randomized to
receive either interferon-alfa or chemotherapy without interferon-alfa for maintenance. The protocol and results of this study have been previously reported.14 All patients were positive for the
Philadelphia chromosome or showed molecular evidence of the BCR-ABL
rearrangement.15 Patients were defined as being in the
accelerated phase according to criteria outlined in Table
1. Blast crisis was defined as more than 20% blasts in the peripheral blood or more than 30% blasts
in the bone marrow. Patients not fulfilling these criteria were
classified as being in the chronic phase.15 Peripheral blood samples were collected from 75 healthy individuals aged between
20 and 60 years.
Mean telomere repeat array measurement
High-molecular weight DNA was obtained using standard methods from
diagnostic peripheral blood leukocyte samples taken from the patients
with CML and from the peripheral blood leukocyte samples taken from
healthy individuals.16 Total white blood counts at
diagnosis were very high (median 210 × 109/L
[range 50-780]). This means that the proportion of DNA derived from
nonclonal (Philadelphia negative) lymphoid cells was very minor and
insufficient to alter the overall Southern blot results. Ten micrograms
of DNA digested with the restriction enzyme HinfI was size
fractionated by electrophoresis through 0.8% agarose gels. The DNA was
transferred to Hybond N (Amersham Int, Amersham, UK), according to
standard procedures for Southern blotting.16 The filters
were hybridized to a 3'-32p-labeled (TTAGGG)4
telomeric probe as previously described.11,17 Telomere
length was measured with an LKB Ultrascan XL densitometer (LKB-Bromma,
CO), with the peak of telomere length in kilobases (peak
TRA) taken as the average telomere length in each patient.7
The TRA was performed with each DNA sample on 2 separate occasions and
the mean value determined.
Analysis of TRA influence on patient outcome
The relationship between the diagnostic age-adjusted TRA loss and
patient outcome were examined. The 3 outcome measures studied were time
to (1) accelerated phase, (2) blast crisis, and (3) death. Linear
regression and Cox-regression analyses, assuming proportional hazards,
were carried out in STATAv6.0 (Stata Corp, College Station, TX).
Univariate, multivariate, and backward stepwise Cox regression analysis
was used to investigate the affect of age, sex, white blood count,
percentage blast count in peripheral blood, platelet count, spleen
size, treatment at trial randomization, and age-adjusted TRA loss on
survival. Variable addition was set at P = .25 and removal at
P = .3.
| |
Results and discussion |
To test the hypothesis that telomere length is a significant
prognostic factor in CML, we have analyzed samples taken from 59 patients who entered into the MRC CML III Trial. As a result of the
therapeutic randomization, 34 patients received interferon-alfa and 24 patients received cytotoxic therapy (hydroxyurea or busulphan). One
patient died before the therapy could be started. The average age at
entry was 48 years (SD 15). At the time of analysis (August 1999),
there had been 51 deaths, with a median follow-up of 3.72 years.
To allow for age-associated telomere shortening, regression analysis
was performed on the observed TRA values for a control group consisting
of 75 healthy individuals aged between 20 and 60 years. This regression
analysis confirmed TRA shortening with increasing age such that
T = 10.669-0.0316 × A (T = TRA in kilobases and A = age
in years) (Figure 1A). Age-related telomere
shortening was hence equivalent to 31.6 base pairs (bp) per year,
similar to that described by other groups.12,18,19
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| Fig 1.
Relationships between mean TRA and age and between TRA
loss and time to acceleration.
(A) Relationship between mean TRA and age. Controls: solid diamonds.
Patients: unfilled squares. (B) Relationship between age-adjusted TRA
loss and time to acceleration.
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The mean TRA at the time of diagnosis for patients with CML was 6.0 kb
(SD = 2.0 kb), which was significantly lower than that for the
control group (mean TRA = 9.7 kb; SD = 1.8 kb)
(P < .001) (Figure 1A). To determine the prognostic
significance of this TRA reduction, the observed TRA loss was adjusted
for age: The age-adjusted TRA reduction was expressed as the difference
between the observed TRA and the regression estimate for TRA for age in healthy individuals: TE-O = Expected TRA Observed
TRA. TE-O represents the difference in kilobases between
the measured TRA and the age-appropriate point on the normal regression
line (Figure 1A).
The age-adjusted TRA (TE-O) was found to correlate with the
time from diagnosis to acceleration (r = 0.502;
P = .008) (Figure 1B). Cox-regression analysis, according to
tertile of TRA reduction, showed a relationship with time
to acceleration, although this just failed to attain statistical
significance; HR3v1 = 4.43 (1.28-15.3); probability of
TE-O effect = 0.054. A date of acceleration was available
for only 22 patients, reflecting the clinical difficulty in defining
the onset of this phase of CML in the remaining patients. The criteria defining these 22 patients as having entered the accelerated phase are
shown in Table 1. The most common criteria are the development of
resistance to therapy (17 patients), increasing spleen size (15 patients), major cytogenetic evolution (7 patients), blast percentage
more than 10% (6 patients), and platelet count more than
1000 × 109/L (5 patients).
We also examined the influence of TE-O on the time to blast
crisis. There was no correlation between TE-O and time to
blast crisis (r = 0.30; P = .18; n = 21)
and regression analysis, according to tertile, failed to show
significant relationship (HR2v1 = 1.22 [0.42-3.55];
HR3v1 = 1.97 [0.64-5.99]; probability of a trend
effect = 0.49).
All diagnostic characteristics, including TRA, were investigated to
identify important prognostic factors influencing survival. In a full
multivariate model, percentage blast count (HR = 2.40, CI 1.13-5.09, P = .02) and white blood count (HR = 0.96; CI 0.93-0.99; P = .03) were significant. Blast count was also significant
in a univariate model. The stepwise multivariate model did not suggest any strong prognostic factors, TRA and TE-O included. The
lack of influence of diagnostic TRA on outcome was confirmed via
tertiles of TE-O.
Eighteen patients within this patient cohort at some point underwent
allogeneic bone marrow transplantation. These patients were excluded
from the same analyses in case the allogeneic procedure had a
confounding impact on survival. A repeat analysis on the 41 remaining
patients again failed to show an impact of TE-O on survival.
The finding that patients with greatly reduced telomere length at the
time of diagnosis enter the accelerated phase relatively early suggests
the existence of a high-risk subgroup of patients with CML who lack an
efficient mechanism of telomere maintenance early on, during disease
onset. In general, telomerase levels have been shown to be normal or
only slightly raised in the chronic phase, but significantly increase
in the blast crisis.8 It would be interesting to measure
telomerase levels in patients with a large telomere length reduction at
diagnosis, as this subgroup might be expected to be just beginning to
show higher levels of telomerase activation than most patients in the
chronic phase with average or long telomeres.
Our observations support the findings of Iwama et al,19 who
have shown that in a group of patients treated with interferon-alfa, those with normal TRA values in the chronic phase had a significantly lower frequency of blast crisis, a significantly higher incidence of
cytogenetic responses, and a favorable prognosis compared with those
with shortened TRA values. Most recently, Brummendorf et al20 have shown that patients with CML, in whom the blast
phase subsequently developed within 2 years, had significantly shorter telomeres than those in whom the blast phase did not develop for at
least 2 years.
The lack of impact of TRA loss on time to blast crisis and overall
survival shown in this study is somewhat surprising. It is possible
that, up until the point of acceleration, the patients were treated in
a fairly uniform manner according to the trial protocol, either
receiving interferon-alfa or chemotherapy (with hydroxyurea or
busulphan). Once the patients entered the accelerated phase, then
therapy varied considerably, and it is possible this may have altered
subsequent survival. It may be that, by increasing the number of
patients included in the study, a prognostic impact of TRA loss could
be detected. Alternatively it may be that progressive telomere
shortening throughout the chronic phase eventually terminates when a
critical threshold is reached and the accelerated phase commences.
After this point, factors independent of TRA may determine outcome.
This situation is analogous to that in SV40-transfected fibroblasts:
Progressive telomere shortening occurs until a critical threshold,
after which telomerase levels rise within some cells that become
immortalized. The threshold telomere length in these fibroblasts is
associated with chromosomal instability.4
In conclusion, diagnostic TRA loss is significantly related to the time
to onset of the accelerated phase. The measurement of diagnostic TRA
may therefore eventually prove to be of clinical importance in the
selection of patients at high risk of disease transformation.
| |
Acknowledgments |
Many thanks to Sarah Cullip at the CTSU, Oxford, for assistance in
providing patient data.
| |
Footnotes |
Submitted December 28, 1999; accepted February 29, 2000.
Supported by the Leukaemia Research Fund of the United Kingdom. The
CMLIII Trial was supported by the UK Medical Research Council.
Reprints: J Boultwood, Leukaemia Research Fund Molecular
Haematology Unit, Department of Cellular Science, John Radcliffe Hospital, Oxford, 0X3 9DU; e-mail: jboultwo{at}worf.molbiol.ox.ac.uk.
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