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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-07-2053.
NEOPLASIA
From the Department of Hematology, University of
Leipzig, Germany; Interdisciplinary Center for Clinical
Research, University of Leipzig, Germany; Department of
Haematology, Western General Hospital, Edinburgh, United
Kingdom; Department of Pathology, University of Leipzig,
Germany; and BMT/Leukemia Center, Oregon Health and
Science University (OHSU), Portland, OR.
Chronic myelogenous leukemia (CML) is characterized by the presence
of a Bcr-Abl fusion protein with deregulated
tyrosine kinase activity that is required for maintaining the malignant phenotype. Imatinib, a selective inhibitor of
Bcr-Abl, induces major cytogenetic remission (MCR) or
complete cytogenetic remission (CCR) in the majority of patients with
CML in first chronic phase. However, thorough re-evaluation of
cytogenetics in a cohort of patients in MCR or CCR demonstrated clonal
karyotypic abnormalities in more than 10% of cases, some of which were
clinically associated with a myelodysplastic syndrome (MDS). Further
analysis identified previous exposure to cytarabine and idarubicin as
significant risk factors for the subsequent occurrence of abnormalities
in Philadelphia chromosome-negative (Ph Almost all patients with chronic myeloid
leukemia (CML) carry a t(9;22)(q34;q11) reciprocal translocation that
results in the formation of a BCR-ABL fusion
gene.1 The chimeric Bcr-Abl protein
derived from the fusion has deregulated protein tyrosine kinase (PTK)
activity, which is sine qua non for cellular
transformation.2 Current thinking holds that the initial
BCR-ABL translocation occurs in a hematopoietic stem cell
that, by virtue of the Bcr-Abl protein, acquires a
proliferative advantage over normal hematopoiesis. There is evidence
that BCR-ABL is both necessary and sufficient to induce CML.
Most notably, lethally irradiated mice receiving transplants of
syngeneic bone marrow infected with a BCR-ABL retrovirus develop a CML-like myeloproliferative syndrome (MDS).3
The fact that Philadelphia chromosome-negative (Ph Treatment with imatinib (formerly STI571) induces major cytogenetic
remissions (MCRs) or complete cytogenetic remissions (CCRs) in more
than 40% of patients resistant or intolerant to
IFN- Patients
Cell separation
Clonality analysis of HUMARA-PCR For DNA extraction the cell pellet was lysed in 40 µL lysis buffer (500 mM Tris [tris(hydroxymethyl)aminomethane]-HCl,10 mM NaCl, 20 mM EDTA [ethylenediaminetetraacetic acid],1% sodium dodecyl sulfate [SDS], pH 8.9). Next, 40 µL phosphate-buffered saline (PBS) and 4 µL proteinase K (Qiagen, Hilden, Germany) were added. The samples were incubated at 56°C for 4 to 6 hours, then proteinase K was inactivated by 10 minutes of incubation at 95°C. The debris was pelleted by 10 minutes centrifugation at 13 000 rpm and the supernatant transferred to a fresh Eppendorf tube. After adding 135 µL isopropanol, the DNA was precipitated by 10 minutes centrifugation at 15 000 rpm, washed once with 70% ethanol, and then dissolved in 50 µL deionized water.DNA (12 µL) was digested with 10 U HpaII (Gibco, Karlsruhe, Germany) overnight. A control reaction was set up under the same conditions but without restriction enzyme. The human androgen receptor (HUMARA) gene was amplified from 5 µL of each sample in a 25-µL reaction containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 200 nM each primer, and 0.2 µL Taq polymerase (Platinum, Gibco). Primers were 5'-CTCTACGATGGGCTTGGGGAGAAC-3' and 6-carboxy-flourescein-labeled 5'-TCCAGAATCTGTTCCAGAGCGTGC-3', respectively. Amplification conditions were 5 minutes at 95°C, 30 seconds at 85°C followed by 27 to 30 cycles of denaturation at 95°C (1 minute), annealing at 68°C (30 seconds), and extension at 72°C. Then, 7 µL polymerase chain reaction (PCR) product was analyzed on an ABI Prism 377 (Applied Biosystems, Foster City, CA). "Blanks" were included at the levels of DNA extraction and PCR to monitor for PCR contamination. X-chromosome inactivation was quantified as described by Delabesse et al.14 Briefly, the peak areas for both alleles were determined. The relative corrected index (RCI) was then calculated for CD3+ and CD33+ cells as the ratio of the 2 undigested alleles divided by the ratio of the 2 HpaII-digested alleles. Nonrandom X-chromosome inactivation in a given population was assumed if the RCI was more than 3:1 or less than 1:3.15 To control for constitutional skewing,16 the ratio RCICD33/RCICD3 was calculated. A ratio more than 2 or less than 0.5 was considered as evidence of clonality.17 For statistical analysis, all values less than 1 were inverted. To assess the sensitivity of the method to detect a clonal population
in a background of polyclonal cells, (polyclonal) CD3+
cells and (monoclonal) CD33+ cells from a CML patient with
100% Ph+ metaphases were mixed at graded concentrations,
and the cell mix subjected to HUMARA-PCR (Figure
1). An RCI more than 3, indicative of
monoclonality, was obtained if the mix contained between 30% and 40%
of CD33+ cells. Thus, monoclonal subpopulations that are
smaller than approximately 30% are not reliably detectable.
FISH and classical cytogenetics Interphase and metaphase preparations were done according to standard protocols. Probes used were LSI ETO/AML1 (for detection of t(8;21)(q22;q22)), LSI BCR/ABL ES (for detection of t(9;22)(q34;q11)), LSI21 (locus-specific probe corresponding to 21q22.13-21q22.2), CEP 8 (centromer probe for chromosome 8), CEPX/Y (centromer probe system for X and Y chromosomes), WCP 3 (whole chromosome paint for chromosome 3), all from Vysis (Downers Grove, IL). In addition, whole chromosome paints for chromosomes 20 and 21 were purchased from Metasystems (Altlussheim, Germany). Hybridization was done with the Hybrite semiautomated system (Vysis) and generally followed the recommendations of the manufacturer. In 2 selected cases, cells sorted by fluorescence-activated cell sorting (FACS) were analyzed by fluorescence in situ hybridization (FISH). R-banding technique was used for classical cytogenetics.Statistical analysis All statistics was done with the SPSS software package (SPSS, Chicago, IL). Comparison of noncategorical variables was done by 2-tailed Mann-Whitney U test. Categorical variables were compared by 2-sided Fisher exact test.
HUMARA-PCR Fourteen females with CML in CCR (nos. 1-9, 10-R, 11-14), 7 females in MCR (10%-35% Ph+ metaphases, nos. 15-21), 6 females with more than 90% Ph+ metaphases (including 1 patient with Ph CML, nos.10-I, 22-26; Table 1) and 10 healthy volunteers (Table 2) were
informative for clonality analysis by HUMARA-PCR (37 of 40 tested = 92.5%). One patient was studied prior (10-I) to imatinib
and after induction of CCR (10-R). Unbalanced X inactivation in
CD33+ cells as defined by an RCI more than 3:1 or less than
1:3 was found in 2 of 14 CML patients (14.3%) in CCR and 1 of
7 patients in MCR (14.3%), in 4 of 6 patients (66.7%) with CML in
NR, and in 1 of 10 healthy volunteers (10%). RCI values were
not significantly different between healthy individuals and patients in
CCR (P = .538) and MCR (P = .435). In
contrast, there was a significant difference between healthy volunteers
and patients in NR (P = .009), between patients in CCR and
NR (P = .017), and between patients in MCR and NR
(P =.045). Applying the criterion of
RCICD33/RCICD3 ratio less than 0.5 or more than
2, evidence of clonality was found in 1 of 14 patients in CCR (6.7%),
2 of 7 (28.6%) in MCR, 5 of 6 in NR (83.3%), and 1 of 10 (10%) of
healthy volunteers. The differences were significant for CCR and MCR
versus NR (P = .002 and P = .015,
respectively) and healthy individuals versus NR
(P = .003), but not for CCR and MCR versus healthy
individuals (P = .747 and P = .204,
respectively) and CCR versus MCR (P = .279). In one
patient, analysis of samples prior to imatinib (no. 10-I) and at the
time of CCR (no. 10-R) was possible and demonstrated normalization of
the RCI of the CD33+ cells (Figure
2). Of note, at the time of the analysis,
there was cytogenetic evidence of clonal evolution in Ph
metaphases in patients nos. 15, 19, and 33, and histologic signs of a
myeloproliferative disease in patient no. 1 (see "Cytogenetics and
FISH"). In patient 33, HUMARA-PCR was performed on PB 6 months after the cytogenetic analysis and demonstrated clonality of the CD33+ cells (Figure 3). At
the time this sample was obtained, BCR-ABL was
undetectable by FISH, but 78% of WBCs were positive for
t(20;21).
Cytogenetics and FISH Cytogenetic preparations were re-evaluated in 28 women (Table 1; patients nos. 1-21, 27-33) and 20 men (Table 3) in CCR or MCR induced by imatinib. In 8 patients (nos. 15, 19, 33, 34, 47, 51, 52, 53), nonrandom chromosomal abnormalities were detected (Table 4).
The karyotypic abnormalities included one case each of trisomy 8, monosomy 7, t(3;21)(q27?;q22) (Figure 4),
del(20q), ider20(q10)t(20;21)(q22;q22), one multiaberrant karyotype
including del(20q) and t(7;17)(q11;q21), and 2 cases with loss of the Y
chromosome. In all patients, archive material was analyzed to see if
the abnormalities were detectable at any time prior to imatinib
treatment. Patients no. 19 and no. 34 had achieved cytogenetic
remission with IFN-
In patients no. 47 and no. 53, FISH detected 2.2% and 3% interphases
with a signal constellation typical of del(20) prior to
iamtinib therapy; however, both values are lower than the laboratory cutoff (3.7%). In 3 patients (nos. 19, 34, and 53), the abnormalities developed after a period of CCR or MCR, without evidence of abnormal Ph
Patients with and without abnormalities in Ph
The chromosomal abnormalities in Ph Of note, profound hematologic and morphologic abnormalities were also
detected in one woman (patient no. 1) with a normal karyotype. This
patient had achieved MCR on IFN-
We show that therapy with imatinib restores polyclonal
hematopoiesis in the majority of patients who achieve CCR. This is indicated by the fact that there is no difference between the RCI
values of CD33+ cells from patients in CCR and healthy
women. Applying the commonly used criterion of RCI less than 0.33 or
more than 3 to diagnose clonality, 18 of 21 patients in CCR or MCR and
9 of 10 healthy women had polyclonal CD33+ cells. In
contrast, 4 of 6 patients without cytogenetic response had a clonal
pattern in their CD33+ cells. Correcting for constitutional
skewing led to similar results. In the one patient, where material was
available, a monoclonal pattern was demonstrable prior to imatinib
therapy and a polyclonal pattern at the time of CCR. Three
considerations should, however, be kept in mind. First, because the
analysis of clonality based on X-chromosome inactivation cannot
distinguish between a truly monoclonal and a polyclonal population with
extreme lyonization,15 it is not capable of diagnosing
clonality with absolute certainty. Second, clearly monoclonal
populations may occasionally show a polyclonal pattern, as demonstrated
by patient no. 24. It has been suggested that in such cases the
malignant transformation leads to unstable X-chromosome
methylation.19 Third, as shown in our dilution experiment
as well as in the patients in MCR, the sensitivity of HUMARA-PCR to
detect a clonal subpopulation in a polyclonal background is limited to
30% to 40%. This is likely to explain our failure to detect clonal
hematopoiesis in most patients in MCR, and patients nos. 15 and 19, where the percentage of interphases carrying the respective
abnormalities is below 30%. However, the example of patient no. 33 (Figure 3) clearly shows that clonality can be diagnosed by HUMARA-PCR
if the majority of the cells carry the cytogenetic abnormality. Thus,
within these limitations, our data provide compelling evidence that,
similar to complete cytogenetic responders to IFN- Surprisingly, we found cytogenetic abnormalities in Ph Perhaps the most intriguing question refers to the time when the
abnormal Ph The second explanation is that a patient's hematopoiesis as a whole
has been subjected to genomic damage, producing multiple abnormal
clones, among them one with a t(9;22). This clonal diversity would not
become apparent as long as the Ph+ clone is at a
proliferative advantage. Application of selective pressure by imatinib
would then shift the balance, allowing Ph The third possibility is that imatinib treatment itself induced or
favored the acquisition of the additional abnormalities. Abl
is known to interact with a number of proteins involved in the response
to DNA damage and in DNA repair such as
p73,31,32 DNA-PK,33
Atm,34,35 and
Rad51.36,37 Thus, its permanent inhibition by
imatinib could contribute to the accumulation of new genetic damage.
This may be particularly relevant in a situation where the
hematopoiesis must be restored from a limited pool of Ph Two considerations are important regarding the frequency of
abnormalities in Ph What are the implications of these findings for the practical
management of patients? One immediate conclusion is that patients on
imatinib should be monitored by classical cytogenetics, even after
achieving CCR. The second point is that the use of cytotoxic agents
other than HU may have to be balanced against an increased risk of
inducing abnormalities in Ph Given the potential clinical implications, a systematic data collection
is required. There are plans to establish an international registry at
Oregon Health and Science University with the aim of collecting data on
patients who develop chromosomal abnormalities in
Ph
While this manuscript was in review, Andersen et al reported data on 3 patients with abnormalities in Ph
We thank Ms Gerlinde Patzer for technical assistance.
Submitted July 17, 2002; accepted October 15, 2002.
Prepublished online as Blood First Edition Paper, October 31, 2002; DOI 10.1182/blood-2002-07-2053.
Supported by a grant from IZKF Leipzig, BMB-F, Interdisciplinary Center for Clinical Research at the University of Leipzig 9504 (K.K.) (01KS 9504, project Z3) and (M.D.) (01KS 9504, project D5).
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.
Reprints: Michael Deininger, OHSU, BMT/Leukemia Center, 3181 SW Sam Jackson Park Rd, Portland, OR 97239; e-mail: deininge{at}ohsu.edu.
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cells in some CML patients in cytogenetic remission to imatinib but
restoration of polyclonal hematopoiesis in the majority
Top
Abstract
Introduction
(IFN-
70°C until used.
