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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2315-2321
A Special Fluorescent In Situ Hybridization Technique to Study
Peripheral Blood and Assess the Effectiveness of Interferon Therapy in
Chronic Myeloid Leukemia
By
Ismael Buño,
William A. Wyatt,
Alan R. Zinsmeister,
Jeanne Dietz-Band,
Richard T. Silver, and
Gordon W. Dewald
From the Division of Laboratory Genetics and Section of
Biostatistics, Mayo Clinic and Mayo Foundation, Rochester, MN; Oncor,
Inc, Gaithersburg, MD; and the Chronic Myeloid Leukemia National Study
Group, Co-ordinating Center, New York Hospital-Cornell Medical Center,
New York, NY.
 |
ABSTRACT |
Using a highly sensitive fluorescence in situ hybridization method
with probes for BCR and ABL1 (D-FISH), we studied 37 paired sets of
bone marrow and blood specimens, collected within 24 to 96 hours of
each other, from 10 patients before and during treatment for chronic
myeloid leukemia (CML). The normal range for 500 interphase nuclei was
 4 (0.8%) nuclei based on 10 bone marrow and 10 blood specimens
from normal individuals. The percentage of neoplastic nuclei was
usually lower in blood than bone marrow. However, changes in the
percentage of neoplastic nuclei in blood and bone marrow tracked
closely over the course of therapy and with the results of quantitative
cytogenetic studies on bone marrow. This result indicates that D-FISH
is useful to test blood from patients with CML to monitor therapy.
Moreover, by analysis of 6,000 nuclei with D-FISH, residual disease was
identified in bone marrow and blood for patients in complete
cytogenetic remission. Consequently, D-FISH analyses of interphase
nuclei from blood could substitute for Q-cytogenetic studies on bone
marrow. Thus, it may not be necessary to collect bone marrow samples so
frequently to monitor therapy in CML.
| |
INTRODUCTION |
CONVENTIONAL CYTOGENETIC studies are used
in clinical practice to monitor the effectiveness of various forms of
treatment for patients with chronic myeloid leukemia (CML), especially
interferon therapy. Considerable evidence exists to show a strong
correlation between changes in percentage of Ph-positive metaphases
after interferon therapy and prognosis.1-3 The best outcome
for survival and prolonged chronic phase seems to be enjoyed by
patients with CML in whom the percentage of Ph-positive metaphases is
reduced to less than 33%.2-5
In clinical practice, physicians usually collect bone marrow aspirates
from patients with CML on interferon therapy at 3- to 6-month intervals
to obtain cytogenetic data. For technical reasons, such as a packed
bone marrow or hypoplasia, it is not always possible to obtain suitable
bone marrow specimens for chromosome studies. Moreover, undergoing bone
marrow aspiration or biopsy is painful and costly. Although peripheral
blood is easier to collect from patients, in our experience, the number
of mitotic cells in blood after treatment is usually inadequate to
accurately quantify disease.
The advent of fluorescence in situ hybridization (FISH) to detect
BCR/ABL fusion in interphase nuclei for patients with CML has become
important to quantify disease.6-8 Until recently, the most
common FISH procedure for CML used different colored probes for BCR and
ABL and detected a single BCR/ABL fusion signal in cells with a Ph
chromosome. For purposes of this paper, we refer to this method as
S-FISH to imply a single BCR/ABL fusion signal. Some investigators have
used S-FISH to show the presence of nuclei with BCR/ABL fusion in
blood, but few data are available to show the efficacy of FISH to study
peripheral blood to monitor therapy in CML.6-12 S-FISH
lacks sensitivity to detect low levels of minimal residual disease and
also lacks precision to quantify disease accurately before and after
treatment.8,13
New FISH strategies are now available that are highly sensitive to
detect BCR/ABL fusion in interphase nuclei.14,15 Recently we investigated the use of one of these new methods called D-FISH to
study bone marrow.15 D-FISH detects double or two BCR/ABL fusion signals in cells with a t(9;22)(q34;q11.2) in most patients with
CML and the false positive and false negative frequency of D-FISH
approaches zero. D-FISH accurately quantifies disease in bone marrow
from patients with CML within a few percentage points at diagnosis and
at all times after treatment including cytogenetic remission. In
addition, D-FISH identifies all known variants of the Ph chromosome
translocation and the percentage of abnormal interphase nuclei
correlates closely with quantitative cytogenetic studies
(Q-cytogenetics) for bone marrow.
The present investigation was designed to test the usefulness of D-FISH
to study peripheral blood for purposes of monitoring the effectiveness
of interferon  -2b therapy for patients with CML. To do this, paired
sets of blood and bone marrow specimens were collected from a series of
patients enrolled in the CML National Study Group who were undergoing
therapy with either interferon -2b alone or interferon -2b and
cytosine arabinoside (ara-C). The results of this investigation show
that changes in the percentage of neoplastic nuclei in blood over the
course of therapy were a good predictor of corresponding changes in
bone marrow. In a previous study, the percentage of interphase nuclei
with BCR/ABL fusion was strongly correlated with Ph-positive metaphases
by Q-cytogenetics.15 D-FISH was also useful to identify
residual disease in both bone marrow and peripheral blood specimens for patients in complete cytogenetic remission.
| |
MATERIALS AND METHODS |
This investigation used D-FISH to study 37 paired sets of bone marrow
and peripheral blood specimens from 10 patients undergoing treatment
for CML, 10 normal peripheral blood specimens, 10 normal bone marrow
specimens, and 4 serial dilutions with known percentages of Ph-positive
nuclei.
Each patient with CML was a participant in the CML National Study Group
clinical trial and was randomly receiving treatment with interferon
-2b with or without ara-C. Each patient was known to have cells with
a Ph chromosome that produced a typical D-FISH pattern for
t(9;22)(q34;q11.2).15 For each patient a paired set of bone
marrow and peripheral blood specimens was collected before treatment
and at two or more times at approximately 4-month intervals during
treatment. Each paired set of peripheral blood and bone marrow
specimens was obtained on the same day except for specimens collected
before treatment in patients 3 (blood and bone marrow were collected 1 day apart), 5, and 8 (blood and bone marrow were collected 4 days
apart).
Uncultured bone marrow and peripheral blood specimens were processed by
conventional procedures for cytogenetic and FISH studies. These
specimens were stored as fixed pellets at  70°C in
methanol:acetic acid (3:1) until FISH studies could be performed. To
prepare specimens for D-FISH, specimens were washed twice with fresh
fixative and cells were placed on microscope slides and allowed to
air-dry in a CDS-5 cytogenetic drying chamber (Thermotron, Holland, MI) adjusted to 50% relative humidity and 25°C.16 Slides
were further dried for 1 hour in a 65°C oven and then treated with
2× standard saline citrate solution (SSC; 300 mmol/L sodium
chloride, 30 mmol/L sodium citrate) for 1 hour at 37°C. Slides were
then dehydrated with a series of 70% to 85% to 100% ethanol at
20°C for 2 minutes each, and air-dried.
Q-cytogenetic studies were performed on each bone marrow specimen by
analyzing 25 consecutive G-banded or Q-banded metaphases in which
chromosomes 9 and 22 could be observed.17 Hypermetaphase studies using S-FISH with probes for BCR and ABL were performed on many
of these specimens.18 The D-FISH procedure was performed according to the method of Dewald et al.15 Chromosomal DNA
was denatured in 70% formamide/2× SSC for 2 minutes at 70°C.
Slides were dehydrated with an ethanol series (70%, 85%, and 100%)
for 2 minutes each and air-dried. The probe was denatured in a water bath at 70°C for 5 minutes. Then 10 µL of BCR/ABL probes were added to each slide, and a 22 × 22-mm coverslip was placed on the slide and sealed with rubber cement. Slides were hybridized for 18 to 20 hours at 37°C in a humidified chamber. After the coverslips
were removed, slides were washed for 2 minutes in 0.4× SSC at
70°C, and then in 1× phosphate-buffered detergent for 2 minutes. Chromatin was counterstained in blue with 10 µL of 1% solution of 4 ,6-diamidine-2-phenylindole in Vectashield
antifade. Representative cells were captured using a computer-based
imaging system (Quips XL Genetics Workstation; Vysis, Inc, Downers
Grove, IL).
D-FISH was performed using directly labeled BCR and ABL1 probes (Oncor
Inc, Gaithersburg, MD) to show two BCR/ABL fusion signals in cells with
a t(9;22)(q34;q11.2), one on the abnormal chromosome 9 and the other on
the abnormal chromosome 22. The ABL1 (400 kb) probe set was directly
labeled with Rhodamine Green (green signal) and included several DNA
sequences that hybridized to 9q34 and spanned the 200-kb breakpoint
region of ABL. The BCR (300 kb) probe-set was directly labeled with
Texas Red (red signal) and included several DNA sequences
that hybridized to 22q11.2 and spanned the common breakpoints in both
the major and minor BCR.
The specimens were studied in random order and in a blind fashion by
two microscopists (I.B. and W.A.W.) using strict scoring criteria for
D-FISH.15 For purposes of this paper, red BCR signals are
referred to as R, green ABL signals as G, and BCR/ABL fusion signals as
F. For scoring purposes, fusion signals were defined as merging or
touching R and G signals. The scoring process was limited to normal
nuclei with 2R2G, and abnormal nuclei with 1R1G2F or 2R2G1F (one Ph
chromosome), and 1R1G3F or 2R2G2F (two Ph chromosomes). For each
specimen, each microscopist scored 250 consecutive qualifying interphase nuclei from different areas of the same slide.
At the conclusion of the study, the intermicroscopist agreement was
sufficient to pool their results on each specimen in subsequent
analyses of the data. Thus, the final statistical analyses were based
on 500 nuclei per specimen.
The normal range for D-FISH was calculated for peripheral blood
specimens collected from 10 patients without any malignant hematologic
disorder and for bone marrow specimens collected from 10 normal bone
marrow transplant donors. The four serial dilutions were prepared by
mixing cells from a normal individual and a Ph-positive specimen to
create a series of specimens determined by repeated blind studies
before this investigation to contain specified mean percentages of
Ph-positive nuclei.
The D-FISH results for each patient's specimens from both peripheral
blood and bone marrow samples were calculated as the proportion of
abnormal cells (number of abnormal cells per 500 scored cells). Because
the proportion of abnormal cells among the specimens ranged from 0 to 1 (ie, 0% to 100%), a sin-1 transformation was used to
stabilize variances and provide a more nearly Gaussian distribution of
values. Then, the differences (delta value) between bone marrow and
peripheral blood in transformed proportions were computed for each
patient's specimens. The proportion of abnormal cells by
Q-cytogenetics was also transformed to sin-1.
The delta values for each paired set of bone marrow and blood specimens
were then analyzed using a repeated measures regression analysis
(PROCEDURE MIXED in SAS).19 To assess the
effects of sampling interval in this analysis, the approximate 4-month
sampling intervals relative to commencement of therapy were considered nominal predictor variables and the transformed proportion from Q-cytogenetics was included as a covariate. The within-patient correlation of delta values among respective specimen collection times
was specified as an autocorrelation structure depending on the actual
number of days between sampling times, ie, smaller correlations between
sequential values for longer times between sampling episodes. To assess
the usefulness of within-subject changes between sampling intervals in
blood specimens as indicators of within-subject changes in bone marrow,
a similar regression analysis was examined. In this analysis the
(within-subject) changes in bone marrow were regressed on corresponding
changes in blood, and a test for an equiangular (y = x) regression line
computed.
The classification scheme for response to therapy was based on
Q-cytogenetics and was similar to the Italian Cooperative
Group,2 ie, no response, minimal, minor, major, and
complete remission when 100%, 99% to 67%, 66% to 33%, 32% to 1%,
and 0% of metaphases are Ph positive, respectively.
| |
RESULTS |
Success of different genetic tests.
The goal for D-FISH was to study 500 nuclei for each bone marrow and
blood specimen. The goal for Q-cytogenetics was to study 25 metaphases
from each bone marrow specimen. The goal for hypermetaphase studies was
to study 200 metaphases from bone marrow. D-FISH was successful on 37 of 37 blood specimens and 37 of 37 bone marrow specimens.
Q-cytogenetics was successful in 32 of 37 bone marrow specimens.
Hypermetaphase was successful in 14 of 24 bone marrow specimens.
Normal range of D-FISH for peripheral blood and bone marrow.
Based on 500 nuclei from each of 10 normal bone marrow specimens, the
mean percentage and standard deviation (SD) of nuclei with false
BCR/ABL fusion was 0.1% ± 0.1% (range, 0 to 1 per 500 nuclei).
Based on 500 nuclei from each of 10 normal peripheral blood specimens,
the mean percentage and SD of nuclei with false BCR/ABL fusion was
0.04% ± 0.08% (range, 0 to 1 per 500 nuclei). Based on this data,
the upper bound of a one-sided 95% confidence interval for observing 1 of 500 (0.2%) neoplastic cells in either bone marrow or peripheral
blood was calculated using the binomial distribution. For both bone
marrow and peripheral blood, this calculation implied a cut-off greater
than 4/500 (>0.8%) nuclei with BCR/ABL fusion to classify any
specimen as abnormal.
Abnormal reference range for D-FISH in untreated CML.
The results of D-FISH for specimens from seven patients (nos. 2 through
7 and 9) that were collected before treatment and that were not mosaic
by Q-cytogenetic studies were used to establish an abnormal reference
range. We believe these specimens generally represent patients with
untreated CML in clinical practice. Among these seven specimens, the
mean percentage of abnormal cells was 97.6% ± 1.38% (range, 95.4 to 99.0) for bone marrow, and 86.1% ± 13.59% (range, 61.6 to
98.5) for blood.
Serial dilutions.
The observed percentage of neoplastic cells in each of the four serial
dilution specimens was 97.6%, 49.2%, 8.2%, and 1.8%. The expected
mean percentage of neoplastic cells in these specimens was 98.2%,
49.1%, 10.7%, and 2.8%, respectively. Thus, the difference between
observed and expected values for each of these specimens was 0.6%,
0.1%, 2.5%, and 1.0%, respectively.
Patient specimens.
Results for Q-cytogenetic studies for bone marrow and D-FISH for bone
marrow and blood for each patient specimen are shown in
Fig 1. Based on Q-cytogenetics, three
patients (nos. 4, 5, and 6) achieved a complete cytogenetic remission,
one patient (no. 3) briefly achieved a major response, and the rest of
the patients were classified as minimal, minor, or nonresponders.
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| Fig 1.
Percentage of Ph-positive cells (y-axis) before therapy
and during treatment at approximately 4-month sampling intervals
(x-axis in days) in bone marrow by Q-cytogenetics and D-FISH, and blood
by D-FISH.
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Each bone marrow specimen that had any abnormal metaphases by
Q-cytogenetics was also abnormal for interphase nuclei by D-FISH in
blood and bone marrow. Six specimens from three patients (nos. 4, 5, and 6) had only normal metaphases by Q-cytogenetics. For patient 6, D-FISH results were abnormal at 357 days in both bone marrow (4.8%
abnormal nuclei) and blood (3.0% abnormal nuclei). For patient 5 at
262 days, the peripheral blood was marginally abnormal (1.0% abnormal
nuclei), but bone marrow was within normal limits (0.6% abnormal
nuclei). Each of the remaining four specimens with only normal
metaphases by Q-cytogenetics were within normal limits for D-FISH in
both bone marrow and blood.
Additional studies were performed to look for minimal residual disease
on the paired sets of bone marrow and blood specimens that were normal
by Q-cytogenetics and D-FISH. In a blind study, D-FISH was used to
score 6,000 nuclei from four of the bone marrow specimens and five of
the peripheral blood specimens in this series (Table 1), and three blood and bone marrow
specimens from normal individuals. In a prior study, the normal range
for D-FISH for 6,000 nuclei was calculated to be
<0.079%.15 Based on this cut-off, each of the normal
blood and bone marrow specimens was correctly classified as normal.
Three of the four patient bone marrow specimens and each of the patient
peripheral blood specimens had minimal residual disease. It was not
possible to perform further studies on bone marrow no. 5 from patient 4 because this specimen had no leftover cells. The paired-blood specimen
for this sampling time was in the abnormal range for D-FISH when 6,000 nuclei were studied and the bone marrow had one Ph-positive metaphase
among 169 metaphases that were examined by hypermetaphase FISH studies.
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Table 1.
Search for Minimal Residual Disease in Specimens That
Were Normal by Q-Cytogenetics for 25 Metaphases and D-FISH for 500 Nuclei
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The actual proportions of neoplastic cells from bone marrow specimens
were plotted against the corresponding proportions from peripheral
blood samples (Fig 2). This plot implied
that the proportion of abnormal cells from bone marrow specimens was
typically greater (above y = x line) than for peripheral blood.
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| Fig 2.
Percentage of neoplastic cells for paired sets of bone
marrow (y-axis) and peripheral blood (x-axis).
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For D-FISH, the mean 4-month intersample differences in percentage of
abnormal nuclei between paired sets of bone marrow and peripheral blood
were not statistically different (P > .3)
(Table 2). These deltas for D-FISH were
associated (P < .05) with the transformed
proportion of abnormal cells based on Q-cytogenetics of the paired bone
marrow specimen. This is important because Q-cytogenetics of bone
marrow is widely recognized as the "gold standard" for monitoring
response to interferon therapy.1-5
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Table 2.
Analysis of Differences in Paired Sets of Bone
Marrow and Blood Over Approximate 4-Month Sampling Occasions
for Patients Undergoing Treatment for CML
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Based on these results, an additional regression analysis was performed
to assess the relationship between within-subject changes (sample
interval) in the proportion of abnormal cells that would be obtained
from bone marrow specimens versus the within-subject changes in
peripheral blood samples. This regression analysis is displayed in
Fig 3 along with an approximate 95%
confidence interval for a new predicted observation (delta bone marrow)
given a (new delta) peripheral blood value (prediction interval). In addition, the expected equiangular regression line (y = x) was calculated and included in Fig 3. This analysis indicated a significant (P < .001) linear relationship but was not different than the equiangular (y = x) line (P > .2).
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| Fig 3.
Linear regression analysis (solid line) of the
within-subject changes in the proportion of neoplastic cells from bone
marrow on the within-subject changes in the proportion of neoplastic
cells for peripheral blood. Dashed lines are the 95% prediction
interval, and the dotted line is the equiangular (y = x)
line.
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DISCUSSION |
In the present investigation, the differences between the percentage of
neoplastic nuclei in bone marrow and blood were consistent over the
follow-up period. This implies that the percentage of neoplastic nuclei
in blood during follow-up tracked the corresponding percentage of
neoplastic metaphases and nuclei in bone marrow over the course of
interferon -2b therapy. In an earlier study, we showed that the
percentage of neoplastic nuclei in the bone marrow strongly correlated
with the percentage of Ph-positive metaphases.15 The
reduction in percentage of Ph-positive metaphases correlates with a
prolonged chronic phase and increased survival in CML, and the results
of D-FISH on blood correlates with Q-cytogenetics. This suggests that
D-FISH is an efficient and sufficiently accurate method to test
periodic peripheral blood specimens from patients with CML to monitor
the effectiveness of interferon therapy.
The analysis of 500 nuclei with D-FISH in bone marrow and peripheral
blood detects less than 1% disease and is at least as sensitive as
Q-cytogenetics. Thus, the use of D-FISH to score 500 interphase nuclei
could substitute for Q-cytogenetics for purposes of monitoring response
to therapy for CML. Moreover, by analyzing 6,000 nuclei it was possible
to identify residual disease in specimens that were normal by
Q-cytogenetics and by initial D-FISH studies (Table 2). Thus, using
D-FISH has considerable potential to detect very low levels of minimal
disease in both blood and bone marrow.
We are aware of only one other report that compares the results of FISH
studies of paired sets of bone marrow and peripheral blood to monitor
therapy in CML. Mühlmann et al11 recently used S-FISH
to study 49 peripheral blood smears and 30 bone marrow specimens from
36 patients in chronic phase CML at different stages of cytogenetic
remission. Although S-FISH is significantly less accurate than D-FISH,
the results of their study also suggest that FISH studies of blood are
useful to monitor the effect of interferon therapy.
In the present investigation, at most times before and after therapy,
the percentage of nuclei with BCR/ABL fusion was usually lower in blood
than in bone marrow. Nevertheless, it was possible to use a simple
linear regression model to predict the changes in percentages of
Ph-positive nuclei in bone marrow using D-FISH data from peripheral
blood. Because only 10 patients were studied, the 95% prediction
interval provided a wide interval estimate of these changes in
neoplastic nuclei in bone marrow. The investigation of a larger series
of patients could produce narrower interval estimates of neoplastic
cells in bone marrow based on studies of blood. This information could
be an important outcome of investigations performed by cooperative
groups that focus their studies on CML.
The present limitation to predict precisely the actual percentage of
neoplastic nuclei in bone marrow based on data from blood should not
limit the use of blood to monitor therapy in clinical practice. The
results of the present investigation indicate that it is best to assess
response to therapy based on changes in percentage of neoplastic nuclei
using the same tissue over time. In other words, assess the changes in
percentage of neoplastic cells using studies of blood as a predictor of
changes in bone marrow samples. This is important because the
percentage of abnormal nuclei in blood and bone marrow vary similarly
within most patients over their course of therapy (Fig 3).
Three patients (nos. 4, 7, and 9) in our investigation had relatively
similar percentages of neoplastic nuclei in blood and bone marrow
before therapy; 1 achieved a complete cytogenetic remission (Fig 1).
The remaining 7 patients had somewhat greater differences in
percentages of neoplastic nuclei in blood and bone marrow and 2 of
these patients achieved complete cytogenetic remission. Although only
10 patients were investigated, these results suggest that chances of
achieving a complete cytogenetic remission may not be affected by
differences in percentage of abnormal nuclei in blood and bone marrow
before therapy.
We wondered why some patients had similar percentages of neoplastic
cells in their bone marrow and blood, whereas other patients did not.
Many investigators have shown that the Ph chromosome occurs in cells of
different hematopoietic compartments in different patients with
CML.20 Perhaps patients with a similar percentage of
neoplastic nuclei in bone marrow and blood have a form of CML that
involves stem cells that give rise to both lymphocyte and myelocytes.
In contrast, patients with CML that have different percentages of
neoplastic nuclei in blood and bone marrow have a form of CML that
involves stem cells of only myeloid cell lines.
Among patients with untreated CML, approximately 90% have a Ph
chromosome in each of their metaphases and the remaining 10% show
mosaicism, ie, a mixture of normal and Ph-positive
metaphases.21 Thus, it was not surprising to find three
patients (nos. 1, 8, and 10) in this investigation who were mosaic
before therapy. The results of D-FISH studies on 43 patients in this
study and an earlier investigation suggest that all patients with CML
may have both normal and neoplastic cells in their bone marrow before treatment; this is not apparent by Q-cytogenetics.15
Most classification schemes for assessing response to therapy are based
on the observed percentage of Ph-positive metaphases. To adjust for
mosaicism when using Q-cytogenetics and D-FISH, it may be useful to
standardize the percentage of neoplastic cells after therapy to the
percentage of neoplastic cells before treatment. One approach is to
divide the percentage of neoplastic cells before therapy into the
percentage of neoplastic cells after therapy and then multiply by 100.
Cells from each patient in this study that had a classical
t(9;22)(q34;q11.2) displayed a D-FISH pattern that matched our strict
scoring criteria. However, some patients with CML have Ph chromosomes
that produce abnormal D-FISH patterns, but the signal patterns are
different than our strict scoring criteria.15 For these
patients, it is necessary to develop special scoring criteria for
D-FISH by first examining signal patterns in metaphase cells. For
patients with atypical D-FISH patterns, the normal and abnormal
reference ranges for nuclei with apparent BCR/ABL fusion are different
than for patients with CML that have typical D-FISH patterns. Moreover,
the quantification of disease is less accurate than it is for patients
with typical D-FISH signal patterns. We have studied patients with CML
who have cells in their bone marrow and blood with atypical D-FISH
patterns and believe that the D-FISH signal patterns do not change in
Ph-positive nuclei over time in these patients.15 Thus, it
should be possible to use modified D-FISH criteria to study blood for
purposes of detecting changes in the percentage of nuclei with BCR/ABL
fusion to monitor therapy.
The results of the present investigation and Mühlmann et
al11 show great potential for using FISH to study blood for
purposes of monitoring the response to therapy in CML. In clinical
practice, we believe that cytogenetic studies on bone marrow should
continue to be performed at diagnosis to identify patients that are Ph positive and to rule out chromosome abnormalities that could indicate neoplasms other than CML. We also believe that it is important to
establish a pretreatment baseline for the percentage of nuclei with
BCR/ABL fusion with D-FISH; this could be performed on bone marrow and
blood. For patients on therapy, D-FISH could then be performed on
peripheral blood at periodic intervals to assess the effectiveness of
therapy. Consequently, bone marrow may not need to be collected to
monitor therapy as frequently as it is in current practice.
| |
FOOTNOTES |
Submitted March 9, 1998;
accepted June 1, 1998.
The Chronic Myeloid Leukemia National Study Group provided specimens
for this study and was supported in part by Integrated Therapeutics
Group Inc. Probes for BCR and ABL1, and salaries for personnel who
performed fluorescence in situ hybridization studies were provided by
Oncor Inc. Research support was also provided by the United Leukemia
Fund Inc and the Cancer Research and Treatment Fund Inc. I.B. was a
fellow visiting Mayo Clinic and was supported by Comunidad
Autónoma de Madrid, Spain.
Address reprint requests to Gordon W. Dewald, PhD, Cytogenetics
Laboratory, Hilton 970, Mayo Clinic, Rochester, MN 55905.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are indebted to the following investigators who supplied bone marrow
and samples that were analyzed in this study as part of the CML
National Study Group: Luis Baez, MD, VA Medical Center; Stephanie
Elkins, MD, University of Mississippi Medical Center; Eric Feldman, MD,
New York Medical College; Bruce Lewis, MD, St Paul, MN; Romeo A. Mandanas, MD, University of Oklahoma; Hussain I. Saba, MD, H. Lee
Moffitt Cancer Center; and Richard Silver, MD, New York Hospital. We
are also grateful to Bernadette Clarke, BSN, at the Chronic Myeloid
Leukemia National Study Group Coordinating Center and Larry Heller from
Integrated Therapeutics Group Inc for assistance with coordinating this
project.
| |
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Abstract
Introduction
Abstract