1. No category

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Quantification of the Breakpoint Cluster Region Rearrangement
for Clinical Monitoring in Philadelphia Chromosome-Positive
Chronic Myeloid Leukemia
By Claire F. Verschraegen, Moshe Talpaz, Cherry1 F. Hirsch-Ginsberg, Rakesh Pherwani, Mary B. Rios,
Sanford A. Stass, and Hagop M. Kantarjian
The purpose ofthis report was to evaluate scintigraphy analysis of Southern blot hybridization as a method to quantify
the breakpoint cluster region (BCR) rearrangement of Philadelphia chromosome (Ph)’ chronic myelogenous leukemia
(CML). Cytogenetic and molecular studies performed simultaneously on 474 bone marrow andlor blood samples from
300 patients treated with a-interferon-based therapy were
compared. Molecular results were expressed asthe percentage of rearranged BCR bands versus the total scintigraphic
signal. The percentage of Ph+ metaphases was calculated
on 25 metaphases. The resultsof molecular studies
obtained
on both peripheral blood and bone marrow samples were
identical. The rank correlation between the BCR quantification and the percentage of Ph positivity in 465 sampleswas
excellent (r = .78). However, of 99 samples with a normal
karyotype, 24% had a BCR rearrangement. Of 86 samples
with no BCR rearrangement, 13% showed a Ph chromosome.
Of 49 sampleswith partial cytogenetic remission (Ph’ metaphases, l%to 34%). 23%had no BCR rearrangement.In samples with a minor or no cytogeneticresponse(Ph’ metaphases, >34%), BCR analysis overestimated the degree of
response in 73 of 326 samples (22%). Nevertheless, survival
analysis by BCR quantificationlevel showed statisticallybetter outcome for patients in complete or partial molecular
response ( P < .OIL Molecular quantificationof BCR was useful in monitoring the course of Ph’ CML. Thismethod, which
can be used on peripheral blood, detected residual disease
not shown by cytogenetic analysis and was prognostically
relevant as a measure of disease suppression.
0 1995 by The American Societyof Hematology.
P
formed on bone marrowaspirates. Samples were incubated overnight
at 37°C in Ham’s F-l0 medium supplemented with 10% fetal calf
serum and Pen-Strep antibiotics. Cultures were harvested and slides
were prepared according to established procedures; 25 metaphases
were analyzed when available.’
Molecular analysis. DNAwaspurifiedwith
standard phenol/
chloroform extractions and ethanol precipitation after proteinase K
digestion.’ Ten micrograms ofDNA was digested to completion
with one of the following restriction endonucleases: EcoRI, BamHI,
Bgl 11, or Xba I. The digests underwent electrophoresis on a Probe
Tech (Oncor, Gaithersburg, MD) with the use of a 0.7% (7 g/L)
agarose gel before being transferred to a nylon filter, in accordance
with the manufacturer’s instructions.’ A solution composed of HindIII and BamHI digests of control DNA in equal proportions and
diluted to approximately 5% of normally loaded sample DNA was
loaded onto each gel as a quality control. Hybridization studies were
performed with either of two oligolabeled DNA probes: theuniversal
bcr probe (Ph-bcr/3), which is a 4.5-kb DNA fragment that contains
sequences from the entire bcr region except for a central Hind111
site that contains repetitive sequences; and a 3’ probe (bcr[PR-l]),
which is a 1.2-kb Bgl 11-Hind111 fragment that contains only sequences 3’ to the central BamHI site.’”
Quantitative molecular analysis. After hybridization, Southern
blots were scanned for 5 to 24 hours with a Betagen scanner (Betagen, Waltham, MA) that detects particle emissions and plots the
origin of the particle to a location on the filter. Molecular weight
HILADELPHIA chromosome (Ph)+ chronic myelogenous leukemia (CML) is a disease of the hematopoietic
stem cell that progresses in a multistep fashion.’ The reciprocal translocation between chromosomes 9 and 22 is the hallmark of CML.’ The breakpoint on chromosome 22 occurs
most often within a 5.8-kb region located between exons 1
and 4 of the breakpoint cluster region (BCR) gene, which
is called the major breakpoint cluster r e g i ~ nIn
. ~patients on
therapy, this translocation has been monitored by karyotype
analysis of bone marrow samples. However, this method is
limited by (1) a requirement for mitotic cells, (2) the presence of translocations below the level of cytogenetic detection, (3) an insufficient amount of metaphase material or a
restricted number of metaphases analyzed in some samples,
and (4) its possible low sensitivity to detect the near disappearance of the leukemic clone under therapy.
Detection of the BCR rearrangement by Southern blot
analysis has been used as a clinical tool in patients with CML
and in patients with acute lymphoblastic leukemia (ALL). To
date, Southern blot analysis has been useful in diagnosing
patients with Ph- CML and BCRrearrangement and patients
with ALL and p210 Ph+ disease not identified by cytogenetic
studies4 The intensity of the rearranged bands shown by
Southern blotting can be quantifiedby densitometric analysis
of autoradiograph e x p ~ s u r e . In
~ . ~this study, we compared
BCR rearrangement quantification with routine cytogenetic
studies performed in a serial evaluation of patients with Ph+
CML on therapy. The objective of our analysis was to assess
whether BCR quantification could be used as an alternative,
or as a superior tool, to monitor the efficacy of therapy in
patients with CML.
MATERIALSANDMETHODS
Patients. Three hundred patients with Ph+ CML treated with ainterferon (IFN-a)-based therapy were included in the study after
informed consent was obtained according to institutional guidelines.
Samples of blood andor bone mamow were submitted for molecular
analysis of BCR quantification. A bone marrow sample was submitted simultaneously for cytogenetic studies.
Cytogenetic analysis. Routine cytogenetic studies were perBlood, Vol 85, No 10 (May 15), 1995: pp 2705-2710
From the Departments of Hematology and Laboratory Medicine,
The University of Texas M.D. Anderson Cancer Center, Houston,
m.
Submitted July 14, 1994; accepted December 29, 1994.
Presented in part at the Meeting of the American Association of
Hematology, St Louis, MO, December 3-7, 1993.
Address reprint requests to Hagop M. Kantarjian, MD, Department of Hematology, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 61, Houston, TX 77030,
The publication costs of this article were defrayed in part by puge
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8510-0029$3.00/0
2705
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VERSCHRAEGEN ET AL
2706
Table 1. Molecular Detection of BCR RearrangementVersus
Cytogenetic Studies in 474 Samples From Patients Treated for Ph'
Chronic-Phase CML
Ph' Metaphases
388
Table 3. Patient Distribution by CML Phases
Newly diagnosed
(3.7-68.8)
30
BCR Rearrangement
Absent
(N = 99)
Present
(N = 375)
Absent
Present
364
75
24
11
Total
86
BCR Quantification
(median percentage, range)
No. of
Patients
CML Phase
28
35
26
46
86
54
25
Early CP (%Ph =(8.2-85)
100) 36
Early CP (%Ph <loo)
(0-39.6)14
Late CP (%Ph =(3.3-59.2)
100) 28
Late CP (%Ph(0-71.1)
<loo) 2
(0-66.7)32
Accelerated phase
Blastic phase (14.7-51.6)
32
Abbreviation: CP, chronic phase.
and counts per minute of the rearranged and germline bands were
determined according to the manufacturer's instructions. Scanned
filters were then exposed to Kodak X-OMAT AR x-ray film (Eastman Kodak, Rochester, NY) for 3 to 14 days at -70°C to obtain a
film record of the rearrangement and to rule out the presence of
faint rearranged bands below the limits of resolution of the Betagen
scanner. If sensitivity bands were not visible at the shorter exposure
period, the filter was exposed to the film for the longer period of
time.
Cytogenetic:molecular ratio. In cytogenetic studies, the presence of one Ph+ chromosome is interpreted as a positive event
(binary system). In molecular studies, the intensity of the rearranged
BCR band is calculated as the ratio of the signal from the rearranged
BCR band(s) to the added signals from the rearranged BCR band
and the normal unrearranged gene. A theoretical 2:l ratio exists
between the cytogenetic and the molecular results. For example, a
cell population that is 100%Ph+ would be expected to have a BCR
rearrangement value of 50% [the abnormal t(9;22) chromosome/(the
same + the other normal chromosome)]; a 50% Ph+ cell population
would have an expected BCR rearrangement of 25% (one quarter
of the DNA carries the 9 2 2 translocation).
Initial studies sought to optimize the ratio of the intensity of the
rearranged band versus the total DNA by analyzing probe and enzyme combinations that yielded a single rearranged band as well as
a single germline band. The reason for this is that more than one
germline band occurs when the probe contains sequences recognizing more than one different portion of the same DNA molecule
that have been separated by enzyme digestion and electrophoresis.
Therefore, adding the intensity of the bands artificially doubles the
contribution of a single DNA molecule to the overall intensity. The
same is true in a majority of the cases of more than one rearranged
band, in which the intensities of the bands are comparable (the
intensity of the rearranged bands obtained on the same sample were
averaged if their difference was no more than 75% for the smallest
band). Cases in which the intensity of the rearranged bands was
different (more than 75%) were thought to possibly represent clonal
variations and the intensities of the bands were added. (Although it
is possible to have diminished intensity for small size bands [<2
kb], most of the rearranged bands were larger and the intensity was
not thought to be affected by the size of the bands.) In the earlier
studies there were often two different enzyme digests present on the
filter, both of which were scanned and analyzed. These analyses
occasionally yielded slightly different ratios. Results obtained on the
same sample with different probes or enzymes were averaged to
obtain one signal per sample per day. Differences obtained on the
same sample with different enzymes or probes did not influence the
final range assignment.
Statistical methods. Analysis of the correlation between molecular quantification and percentage of Ph+ metaphases was performed
using the nonparametric Spearman's Rank correlation coefficient."
...
.
.
r=.78
i
.
a
..
i
..
.
Table 2. Comparison of Cytogenetic Versus Molecular Analyses
(465Samples)
Percent Ph+ Metaphases
total, % columnl)
(no. of samples I%
Percent BCR
(luantification
0
35
1 to 34
to 95
0
1 to c17.5
17.5 to 47.5
247.5
75 (16, 83)1 1 (2, 23)
9 (2, 10)32 (7,65)
5 (1, 10)
6 (1,7)
0
1 (0.2)
0
44
41
3
Total
90
88
238
49
>95
0
29 (6, 12)
50)
(10,
76)
(9, 47)181 (39.
(1.3)
28 (6, 12)
0
20
40
60
80
100
Ph-Positive Mabphases (YO)
Fig 1. Correlation between BCR quantificationand percentage of
PhCmetaphases in 465 samples.
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BCR QUANTIFICATION IN CML
2707
...........!.L .....!l....
1
1".
.!
I
!:!.....!.;!.L!.L!.d!.-.!.L.!J
"qy,
I
11-.r
80-
60 -
il..ll.....l1
40 -
20 Fig 2. Survival analysis by cytogeneticresponseinpatients
with Ph+ chromosomeCML. Survival is calculated fromthe time
of the bost cytogenetic
rasponse.
.............
.............
-.e.-
Total
49
29
50
172
04
24
0
l
18 6
30
12
male. Twenty-eight patients were studied at diagnosis, and
272 patients were studied during therapy with IFN-a.
There were 221 patients analyzed in chronic phase: 89
were within 1 year of diagnosis (early chronic phase) and
132 were diagnosed for more than l year (late chronic
phase). Seventy-nine patients were investigated in CML
transformed phases: 54 were in accelerated phase and 25
in blastic phase.
At the time of blood sampling, 4 patients had a double
Ph chromosome in 24% to 100% of the analyzed metaphases.
Three patients were in blastic phase and 1 was in accelerated
RESULTS
Patient characteristics. All 300 patients had Ph'
CML. Their median age was 42 years and 40% were fe-
I
U '
6
36
Months
Results obtained from bone marrow and blood samples were analyzed with the two-tailed paired Student's t-test. Survival was calculated from the date of the sample analysis by the modified Wilcoxon
test. Differences in survival were assessed by the log-rank test.'*.''
When serial studies were performed, the patient was categorized by
the best cytogenetic response; survival was calculated from the date
of the sample analysis with the best cytogenetic response.
0
Dead Cytogenetic
Response
2
CR
PR
1
Minor
8
43
None P=.OO1
12
18
Months
24
30
~~~
I
36
Fig 3. Survival
analysis
according to the BCR quantification in patients with Ph*chrois
mosome CML. Survival
calculated from the time of the
best cytogenetic response.
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
VEASCHRAEGEN ET AL
2708
phase. The presence of a double Ph chromosome was not
consistently associated with a second rearranged band.
Comparison of cytogenetic and molecular studies, Four
hundred seventy-four samples from 300 patients were analyzed. Table l shows the distribution of samples by the
percentage of Ph+ metaphases and the BCR quantification.
Ninety-nine samples were from patients who had achieved
a complete cytogenetic response (Ph' metaphases, 0%); 75
samples had no BCR rearrangement; and24 (24%) had a
rearrangement. These rearrangements were quantified for 15
samples and ranged from 2.1% to 38.5% (median, 12.4%).
In contrast, 1 1 (3%) of 375 samples with Ph+ metaphases
had no BCR rearrangement. In these 1 1 samples, the percentage ofPh' metaphases ranged from 4%to24% (median,
5%).
Table 2 shows the distribution of samples when categorized by the percentage of Ph+ metaphases and the BCR
quantification. The cut-off values were chosen to reflect cytogenetic response categories, as previously described.I4 A
complete cytogenetic response would correspond to a BCR
quantification of 0%. A partial cytogenetic response (1% to
34% Ph+ metaphases) would correspond to a BCR quantification of1% to less than 17.5%. A major cytogenetic response includes both complete and partial cytogenetic responses. A minor cytogenetic response (35% to 95% Ph+
metaphases) would correspond to a BCR quantification of
17.5% to 47.5%.
Of the 238 samples with no cytogenetic response (>95%
Ph+ metaphases), 29 (12%) had a BCR value less than
17.5%. However, in samples with a minor cytogenetic response (35% to 95% Ph+ metaphases), 44 of 88 (50%) had
a BCR quantification less than 17.5%. In contrast, in patients
with 1% to 34% Phi metaphases, only 6 of 49 (12%) had
BCR quantification 2 17.5%. A good correlation was found
between the percentage of Ph+ metaphases and the BCR
quantification (Fig 1; r = .78; P < .OOl).
The BCR quantification level was similar in patients investigated at diagnosis, in those in chronic phase with 100%
Ph' cells, and in those in accelerated or blastic phases (Table
3). The lower values seen in patients with less than 100%
Ph positivity correlates with the values shown in Fig 1.
Prognosis by measurements of the Ph abnormality. Figs
2 and 3 show the survival of patients according totheir
cytogenetic response and BCR quantification. Both cytogenetic and molecular studies were significant prognostic determinants of survival.
One important question was which of these two studies
was a better prognostic determinant in discordant situations.
Because the cytogenetic study is currently the gold standard
for prognostic monitoring, we evaluated the survival of patients in specific cytogenetic categories by different BCR
quantification levels.
Table 4 shows the survival of all patients by both molecular quantification (below or above 17.5%) and cytogenetic
response. The survival of patients who did not respond to
therapy (>95% Ph" metaphases) is depicted in Fig 4, according to the molecular quantification. There is a trend for
higher incidence of survival when the BCR quantification is
less than 17.5% ( P = .05).
Table 4. Survival From the Date of the Best Cytogenetic Response
by BCR Quantification Within Cytogenetic Response Categories
% Ph '
YO BCR
Metaphases
Quantification
Patients
0
4
1 to 34
No. of
Abbreviation:
P Value
0
1 to <17.5
217.5
41
4
98
100
75
NS
<l75
217.5
25
4
95
100
NS
21
29
84
75
NS
217.5
<17.5
217.5
.05
151
85
67
35<17.5
to 95
>95
21
Probability of
Survival at
2 yr ~ % )
NS, not significant.
Signijicance of BCR quantijication at diagnosis. All but
l of the 28 patients tested at diagnosis had greater than 90%
Ph' metaphases. Among them, 5 had a BCR quantification
less than 17.5%.Patients were divided into two groups (BCR
levels <17.5%, 5 patients; >17.5%, 23 patients). At this
time of follow-up, only one death was recorded among the
patients of the latter group. Therefore, no significant conclusion can be drawn regarding differences in survival by BCR
values at diagnosis.
BCR quantGcation in bone marrow versus blood samples.
Samples obtained simultaneously from peripheral blood and
bone marrow were compared for 79 cases (Table 5). BCR
quantification was performed in 72 of these samples. Figure
5 shows the correlation between results obtained on the bone
marrowand the peripheral blood. Molecular studies were
negative in both for 35 samples of blood and bone marrow
and positive in both for 42 samples. A BCR rearrangement
waspresentin 1 sample of peripheral blood, whereas the
corresponding sample of bone marrow had none;the cytogenetic analysis showed 5% Ph' metaphases. In 1 case of a
patient with a normal karyotype, the marrow sample showed
a questionable BCR rearrangement whereas the peripheral
blood sample showed none. The Pearson correlation coefficient for the samples that were quantified was .94.
DISCUSSION
Molecular analysis of the BCR rearrangement is currently
used in clinical practice to diagnose Ph+ CML and ALL.
However, veryfew studies have analyzed the correlation
between the conventional cytogenetic analysis and the newer
molecular technique for monitoring the courses of these diseases.','
Our results in a large number of patients investigated indicate a good correlation between the BCR quantification and
the percentage of Phi metaphases (Fig 1). However, a significant discordance was found in 50% of samples with a
minor cytogenetic response in whom quantification of cells
withtheBCR rearrangement overestimated the degree of
response (ie, BCR rearrangement < 17.5%). This difference
may be explained by the proliferative advantage of the Ph+
clone, which results in a higher detection of Phi metaphases
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
BCR QUANTIFICATION INCML
2709
n
Fig 4. Survival analysis according to the BCR quantification in patients with Ph+chromosome CML who did not have
a response despite therapy. Survival is calculated from the time
of the best cytogenetic response.
g
0
18 6
36
24
12
in dividing (cytogenetic studies) versus clonal and total cell
populations (molecular studies). BCR rearrangement analysis is performed on a mixed pool of myeloid and nonmyeloid
cells, and some of the latter (T-cell lymphocytes as well as
some B cells) may not have BCR rearrangements. Thus, a
better correlation of BCR quantification with cytogenetic
analysis may be found in studies that isolate myeloid and
B- and T-lymphoid elements and quantify the rearranged
BCR value in subsets (eg, myeloid cells). However, such
approaches may be tedious and may not be more practical
than current cytogenetic studies.
The comparison between bone marrow samples and peripheral blood samples shows that results are interchangeable, a finding also reported by Stock et al.'5 DNA analysis
on peripheral blood samples could replace bone marrow
samples analysis if the equal or superior prognostic value in
discordant cases to cytogenetic studies is confirmed. Furthermore, the technical cost of cytogenetic analysis is 2.8 times
higher than the cost of molecular analysis performed on
peripheral blood.
Several additional questions are of importance. (1) Can
BCR quantification detect residual disease in patients for
whom cytogenetic studies show no Ph' metaphases? (2) Can
BCR quantification be used for prognostic purposes? (3)
In situations of discordant results between cytogenetic and
molecular analyses, is BCR quantification a better prognostic
determinant than cytogenetic analysis?
It was interesting that BCR rearrangement studies were
positive in 24 of 99 samples (24%)in which Ph+ metaphases
were not detected by cytogenetic analysis. This finding suggests that molecular studies can identify residual disease that
is undetected by cytogenetic studies. Conversely, 1 1 of 86
l
70
60
Table 5. Percentage of BCR Quantification on Bone Marrow and
Blood Samples Drawn on the Same Day
35
1
1
6
3
4
16
8
5
Ph%
BCR% Bone Marrow
Samples
(median, range)
0
0
0
Positive
5
0
Positive
3.11 (2.79-3.31)
6.08 (3.16-9.93)
16.21 (3.16-39.49)
24.01 (18.34-55.05)
14.97 (4.71-17.1)
0
0
1-34
35-95
>95
No results
BCR% Blood
Samples
(median, range)
0
Negative
6.31
Positive
5.01 (0.91-6.31)
15.72 (4.03-25.75)
10.17 (2.49-43.23)
20.72 (14.97-66.32)
12.38 (10.65-16.33)
r=.94
0
50
40
No. of
Samples
30
Months
0
0
a
30
20
10
0
0
10 20 30 40 50 60 70
% BCR Bone Marrow
-
Fig 5. Comparison between BCR quantification performed on
bone marrow samples versus blood samples.
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
2710
VERSCHRAEGEN ET A L
samples (13%) without BCRrearrangementshowedPh+
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ACKNOWLEDGMENT
We are indebted to Kimberly Hemck for her editorial help in the
preparation of the manuscript.
15. Stock W, Westbrook C, Peterson B, Szatrowski T, Silver R,
Schiffer C, Bloomfield C: Molecular monitoring of bcdabl fusion
gene in chronic myelogenous leukemia: Peripheral blood analysis is
comparable to a bonemarrow examination. Blood 82:331a, 1993
(abstr, suppl 1)
From www.bloodjournal.org by guest on December 29, 2014. For personal use only.
1995 85: 2705-2710
Quantification of the breakpoint cluster region rearrangement for
clinical monitoring in Philadelphia chromosome-positive chronic
myeloid leukemia
CF Verschraegen, M Talpaz, CF Hirsch-Ginsberg, R Pherwani, MB Rios, SA Stass and HM
Kantarjian
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