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Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2038-2042
Spectral Karyotype Analysis of T-Cell Acute Leukemia
By
Janet D. Rowley,
Shalini Reshmi,
Katrin Carlson, and
Diane Roulston
From the Section of Hematology/Oncology, Department of Medicine,
University of Chicago, Chicago, IL.
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ABSTRACT |
Analysis of 15 cases of T-cell acute lymphoblastic leukemia with
spectral karyotyping (SKY), which can identify all chromosomes simultaneously, clarified the chromosome rearrangements in 3 cases and
confirmed them in 11 others; no abnormal cells were identified in 1 case, which had only 10% abnormal cells. Five of the latter cases had
a normal karyotype. Thus, the use of SKY substantially improves the
precision of karyotype analysis of malignant cells, which in turn leads
to a more accurate assessment of the genotypic abnormalities in those cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ACUTE LYMPHOBLASTIC leukemia
(ALL) affecting T cells has been associated with a normal karyotype in
30% to 40% of patients.1-3 This is in contrast to B-cell
ALL, in which about 10% to 25% of patients have a normal karyotype in
their leukemic cells.2 One concern regarding such data is
the possibility that a recurring translocation may have been
overlooked. This concern is reinforced by the observation only a few
years ago of the presence of the t(12;21) in 25% of childhood
pre-B-cell ALL. This translocation was identified by Berger et
al,4 using a chromosome 12 painting probe. The
translocation breakpoints were subsequently cloned and shown to involve
TEL (ETV6) on chromosome 12 and AML1
(CBFA2) on chromosome 21.5,6
We therefore undertook a study of T-cell ALL to determine whether there
was an overlooked recurring translocation analogous to the t(12;21). We
used spectral karyotyping (SKY) to perform this study because we wanted
to examine the entire genome in one hybridization.7-9 In
our present study, SKY clarified the karyotype abnormalities in a
number of cases, but it did not detect any unexpected recurring aberrations.
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MATERIALS AND METHODS |
We have karyotyped 33 patients with T-cell ALL from 1988 to 1997. Of
these, 7 had a normal karyotype in the bone marrow sample obtained at
diagnosis, 4 had known recurring abnormalities [t(8;14), t(11;14)],
and 22 had random, often complex, karyotype rearrangements. We had
material for SKY analysis for 17 patients, 5 normal and 12 abnormal.
Cells from 2 of the abnormal samples had such poor morphology that
precise analysis was impossible, and these samples are excluded from
the summary that follows. Therefore, this report is based on 15 patients. There were 11 males and 4 females; 11 were children 16 years
of age or under, and 4 were older than 16. The diagnosis of T-cell ALL
was based on immunophenotypic markers, including positivity for CD2,
CD5, and/or CD7 and absence of B-cell markers (eg, HLA-DR or
CD19) or myeloid markers. The samples were obtained with informed
consent. They were obtained at diagnosis in 12 cases and at relapse in
3 cases; the analysis was performed using bone marrow in 12 cases and
peripheral blood in 3 cases.
We used previously prepared slides from 13 patients; for 2 patients, we
used freshly prepared slides from material stored in fixative for up to
8 years. We hybridized one or two slides for each case, using the SKY
fluorescence in situ probe according to the protocol recommendations by
the manufacturer (Applied Spectral Imaging, Carlsbad, CA). For each
case, between 4 and 12 (average, 8) metaphase cells were captured and
analyzed, using the SD200 system (Applied Spectral Imaging). Each case
that presented questionable or nonobvious G-banded chromosome
rearrangements was then analyzed using the appropriate single- or
dual-color chromosome paints (WCPs; Vysis, Downers Grove, IL).
Chromosome probes for 2, 5, 8, 12, 14, 16, and 21 were labeled with
Spectrum OrangeTM (SO); those for 4, 7, 13, X, and Y were labeled with
Spectrum GreenTM (SG), and both fluorochromes were used for chromosomes
1, 6, and 9. Coatasome 17 (Oncor, Gaithersburg, MD) was labeled with digoxigenin.
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RESULTS |
The clinical data, as well as the conventional cytogenetic analysis and
the summary of the SKY results, are included in Table 1.
Case 1.
The G-banded karyotype showed complex rearrangements including an
interchange involving chromosomes 1, 5, and 6. SKY was not informative
with regard to the inverted X chromosome, but in cells with adequate
DAPI banding, it could be identified because of the
abnormal position of the centromere. The del(9p) was confirmed. SKY was
very helpful in sorting out the 1, 5, 6 rearrangement (Fig
1, upper portion). The der(1) had part of
chromosome 5 translocated to the long arm; the der(5) had part of
chromosome 1, and these could represent a balanced t(1;5)(q31;q32). One
chromosome 6 also had material from 5, most likely from 5q, because it
seemed to be too large to be from 5p. In addition, there is one normal
chromosome 5. The chromosome 1, 5, and 6 abnormalities were confirmed
using painting probes for these three chromosomes. The genetic
consequences of these changes are loss of part of 6q and 9p and gain of
5, possibly 5q.
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| Fig 1.
Examples of the analysis of cells from 2 patients showing
the DAPI, SKY, and classified images as well as the results using
painting probes. (Top two rows, Case 1) (Left) Reverse DAPI image with
the der(1), der(5), and der (6) chromosomes as well as normal 9 and del
(9p) identified by arrows. (Middle) SKY image; (right) classified
image. (Second row right) The classified karyotype of this cell.
(Second row) Three panels show the results with painting probes for
chromosomes 1, 5, and 6, confirming (left) that chromosome 5 material
is on both the der(1) and der(6), (middle left) that the t(1;5) is
reciprocal, and (middle right) that there is no material from
chromosome 6 on either the der(1) or the der(5). (Bottom two rows, Case
3) (Left) Reverse DAPI image with the abnormal chromosomes including
two Y chromosomes and dup(1p), del(7p), der(14), der(18), and der(19),
are identified by arrows. (Middle) SKY image of same cell; (right)
classified image. (Bottom row right) Classified karyotype. (Bottom row)
Three panels showing the results of painting probes for chromosomes 4, 7, and 9, confirming (left) that part of chromosome 4 is on the
der(18), (middle left) that part of 7, presumably 7p, is on the
der(14), and (middle right) that part of 9 is on the der(19).
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Case 2.
A complex pattern of abnormalities was noted with G-banding, which
included loss of the Y chromosome, a 12;13 rearrangement, and
additional unidentified material on chromosomes 14, 17, and 21, as well
as a second clone with deletion of 6q added to these abnormalities.
Using SKY, we confirmed the 12;13 rearrangement and clarified the
origin of the other rearrangements (Fig 2,
upper right). The large chromosome 14 is probably a duplication,
because there was no evidence for a translocation of other chromosomal material. The add(21) was a derivative (Y;21); the origin of the centromere was unclear. In addition, there was an apparently balanced undetected translocation involving chromosomes 6 and 17, with material
from 17q translocated to 6q and 6q to 17q, resulting in the der(17)
chromosome. The involvement of chromosomes 6, 17, 12, 13, 21, and the Y
was confirmed with the appropriate sets of painting probes. The
consequences of the rearrangements were loss of 12p, probable
duplication of the telomeric portion of 14q, and loss of the
centromere-short arm region of 13 and material from either the Y or
chromosome 21.
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| Fig 2.
Partial karyotypes of cells with rearranged chromosomes;
the involved chromosomes from the G-banded karyotype on the left and
the classified images on the right. The chromosome origin of the
classified image is listed for each chromosome. In panel 1, note that
for Case 4, the left chromosome 9 has a deletion of the p arm. Panel 2 is Case 2, panel 3 is Case 6, and panel 4 is Case 5.
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Case 3.
Standard G-banding identified a hyperdiploid karyotype with a probable
gain of a Y chromosome, a duplication of 1p, possibly additional
material on 14q, a 7;18 translocation, and a second clone with an add
(19p or 19q) in addition to these abnormalities. SKY confirmed that the
extra small chromosome was a Y and the duplication 1p contained only
material from chromosome 1 (Fig 1, lower portion). The add(14) was
shown to result from a translocation from 7p. The short arm of
chromosome 18 was involved with chromosome 4, possibly the short arm of
chromosome 4. Involvement of chromosomes 4, 7, 9, and 14 was confirmed
with the appropriate painting probes; we could find no evidence of
chromosome 14 on chromosome 7. Therefore, the consequences of these
rearrangements is an extra Y, duplication of 1p, loss of part of 14q,
and an extra copy of some portion of chromosomes 4 and 9, both as yet undefined.
SKY confirmed conventional G-banded karyotype analysis in 6 abnormal
cases. In one case (Case 10), no abnormal cells were detected; however,
abnormal cells were identified in fewer than 10% (2 of 23) of the
cells in the bone marrow (BM) sample analyzed in the
clinical laboratory. G-banded analysis of Case 4 showed t(X;7), t(8;9),
and del(9p), all of which were confirmed by SKY; no additional
abnormalities were identified (Fig 2, upper left). The 8;9
translocation was also confirmed with painting probes. The genetic
consequence was loss of 9p. With G-banding, 29 cells of Case 5 had a
t(1;16), which was confirmed using SKY (Fig 2, lower right). The
translocation was also confirmed with painting probes for chromosomes 1 and 16. The G-banded karyotype in Case 6 showed a t(7;11) in 19 of 23 cells. This was confirmed by SKY, which also confirmed that it was a
balanced translocation (Fig 2, lower left). The G-banded analysis of
Case 7 showed a del(9p) in all five cells and a del(6q) in three of
them. Thus, the genetic consequence was loss of part of 9p in all cells
and of 6q in some cells.
The G-banded karyotype in Case 8 showed a deletion of 1p in all
abnormal cells and an inv(7) in most of the abnormal cells. SKY
confirmed del(1p) and inv(7) in two cells.
We studied a sample from case 8 obtained in first relapse, 3.5 years
after the initial sample. The original abnormalities were present as
well as several new ones. The cells had a t(2;15)(q37;q15) that was
confirmed by SKY. The breakpoints were modified based on SKY to 2q3?3
and 15q1?5. The karyotype contained an add(1)(p21) as well as a
der(17)t(1;17)(q23;p11). SKY showed that the add(1) contained only
chromosome 1 material. The banding pattern of the dark band at the end
of the short arm appeared to be 1q31. In addition, the der(17) lacked
any evidence of 1q31. Therefore, a plausible explanation is that there
was a break in 1q23 with 1q23 to 1q32 being translocated to the add(1p)
and 1q32 to 1qter being translocated to the der(17). Deletions of 5q
and 6q were confirmed by SKY. In addition, an undetected translocation
involving the long arm of both chromosomes 12 and 14 was observed; this rearrangement was confirmed using a painting probe for chromosome 12 and a telomere probe for 14q.
This relapse sample was obtained after extensive chemotherapy,
radiotherapy, and a bone marrow transplant; a deletion of 5q is common
in this situation. The additional structural rearrangements could also
be secondary to exposure to these mutagenic agents. The results of the
chromosome rearrangements are loss of material from 5q, 6q, and 17p and
triplication of genetic material from most of 1q. The genetic
consequence of the 12;14 translocation is unclear.
An interchange between both chromosomes 9 as well as a deletion of one
chromosome 14 was seen on conventional karyotyping in Case 9. These
abnormalities were confirmed with SKY. Case 10 had a t(11;14) seen in 2 of 23 cells. Only 7 cells were analyzed using SKY, and all had a normal karyotype.
We examined cells from 5 patients (Cases 11 through 15) said to have a
normal karyotype; four of these patients were younger than 17 years of
age. Based on SKY analysis of about 7 cells per patient (range, 4 to 9 cells), all of the cases seemed to have a normal karyotype. This number
of cells is too low for our results to be considered more than
preliminary. However, considering that all 5 were bone marrow samples
at diagnosis, which for Cases 12 and 13 contained 99.5% and 94%
blasts, respectively, our data suggest that leukemia cells of some
patients may have a normal karyotype.
| |
DISCUSSION |
SKY clarified the abnormalities in three patients with complex
karyotypes. In each case, the standard G-banded analysis showed chromosomes that had added unidentified material. SKY confirmed the
analysis of G-banded chromosomes in 8 cases that had balanced translocations or deletions alone or in combination. SKY also confirmed
the presence of a normal karyotype in five patients. We did not
identify any recurring translocations in the patients with a normal
karyotype analogous to the recently discovered t(12;21) in B-cell ALL.
However, the number of patients with normal karyotypes examined,
especially of children, was small; moreover, only about seven cells
were analyzed for each of these patients.
The most common abnormality seen in three of our patients was a
del(9p), accompanied in all three by a del(6q); these abnormalities were identified by standard G-banding. We identified two balanced and
one unbalanced translocations involving chromosome 9, one on the short
arm and two on the long arm. All of these occurred in cells with 6q
deletions, and in two patients, the del(6q) was clearly a secondary
event. One patient had an unbalanced translocation involving 12p,
leading to a deletion of part of 12p. Deletions of 6q, 9p, and 12p are
well recognized recurring abnormalities in ALL of both the B- and
T-cell lineages.1-3 In the recent report from
CCG,2 31 of 169 children with T-cell ALL had
del(6q), and 15 had del(9p). In the past, we and others have suggested that, in fact, del(9p) may be more common in T-cell ALL than in B-cell
ALL.10-12 The analysis of karyotype and survival of
children with T-cell ALL showed that there was no statistically
significant difference among various cytogenetic
subgroups.2
There has been a significant improvement in the software used to
analyze the spectral images, although there are still technical problems that can interfere with an optimal analysis. For example, the
results vary considerably depending on the amount of cytoplasm remaining after the hybridization, which may lead to background and
misclassification of some chromosomes. In addition, new software that
produces an improved reverse DAPI image allows more accurate G-banded
analysis and thus, better identification of deletions, duplications,
and inversions. However, at present, SKY should be used as an adjunct
to classical cytogenetics. No matter how good the resolution may seem,
it is still not as precise as a G-banded metaphase cell because of the
harsh treatment of the chromosomes during the hybridization process.
At present, the type of therapy administered to patients is often based
on the genetic aberrations identified in the patient's cells, as
defined by specific chromosome abnormalities. There seems to be no
correlation between cytogenetic subgroups using conventional
cytogenetic analysis and survival in children with T-cell
ALL.2 Accurate cytogenetic analysis is the best, and in
fact, for some aberrations, it is the only means of detecting these
changes. Thus, for the foreseeable future, any technique that enhances
our ability to define the genetic changes in cells, and therefore,
results in more accurate karyotypic diagnosis, may potentially have a
measurable impact on treatment. As we have shown in this report, the
cytogenetic findings in 3 of 15 patients were modified based on SKY.
All 3 patients had abnormalities that were recognized by the
cytogeneticists as being incompletely characterized. Except for one
case (No. 10) which had only 10% abnormal cells, SKY confirmed the
original analysis in the other 11 cells. Thus, SKY provides an
additional tool that will be important to clarify the karyotype in
patients in whom complex and incompletely defined abnormalities were
identified on standard karyotype analysis.
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ACKNOWLEDGEMENT |
The authors thank Dr Michelle LeBeau for providing the conventional
cytogenetic data, Margie Isaacson for data management and Elizabeth
Davis and Raphael Espinosa for assistance with SKY hybridization and
preparation of the figures.
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FOOTNOTES |
Submitted July 9, 1998; accepted November 2, 1998.
Supported by National Cancer Institute Grant No. CA42557 (J.D.R.);
Purchase of the SKY equipment was partially funded by Amgen, Inc,
Thousand Oaks, CA.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Janet D. Rowley, MD, DSc, University
of Chicago, Medical Center, Section of Hematology/Oncology,
5841 S Maryland Ave, MC 2115, Chicago, IL 60637-1470; e-mail:
jdrowley{at}mcis.bsd.uchicago.edu.
| |
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ABSTRACT
INTRODUCTION