1. No category

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1993 82: 904-913
Detection of monosomy 7 and trisomy 8 in myeloid neoplasia: a
comparison of banding and fluorescence in situ hybridization
RE Kibbelaar, JW Mulder, EJ Dreef, H van Kamp, WE Fibbe, JW Wessels, GC Beverstock, HL
Haak and PM Kluin
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Detection of Monosomy 7 and Trisomy 8 in Myeloid Neoplasia: A Comparison
of Banding and Fluorescence In Situ Hybridization
By R.E. Kibbelaar, J.W.R. Mulder, E.J. Dreef, H. van Kamp, W.E. Fibbe, J.W. Wessels, G.C. Beverstock,
H.L. Haak, and Ph.M. Kluin
Fluorescence in situ hybridization (FISH) is a powerful tool
for detection of numerical and structural chromosomal
aberrations. We have compared conventional banding
techniques and FISH for the detection of monosomy 7
( 7)and trisomy 8 ( 8) in 89 patients with myeloid malignancies. Of these patients, 21 had 7,30 had 8,four
had both, and 34 had no aberrations or aberrations other
than
7 or
8 as assessed by banding techniques. Sequential samples were available in 23 patients. Alphoid
DNA probes specific for chromosomes no. 7 and 8 were
used for FISH. As controls, 10 normal bone marrow (BM)
samples were hybridized with the chromosomes no. 7 and
8 probes, and in addition all tumor samples were hybridized with a chromosome no. 1 specific probe. The cut-off
value for 7 was 18%one-spot cells, and for 8 was 3%
three-spot cells. FISH analysis of 44 samples with 7 or
8, and at least 10 metaphases evaluated, showed that
the proportions of aberrant metaphase cells mirrored the
interphase clone sizes. Most samples with nonclonal metaphase aberrations, including those with only a few metaphases, had increased numbers of aberrant interphase
cells: 20% to 80%for - 7, and 3% t o 43% for
8. Interphase cytogenetics of the 3 4 samples without 7 or 8
did not show significant cell populations with 7 or 8.
In four patients, 7 or 8 could not be confirmed by FISH
due t o additional structural aberrations, marker chromosomes, or wrongly interpreted banding results. As FISH
will be used more and more in cytogenetic diagnosis, clinical follow-up, and therapy monitoring, it will be necessary
to standardize FISH procedures and supplement the
Standing Committee on Human Cytogenetic Nomenclature (ISCN) definitions of a clone with criteria specifically
for in situ hybridization.
0 1993 by The American Society of Hematology.
M
ANY HEMATOLOGIC malignancies are characterized by specific numerical or structural chromosoma1 abnormalities that have prognostic, therapeutic, and
biologic implications.’.’ Two of the most common numerical aberrations in myelodysplastic syndromes (MDS) and
acute myeloid leukemia (AML) are monosomy 7 (-7) and
trisomy 8 (+8). These and other chromosomal aberrations
can be detected by banding and fluorescence in situ hybridization (FISH) techniques, both having their own specific
advantages and disadvantages.
For banding techniques, metaphase cells must be obtained and the metaphase cells harvested must have a certain quality for proper banding and evaluation. As the metaphase cells are only a small subset of the cells present in the
cell suspension, the qualitative and quantitative potentialities of banding techniques may be limited. In addition, the
quality constraint may contribute to biased qualitative and
quantitative analysis. Recent developments in DNA in situ
hybridization probes and protocols, in particular FISH,
have made this technique widely available for cytogenetic
research and diagnostic^.^-^ The molecular in situ hybridiza-
tion approach can supplement morphologic banding analysis in several ways: (1) marker chromosomes can be identified, that is, the morphologic identification can be
validated7,*;(2) breakpoints caused by deletions, inversions,
or translocations can be delineated more preci~ely;~*’~
(3)
metaphase cells of poor quality or those obtained from archival material can be analyzed; and (4) interphase cells can
be studied (interphase cytogenetics), hence the in vivo occurrence of chromosomal aberrations can be assessed and
quantified. Only a few reports have been published on the
benefit of FISH versus banding cytogenetic analysis in large
series of patients. For example, Jenkins et all’ examined
material of 60 patients with +8, 13 with nonclonal +8, and
144 with normal karyotypes or aberrations other than +8.
In our study, banding and in situ hybridization cytogenetics were compared, both qualitatively and quantitatively, on bone marrow (BM) or peripheral blood (PB) cells
of 5 5 patients with myeloid malignancies and -7 or +8
identified by banding techniques. Furthermore, material
obtained from 34 patients who had normal karyotypes or
aberrations other than -7 or +8 was also studied.
From the Departments of Pathology, Hematology, and Human
Genetics, University of Leiden, Leiden; and the Department of Hematology and Cytogenetics, Leyenburg Hospital. The Hague, The
Netherlands.
Submitted December 16, 1992; accepted March 31, 1993.
Supported by Grant No. 6.2.3fvom the J.A. Cohen Institute, Leiden, The Netherlands.
Address reprint requests to R.E. Kibbelaar, MD, Laboratory of
Pathology, State University of Leiden, PO Box 9603, 2300 RC Leiden, The Netherlands.
The publication costs of this article were defiayed 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.
0 1993 by The American Societ.v of Hematology.
0006-49 71/93/8203-0041$3.00/0
Patients. BM or PB cells of 5 5 patients with myeloid malignancies, mostly AML and MDS, and -7 or +8 as assessed by banding
analysis, were available for in situ hybridization studies. This included patients with nonclonal abnormalities according to the specifications of the Standing Committee on Human Cytogenetic Nomenclature (ISCN).” Cell suspensions were stored for up to 4 years
before in situ hybridization was performed. Data of nine patients
have been previously reported in part (Table 2, no. 4. 6 , 7 , 10, 15,
19,20, 22, and 25).9.’3Additionally, material from 34 patients with
MDS or AML and normal karyotypes or aberrations other than -7
or +8 was investigated with FISH. Additional hematologic data,
including the differential, were obtained for each sample, and clinical follow-up data were collected. These are not represented in the
tables, but can be made available by the authors. BM cells of 10 BM
transplantation donors were used as negative control samples.
Banding analysis. Metaphases were obtained after a 30-minute
+
-
+
+
-
-
+
-
+
-
+
-
+
-
+
+
MATERIALS AND METHODS
904
Blood, Vol 82, No 3 (August 1). 1993: pp 904-9 13
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905
-7 AND +8 DETECTION BY FISH
mitotic arrest from BM and PB, either directly or after 24-, 48-, and
72-hours cultures, hereafter referred to respectively as BMD, PBD,
BM24, PB24, etc. Cells were harvested and processed according to
standard techniques, after which GTG-bandingwas perf~rmed.’~
If
possible, at least 20 metaphases were analyzed, and five metaphases
were fully karyotyped. Karyotyping was performed following ISCN
specifications.12Sampleswere classified as -7 if at least three metaphases were found with a deleted chromosome no. 7. Trisomy 8 was
diagnosed if at least two metaphases were found with an extra chromosome no. 8. If these criteria were not met, the sample was classified as nonclonal -7 or +8.
FISH. FISH was performed on BM and PB cell suspensions
that had been used for banding analysis stored in methano1:acetic
acid (3: 1 vol/vol) at -20°C or +4”C. Also, BM aspirates (BMA),
used for cytologic diagnosis and obtained simultaneously with the
samples used for banding, were fixed and stored for FISH studies.
Alphoid repetitive DNA sequences specific for chromosomes no. 7
(p7tl for locus D7Z1)I5and 8 (ATCC, D8Zl),” hereafter referred to
as ap7 and ap8, respectively, were used as probes. As a control
probe, satellite DNA specific for the lq12 heterochromatic region
(PUC1.77) was used.I6 In some cases, additional hybridizations
with chromosome-specificlibraries were performed.” Fixation of
cells, preparation of slides, predigestion of cells, hybridization conditions, posthybridization washings, and immunodetection were all
performed according to standard protocols as previously deVisualization of the hybridization reactions was done by
fluorescein isothiocyanate (FITC). Hybridization signalswere evaluated on a LEITZ Diaplan fluorescence microscope (Emst Leitz
Wetzlar GmbH, Germany) equipped with a K3 filter for simultaneous detection of FITC signals and propidiumiodide (PI) DNA
counterstaining. All slides were analyzed by at least two observers.
From each preparation, 200 interphase nuclei and all available
metaphases present on one slide were evaluated. As the differences
between the observers varied in general between 0%and lo%, the
results were summed and averaged.
RESULTS
Controls. Two types of controls were used for determination of the cut-off values for detection of -7 and +8: (1)
the test probes specific for chromosomes no. 7 and 8 were
hybridized onto control BM cells with normal karyotypes,
and (2) the tumor cells were hybridized with a control probe
specific for chromosome no. 1 [N = 82, samples with structural or numerical abnormalities of chromosome no. 1, eg,
+t( 1;7) or +t( 1;9) were excluded]. The first type of control
gave an estimation of the specificity of the test probes, and
the second control provided an estimation of the overall
hybridization quality of the test material.
The results of the control studies are listed in Table 1. For
detection of -7, the control samples showed a cut-off level
of 1 1.7% (mean + 2 SD), and the control probe showed a
cut-off level of 17.7%. Therefore, a threshold value of 2 18%
was used for detection of -7. To distinguish true -7 from
suboptimal hybridization, all samples with more than 15%
null spot cells were rehybridized with prolonged predigestion times; if the results did not improve, these data were
discarded. For detection of +8, the studies of the control
samples and control probe showed cut-off levels of 2. I % and
2.9%, respectively. Therefore, a threshold of 23%was used
for detection of +8 by interphase in situ hybridization.
Detection of - 7 and +8 with FISH. Clinical and cytogenetic data from the -7 and +8 patients are listed in Tables 2
Table 1. Distribution of Spots in Controls
% of Cells (mean and SD): No. of Spots/Cell
Probe
Material
ap7
BMT donor (N = 10)
Mean
SD
Mean 2 SD
BMT donor (N = 10)
Mean
SD
Mean 2 SD
-7 and +8 study
group (N = 82)
Mean
SD
Mean 2 SD
+
ap8
+
spl
+
0
1
2
3
4
0.9
0.8
6.5
2.6
11.7
92.5
2.9
0.1
0.2
0.0
0.7
0.7
2.6
1.7
96.0
2.4
0.7
0.7
2.1
0.0
1.1
2.3
8.9
4.4
17.7
88.8
5.8
0.9
1.0
2.9
0.3
1.3
0.0
0.0
Two groups of controls for determination of the levels of false-positive or false-negative FISH hybridization signals: (1) karyotypically normal BM cells hybridizedwith the test probes ap7 and ap8; (2) test samples from the -7 and +8 group hybridizedwith a control probe specific
for chromosome no. 1 [samples with structuralor numericalaberrations
of chromosome no. 1 like +t(l;7) or +t( 1 ;9) were excluded]. See text for
further discussion and interpretation of the results.
Abbreviations: ap7, alphoid DNA probe specific for chromosome 7;
ap8, alphoid DNA probe specificfor chromosome 8; BMT, bone marrow
transplantation; spl. satellite DNA probe specific for chromosome 1.
and 3, respectively. Twenty-one patients had -7 (clonal or
nonclonal), 30 had +8 (clonal or nonclonal), and four had
combined -7 and +8 (no. 22 through 25 in Table 2, and no.
31 through 34 in Table 3). Sequential samples were available in 23 patients. In four samples with +8, parallel BMD
and BMA specimens processed separately in different laboratories demonstrated similar percentages of three-spot interphase cells (Table 3, patients no. 4, 14, and 19). These
results corroborate the reliability of FISH procedures, both
for the detection of low and high percentages of +8 cells.
In 51 of 55 patients, FISH confirmed the presence of -7
or +8 as detected by banding (Fig 1). The following four
patients showed a discongruency between banding and
FISH: (1) patient no. 5 of the -7 group showed 1 I of 11
metaphases with a -7 and three marker chromosomes by
banding (PB, there was no material left for FISH); the parallel BM culture showed five of five metaphases with two
spots, and no significant number of one-spot interphase
cells by FISH (no assessable metaphases in the banding analysis). Most probably, the marker chromosomes contained
chromosome no. 7 DNA (no material left for chromosome
painting). (2) Patient no. 7 had a complex karyotype with a
t( l;?) and multiple markers. Chromosome painting showed
multiple fragments of chromosomes no. 1 and 7 in metaphase cells without a t ( 1;7).9(3) Patient no. IO had a -7 and
an additional t(7;16) in I 1 of 12 metaphases. FISH with the
ap7 probe did not show a significant increase of one-spot
cells. Chromosome painting demonstrated the presence of
one complete and one partial chromosome no. 7 (Fig 2). (4)
Patient no. 11 of the +8 group had 26 of 29 metaphases with
a +8. FISH with the ap8 probe and with a chromosome no.
8-specific library could not demonstrate any additional
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906
KIBBELAAR ET AL
Table 2. Monosomy 7, Clinical and Cytogenetic Data
Patient
Age/
No.
Sex
1
61/F
2
69/F
3
4
72/M
44/M
GTG Banding
Diagnosis
5
8O/F
AML M2
Feb 86
Feb 86
Feb 86
Jul86
Aug 87
Sep 86
Apr 88
Dec 88
Sep 88
6
82/M
RAEB-t
Mar 89
RAEB-t
AML M4
CMML
Anemia
MDS
RAEB-t
RAEB
, RA
MDS/AML
AML
RAEB-t
CMML
MDS
AML M2
7
8
9
10
11
12
13
65/M
73/F
29/M
72/F
61/F
52/F
49/F
14
15
16
5 1 ~
73/M
88/F
RAEB-t
AML M5a
AML M1
17
18
75/M
62/F
AML M2
AML M5b
19
21
68/M
7O/F
72/F
AML M2
AML M4
CMML
22
55/F
AML
23
49/M
AML M4
20
24
25
78/F
87/F
M PS
MDS
(proportion of -7 cells)
Date
May 90
Aug 9 0
Oct 89
Jun 89
Apr 91
Jun 88
May 86
Oct 90
Oct 9 0
Oct 89
Sep 86
Jul87
Sep 87
Dec 89
Nov 87
Feb 88
Jun 88
Jun 88
May 91
May 89
BM24 (212)
PB24 (617)
PB (617)
PB24 48 72 (213)
BM24 ( 1/20)
PB72+t (2114)
BM24 (29129) PB48 (-) PB48+b (214)
BM24 (13116)
PB+t (1111 1)
BM24 I-)
BMD (14114)
BM24 (not analyzed)
+
+
BMD (8116)
PB24 (14/ 14)
BM24 (20120)
BMD (1 1/12)
PB24 + 48 (1 8118)
BM24 (9113)
PB72 (22122)
May 88
Nov 88
Feb 86
BM24 (1511 5)
BM24 (25132)
PB48 (313)
BM24 (818)
BM24 (10115)
BM24 (14/33)
BM24 (0116)
PB24 (313)
BM24 (718)
BMD (9120)
BM(12/12)
PB72 (-)
BM24 (0115)
PB24 + 48 + 72 (919)
PB24 5 abnormal metaphases
Aug 86
Apr 87
Nov 87
6/57)
PB48 10 HAM$ a.0. 5q-, -7
BM24 (1110)
PB24 (111) PB48 (NE)
BM24 (2115)
FISH
Material
Metaphases'
With One
interphases (%I
With One Spot
spot
SP 1
ap7
BM24
PB24
12/25
11/29
6
10
29
30
PB72
BM24
010
0110
8
9
82
9
PB48
BM24
1I 1
23/35
12
8
55
89
BM24
015
9
9
BM24
BMA
BMD
0150
PB24
BM24
BMD
PB48
BM24
PB72
BMA
BMD
BM24
PB48
BM24
BM24
BM24
213
5/16
0143
12/16
17/27
29/40
20137
411 8
26/36
40148
12/44
29/30
11
7
4
9
10
5
12
12
22
4
7
12
6
6
10
19
11
64
11
10
15
3
89
66
72
70
71
21
75
74
53
89
PB24
BM24
BMD
414
29/29
34/50
7
8
3
83
93
69
PB72
BM24
PB24
PB24
010
3117
13/19
1 /7
12
4
17
9
56
12
57
14
BM24
PB48
BM24
0144
3115
9
9
21
78
12
14
1/46
111
Myelodysplastic syndromes (MDS)and acute myeloid leukemias (AML) were classified according to the subdivisions of the French American British
(FAB) cooperative g r o ~ p .It~was
~ . not
~ ~possible to subclassify all individual samples, in these cases the diagnosis MDS or AML NOS was used.
Abbreviations: RA, refractory anemia; RARS. RA with ringed sideroblasts; RAEB, RA with excess of blasts; RAEB-t, RAEB in transformation; CMML,
chronic myelomonocytic leukemia; BMA, bone marrow aspirate; BMD. bone marrow direct culture; BM24.24-hour culture, etc; ND, not done; NE, not
evaluable; a.0.. among others; PB, peripheral blood; PBD, peripheral blood direct culture; PB24, 24-hour culture, etc.
Number of aberrant metaphases versus total of metaphases analyzed.
t Peripheral blood samples were cultured with phytohemagglutinin.
t Highly abnormal metaphases.
chromosome no. 8 DNA. Therefore, the identification of
the extra chromosome with standard GTG-banding was not
correct.
Material of 34 AML or MDS patients with aberrations
other than -7 or +8 was investigated by FISH. Twenty-five
had normal karyotypes as assessed by banding analysis. In
33 patients, at least 20 metaphases and in one patient six
metaphases were karyotyped (data not shown). Using the
criteria outlined above, a -7 or +8 was not found in any of
the samples using interphase cytogenetics. The average number of cells with one spot using the ap7 probe was 4.8 (maximum, 16; minimum, 0; SD = 3.44), and the average number of cells with three spots using the ap8 probe was 0.8 1
(maximum, 2.5; minimum, 0; SD = 0.81).
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-7 AND +8 DETECTION BY FISH
Quantitative comparison of banding and FISH. The
proportions of aberrant metaphase cells, both by banding
and FISH, were compared with the proportions of aberrant
interphase cells. To ascertain any quantitative significance
of our conclusions,all samples with fewer than 10 assessable
metaphase cells were excluded. Using this arbitrary criterion, 16 samples of the -7 group could be compared for
metaphase FISH versus interphase FISH (Fig 3A) and 10
samples could be compared for banding versus interphase
FISH (Fig 3B). All samples but one had a clonal -7 according to the ISCN criteria. A good correlation between metaphase FISH and interphase FISH was found (Y = 37). Comparison of interphase FISH with banding corroborated the
presence of a -7 clone, but did not show any quantitative
relation (Fig 3B).
In the +8 group, 28 samples could be compared for metaphase and interphase FISH (Fig 4A) and 26 samples for
banding versus interphase FISH (Fig 4B). The FISH metaphase analysis included six samples with a nonclonal +8. A
high correlation ( r = .89) between metaphase FISH versus
interphase FISH was found. In contrast to the -7 study, a
high correlation between banding and interphase FISH was
also found ( r = .93). Nine samples had a nonclonal +8 in
the banding analysis; in these samples, 3% to 10%three-spot
interphase cells were found.
To obtain further insight into the quantitative aspects of
banding and FISH on metaphase cells of patients with
clonal abnormalities, we extracted relevant data from Tables 2 and 3 (Table 4). Samples with fewer than 10 assessable metaphase cells were also included, but grouped separately. Although, in contrast to banding, FISH was
performed on only one slide, generally more metaphase
cells could be evaluated by FISH than by banding. This
emphasizes the practical significanceof FISH. Using a 25%
difference as a criterion, the clone size as estimated by FISH
analysis of metaphases was smaller than as assessed by
banding in approximately half of the -7 samples (Table 4).
In case of +8, no gross differenceswere found between both
techniques. The data showed that the discrepancies for -7
were relatively independent of the total numbers of cells
analyzed, ie, they apparently were not caused by quantitative inaccuracies.
FISH analysis of nonclonal samples. We investigated
whether it was possible to detect a clone of aberrant cells
with FISH in cases with nonclonal (by ISCN criteria) -7 or
+8. Samples with inconclusive banding analysis, eg, two of
two aberrant metaphases, were also included. Seven samples of the -7 group (Table 2, patient no. 1 [BM24], 2
[PB72], 3 [BM24], 4 [PB48], 23 [BM24], 24 [PB48], and 25
[BM24]) were assessable. FISH metaphase analysis of patient no. l showed a -7 clone (12/25 one-spot metaphase
cells). FISH interphase analysis of patients no. 1, 2, 4, and
23 demonstrated 29%, 82%, 55%, and 78% one-spot cells,
respectively, also indicative of -7 clones. The presence of
these -7 clones was confirmed by banding of other samples
of the same patients.
In three patients (no. 3, 24, and 25), no -7 clone was
found by FISH. Follow-up of patient no. 3 showed intravascular hemolytic anemia due to a heart valve prosthesis, in-
907
stead of myeloid malignancy. Banding analysis of patient
no. 24 showed one metaphase with a complex karyotype
including -7, +8, and marker chromosomes in the PB24,
while the PB48 was not assessable. No PB24 cells were left
for FISH, and a -7 clone could not be detected in the PB48.
This leaves several possibilities: the metaphase with -7 was
an artifact, the aberrant clone was lost during the prolonged
culture, or the two spots per cell in 87% of the cells resulted
from one chromosome no. 7 and one marker chromosome.
Patient no. 25 had two of 15 metaphases with 5q-, -7, and
one marker chromosome. No increase of -7 interphase
cells was detected by FISH, but metaphase analysis suggested a clonal aberration (3/15 one-spot cells). However,
these FISH data are difficult to interpret, especially since the
chromosome no. 1 control probe showed 2 1% of interphase
cells with one spot, indicating suboptimal hybridization
properties of the material.
The first 10 +8 patients listed in Table 3 had one or more
samples with a nonclonal +8. Four of them have been reIn the first nine
ported previously (no. 4, 6, 7, and 10).9,'3
patients, FISH demonstrated 3% to 14% three-spot interphase cells, indicating that the samples contained small populations of +8 cells. Note that other samples of these patients did not show +8 by banding, but consistently
demonstrated a significant proportion of +8 cells as assessed
by FISH. Patient no. 10 had +8 in one of five cells analyzed
by banding, but 43% three-spot interphase cells. The high
number of three-spot cells with the chromosome no. 1 control probe was caused by a +t( 1;7).9
Quantitative discrepancies: Selected cases. To determine the biologic significance of the quantitative differences between banding and interphase cytogenetics, we
compared all available samples, especially the follow-up
samples of several patients. (1) The BMD of patient no. 6 of
the -7 group had 14 of 14 aberrant metaphases, but no -7
interphase clone was observed in the same sample cultured
for 24 hours (no BMD cells available for FISH). However, a
BMA taken 14 months later showed 64%one-spot cells. The
negative FISH results in the first BM24 sample might have
been caused by selective loss of the aberrant myeloid cells
during culture, or by interchange of patient samples. (2) The
PB sample of patient no. 8 of the -7 group had 14 of 14
metaphases with -7 by banding, two of three one-spot cells
by metaphase FISH, but only 10%one-spot interphase cells.
This discrepancy might be explained by the presence of 66%
nondividing lymphoid cells (differential not shown) in the
PB sample. We, and others, have shown that these genetic
alterations in myeloid malignanciesare restricted to the myeloid c ~ m p a r t m e n t . ' ~
Taking
- ~ ~ into consideration the aforementioned cut-off value of 18%for one-spot cells, it would
be impossible to detect a -7 clone in interphase cells of this
sample. (3) Patient no. 9 of the -7 group had 20 of 20
metaphases with -7, whereas FISH showed 15 of 16 onespot metaphase and 15%one-spot interphase cells. This discrepancy cannot be explained (no follow-up available). (4)
The data of patient no. 22 of the -7 group suggest that with
FISH metaphase analysis the -7 clone could have been detected 6 months before it was found by banding analysis. In
the second sample, the clone size as measured by interphase
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908
KIBBELAAR ET AL
Table 3. Trisomy 8, Clinical and Cytogenetic Data
Patient
No.
1
Age/Sex
71/M
Diagnosis
2
31/F
RAEB
RAEB-t
AML
AML
ET/PV
3
72/M
AML M2
4
66/F
MDS
5
6
58/M
67/M
RAEB
RARS
Date
Apr 89
Aug 89
Nov 89
Feb 90
Oct 81
Aug 86
Mar 90
Apr 90
May 90
Jul90
Aug 90
Oct 89
Jul90
Jun 89
Sep 89
Oct 89
GTG Banding
(proportion of +8 cells)
BM24 (0118)
BM24 (0113)
BM24 (1116)
BM24 (1111)
PBD (0120)
BM24 (1156)
BM (-) PB (-)
PB72 (011)
PB24 (818) PB48 (214)
BM24 (1116)
BM24 (0116)
BM24 (1136)
BM24 (1130)
BMD (0120)
BMD (1120)
BM24 (1?/20)
7
8
55/F
67/M
MDS
MDS
Nov 89
Feb 90
May 90
BMD (1130)
BMD (-)
BM24 (0110)
Sep 90
RAEB
11
12
42/M
74/M
AML M2
RAEB-t
AML M6
RA
13
61/M
MDS
Nov 88
Oct 89
Jan 90
May 90
May 86
Sep 86
Jun 91
Mar 90
Jun 90
Aug 91
Sep 88
14
49/M
RAEB-t
Aug 91
9
68/M
RAEB-t
10
44/M
BMD (1122) BM24 (0/30)
BM24 (1120)
BM24 (018)
BM24 ( 1/20)
BM24 (012)
BM24 (114)
BM24 ( 115)
BMD (26129)
BM24 (2122)
BM24 (-)
BM24 (718)
PB24 (-) PB48 (--)
PB72+t (215)
BMD (1 13)
AML
15
16
81/F
79/F
RAEB-t
AML M2/M4?
17
78/F
RAEB-t
18
19
78/M
43/M
AML M1
RARS
Sep 91
Oct 91
Feb 89
Nov 90
May 91
Jun 91
Jun 89
Sep 89
BM24 (20/20)
BM24 (014)
PB24 (414)
BMD (14114)
BM24 (2?/9)
BMD (20121)
Nov 89
Jan 90
RAEB-t
20
73/F
RAEB
Jun 90
Mar 91
Jan 90
Jan 90
BMD (13120)
BMD (20120)
BM24 (20120)
(Continuedon followingpage)
FISH
Material
BM24
BM24
BM24
BM24
BM24
BM24
PB48
BM24
BM24
BMA
BM24
BM24
BMD
BMA
BMA
BMD
BM24
BMA
BMD
BMA
Metaphases'
With Three
spots
1/22
Interphases (%)
With Three
spots
1
1
0
0
66
5
6
11
2
1
7
14
0
1
1
0
ND
1
1
1
ND
ND
ND
1
0
25
3
4
3
3
3
3
3
6
4
4
4
3
7
o/ 1
3
0
7
5
0117
0115
3
0
3
4
012
0150
0126
0
411 9
3/50
34
3
2
2
1
2
43
3
2
3
35
11
2
9
46
4
2
1
1
0
3
0
ND
ND
ND
ND
ND
8
6
19
2
64
1
29
48
67
54
87
62
47
80
76
1
ND
ND
78
83
81
319
3/10
012
011 1
014
015
015
0127
111 1
1/25
1/40
1
1/20
1/20
2/20
-
BM24
BMA
BM24
BM24
-
BM24
BMD
BM24
BM24
BM24
PB24
BMA
BMD
BMA
BMA
BM24
BM24
PB24
BMD
BMA
BM24
BMA
BMD
BMA
BMA
BMD
BMA
BM24
BMA
7/50
20120
014
NE
012
28/29
10/10
010
313
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
-7 AND +8 DETECTION BY FISH
909
Table 3. Trisomy 8, Clinical and Cytogenetic Data (Cont'd)
GTG Banding
Patient
NO.
Age/Sex
21
62/F
AML M5a
22
55/M
71/F
RA
AML M3
23
61/F
24
52/F
75/M
25
26
Diagnosis
MDS
MDS
AML M2
Date
Aug 86
Sep 86
Oct 86
Jul89
Aug 90
Sep 90
Oct 90
Aug 90
Dec 90
Apr 89
Sep 90
Oct 90
27
67/M
RAEB
28
65/M
CMML
29
30
77/M
81/M
RA
MPS
31
55/F
AML/PV
32
49/M
AML M4
May 88
Nov 88
Feb 86
33
34
78/F
871F
MPS
MDS
Aug 86
Apr 87
Nov 87
Mar 91
Sep 91
Jul88
Jul89
Apr 88
Feb 86
(propoflion of +8 cells)
BM24 (10116)
Em24 (20125)
BM24 (0115)
BM24 (7127)
PB24 (47##=1[+8?/1])
BM24 (818)
BM24 (318)
PB24 (12112)
BMD (not analyzed)
BM24 (13125)
*" ( 15?/20)
BM24 (-)
PB24 (414)
BM24 (13116)
BMD (24129)
BMD (23128)
8M24 (6120)
BM24 (2116)
PB24 (010)
PB48 (4118)
BM24 (0115)
PB24 + 48 + 72 (919)
PB24 5 abn. metaph. (5?/5)
PB48 10 HAMS a.o.5q-, -7
BM24 (1110)
PB24 (111) PB48 (NE)
BM24 (311
. . 5).
FISH
Material
BM24
BM24
BM24
BM24
PB24
BM24
BM24
PB24
BMA
BMD
BM24
*
BM24
PB24
BM24
BMA
8MD
-
BM24
BM24
PB24
PB48
BM24
PB24
PB24
Metaphases.
With Three
spots
Interphases (%)
With Three
spots
SPl
np8
47/50
0
0
0
2
1
1
2
0
0
0
3
66
0
0
0
1
2
55
60
7
15
44
17
69
69
23
40
38
57
66
62
41
86
73
416
5/50
0119
0136
0150
14/16
16/21
0
1
3
0
40
29
0
35
7
9
3
8
37
45
40150
0114
0
0
67
12
34/50
37/50
0150
6/18
26/42
16/26
10113
32/48
20150
NE
010
010
516
48/50
PB48
BM24
* t S See legends to Table 2.
11 BM cells were deep frozen, thawed, and cultured for 4 days stimulated with granulocyte-monocyte colony-stimulating factor (GM89-107, Sandoz).
-
Fig 1. FISH detection of 7
8. Hybridization signals
and
were visualized with FlTC and
DNA was counterstained with
PI (original magnification X
63). (Top) Interphasecellsof patient no. 19 from the - 7 group,
hybridized with ap7, and demonstrating one spot per cell, indicative of monosomy. (Bottom) Cells of patient no. 26
from the
8 group, hybridized
with up8. One interphase and
one metaphase cell demonstrate three spots, indicative of
+8, and one interphase cell
shows two spots.
+
+
FISH probably was underestimated because of the presence
of 30% contaminating lymphoid cells (differential not
shown).
As reported above, a good quantitative correlation between banding and interphase FISH was found in the +8
samples. Two additional observations were made: ( I ) in
samples with a high proportion of aberrant metaphase cells,
interphase FISH generally showed somewhat lower proportions than banding (Fig 4B,48%to 8370 three-spot cells in
the four samples with 100%+8 cells by banding). This dif-
Fig 2. Metaphase spread of
patient no. 10of the - 7 series,
hybridized with the chromosome no. 7-specific library.
One complete chromosome no.
7 and one partial chromosome
no. 7 is seen. The latter is derived from the +t(7;16) and
caused a normal distribution of
spots per cell using the up7
probe with 97% two-spot cells
and 3% one-spot cells.
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910
KIBBELAAR ET AL
involvement of chromosome no. 8 found with chromosome
painting. The +8 was subsequently confirmed by follow-up
FISH analysis.
K d 1-*pot intamhaw ails
80-
DISCUSSION
70-
In this study, banding and FISH cytogenetics of -7 and
+8 in were compared qualitatively and quantitatively in a
large series of patients with myeloid malignancies. Several
features, including advantages and disadvantages of the
FISH methodology, are demonstrated.
A critical problem in interphase FISH methodology is
determination of the cut-off values for establishing the diagnosis of aberrant clones. This is important for quantitative
applications, eg, the detection of minimal residual disease
8050-
4030-
20,
0
10
io
3b
40
I
eb
70
Q
sb
lb
% 1-spot metaphaw calls
H of 1-
lntsrphgs at8
100
I,
901
70
do
4
50
20
10
10
% d -7 muaphnsemUa
Fig 3. Comparison of metaphaseversus interphase cell analysis
in the - 7 group. Only those samples of which at least 10 metaphases could be analyzed were included. (A) Metaphase FISH (xaxis) versus interphase FISH (y-axis). A linear regression line was
calculated (slope, .91;r = .87; = .75). (B) Banding versus interphase FISH. All but one of the samples (a nonclonal - 7) are also
represented in Fig 3A. The nonclonal sample is indicated with a
filled box. No significant correlation was found. The range of onespot cells (maximum, 16%) observed in the 34 samples with
aberrations other than 7 or 8 is indicated by a bar at the y-axis.
- +
ference might be explained by intrinsic problems of FISH,
ie, suboptimal hybridization properties of the cells, but also
by admixture with lymphoid cells. (2) In seven samples with
few (zero to five) metaphases or poor banding quality of
metaphase cells, significantly increased numbers of interphase cells (6%to 67%)with three spots were found by FISH
(patients no. 3 [March 19901, 13 [September 19881, 14 [August 1991],23 [August 1990],30 [PB24, February 1986],32
[February 19861, and 33 [April 19871). The same (7% and
8% three-spot cells) was found in two samples with sufficient metaphase cells without any indication of +8 (patient
no. 21 [October 19861 and 31 [May 19881). In contrast, in
one patient (no. 12), banding analysis of the first sample
showed two of 22 cells with +8, but no increase ofthree-spot
cells by FISH, nor were trisomic cells or translocations with
8070 W50-
Fig 4. Comparison of metaphaseversus interphase cell analysis
in the
8 group. Only those samples of which at least 10 metaphases could be analyzed were included. (A) Metaphase FISH (xaxis) versus interphase FISH (y-axis). A linear regression line was
= .79). ( 8 )Banding versus intercalculated (slope, .62;r = .89;
phase FISH. Nonclonal samples are indicated by a filled box. A linear regression line was calculated (slope, .69; r = .93;
= .87).
The range of three-spot cells (maximum, 2.5%) observed in the 34
samples with aberrations other than - 7 or 8 is indicated by a
bar at the y-axis.
+
+
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91 1
-7 AND +8 DETECTION BY FISH
Table 4. Bandina Versus In Situ Hvbridization on Metaohase Cells
Trisomy 8
Monosomy 7
Patient
No.
18
11
13
20
4
12
14
17
15
Material
FISH
Bandina
Patient
No.
Material
FISH
Banding
BM24
PB48
PB72
BMD
BM24
BM24
BMD
BM24
BM24
0.97 (29/30)
0.75 (12/16)
0.73 (29/40)
0.68 (34/50)
0.66 (23/35)
0.63 (17/27)
0.54 (20/37)
0.27 (12/44)
0.22 14/18)
r 0 . 4 2 (14/33)
tl (18/18)
<1 (22/22)
>0.45 (9/20)
0.81 (13/16)
0.69 (9/13)
<1 (15/15)
<0.67 (10/15)
<0.78 (25/32)
15
19
26
27
21
21
26
22
29
BM24
BMD
BM24
BMD
BM24
BM24
BM24
BM24
BM24
1 (20/20)
1 (10/10)
0.96 (48/50)
0.94 (47/50)
0.74 (37/50)
0.68 (34/50)
0.67 (32/48)
0.33 (6/18)
0.1 (5/50)
1 (20/20)
0.95 (20/21)
0.81 (13/16)
0.83 (24/29)
0.8 (20/25)
0.63 (10/16)
<1 (12/12)
0.26 (7/27)
0.13 (2/16)
1 (29/29)
1 14/41
0.83 (40/48)
0.72 (25/36)
0.69 (13/19)
0.38 (11/29)
0.88 (7/8)
1 (3/3)
1 18/81
<1 13/31
< l (9/9)
<0.86 (6/7)
20
18
31
26
23
28
23
BM24
BM24
PB24
BM24
BM24
BM24
BM24
1 (3/3)
0.97 (28/29)
0.88 (14/16)
0.83 (5/6)
0.77 (10/13)
0.67 (4/6)
0.62 (16/26)
1 (20/20)
>0.22 (2/9)
1 (9/9)
1 (4/4)
>0.38 (3/8)
>0.3 (6/20)
il (8/8)
Fewer than 10 metaphases
19
18
16
16
22
1
BM24
PB24
BM24
PB48
PB24
PE24
Quantitative comparison of metaphase FISH and banding. The samples are ordered according to the proponions of aberrant cells found with FISH.
The samples are divided into one group based on at least 10 metaphases analyzed both by FISH and banding, and one group where less than 10 cells
could be analyzed. If two ratios differed >25%, this was arbitrary taken as a difference, and these samples are marked with a > or < sign.
or early relapse. Due to variables related to the quality of
cells, probe features, hybridization conditions, immunoreagents, and observers, FISH will result in a certain number
of cells of which the true number of targets (chromosomes)
is not properly assessed. This problem is especially relevant
for determination of loss of chromosomes. Under optimal
conditions, FISH applied to normal diploid cells will result
in values of 5% to 10% one-spot cells (Table 1). We have
developed two types of controls for determination of the
cut-off values for -7 and +8 detection: (1) normal cells
hybridized with the test probes specific for the test chromosomes no. 7 and 8, and (2) a control probe for which the
tumor cells were assumed to be disomic, hybridized onto
the test samples. Cut-off values were determined by calculation of the mean 2 SD for one-spot cells and three-spot
cells, respectively. This procedure resulted in a 18% onespot cell value for -7 detection, and a 3% three-spot cell
value for +8 detection. The relative high -7 cut-off level is
caused in part by the chromosome no. 1 data obtained from
the test cells. However, this high level may be too conservative, in particular as nonrandom loss of chromosome no. I
in the test cells cannot be excluded. For trisomy detection, a
cut-off value of 3% was established, which is similar to the
value reported by Jenkins et al.'' The similar results obtained from parallel BMAs and directly cultured cells validate the conclusion that values as low as 3%cannot be attributed to technique variables.
The main advantage of FISH is the possibility to quantitate aberrant cells. Both in the -7 and the +8 group, we
found that by applying the FISH methodology the relative
numbers of aberrant metaphase cells were proportional to
the relative numbers of interphase cells (Figs 3A and 4A).
+
This indicates that the percentage of aberrant cells in mitosis is representative for the in vivo clone size, and that there
is no mitotic selection. Interestingly,the same was found for
banding versus FISH interphase analysis of +8 (Fig 4B), but
not for -7. Banding analysis generally resulted in higher
proportions of -7 cells compared with FISH (Fig 3B and
Table 4). This may be explained by positive observer bias in
the banding analysis, which is understandable as -7 metaphases are often difficult to evaluate.
We have tried to determine the biologic significance of
nonclonal abnormalities as defined by the ISCN criteria. 12,24,25 One major handicap of the ISCN criteria is that no
minimum number of analyzed metaphases is required. In
our study, we regarded IO metaphases as a minimum for
quantitative analysis, but we also included samples with
lower numbers for qualitative evaluation. Interphase FISH
proved its value especially, but not exclusively, in those
cases with few assessable metaphase cells. Thus, four of
seven samples with a nonclonal -7 had significantly increased numbers of -7 cells by FISH. In the +8 group, all
but one sample demonstrated significant increases of threespot cells (3% to 25%). In most cases, these elevations centered just above the threshold value of 3%. Jenkins et all'
found similar small increases in four of 13 nonclonal +8
patients.
FISH analysis of our 34 patients with normal karyotypes
or aberrations other than -7 or +8 as assessed by banding
analysis showed no percentages of aberrant cells above the
stipulated thresholds. This validates the reliability of the
banding procedures, and provides additional evidence that
levels as low as 3%trisomic cells reflect biologic phenomena
and not artifacts. Using a similar approach in a series of 144
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912
KIBBELAAR ET AL
patients with a myeloid malignancy without +8, Jenkins et
all' reported an increase of three-spot interphase cells by
FISH in seven patients. The percentages of these three-spot
cells were low (2.7% to 3.4%). Interestingly, a comparable
problem concerning low percentages of aberrant cells was
observed in our study on trisomy 12 in chronic lymphocytic
leukemia.26 A considerable part of the patients had 3% to
5% of cells with three spots. The biologic and clinical significance of these low percentages of aberrant cells as determined by FISH is not known.
Several of our -7 and +8 samples demonstrated two potential pitfalls in interphase cytogenetics: ( l ) admixture with
nontumor cells (Table 2, patient no. 8). We and others have
shown that lymphoid cells do not contain the here studied
genetic aberrations -7 and +8.'8-22The same is reported for
the +t( 1;7),2' and for 5q deletion^.'^ In view ofthe relatively
high threshold value for monosomy detection, it would
have been almost impossible to make an -7 interphase
diagnosis in the sample of patient no. 8. (2) This and other
studies demonstrated that the presence of translocations or
marker chromosomes can lead to misinterpretations of
patient
(FISH) spots per cell d i s t r i b ~ t i o n s . ~For
, ~ , example,
'~
no. 10 of the -7 group had I 1 of 12 -7 metaphase cells in
the banding analysis, but due to an additional t(7;16), the
-7 could not be detected with the ap7 probe.
In conclusion, this study has shown the feasibility and
potential of FISH analysis in a large series of MDS and
AML patients. FISH may be more sensitive than conventional cytogenetics, especially in samples with nonclonal abnormalities and with few or poorly assessable metaphases.
However, the potential of FISH must not be overestimated,
which was illustrated by the fact that we did not identify any
additional case of (low percentage) -7 or +8 in 34 patients
without a -7 or +8 established by banding. Moreover, the
threshold for -7 was relatively high. As FISH will be used
more and more in cytogenetic diagnosis, follow-up, and
therapy m~nitoring,'~
it will be necessary to standardize
FISH procedures and supplement the ISCN definitions of a
clone with criteria specifically for FISH.
ACKNOWLEDGMENT
We are grateful to M.H.H. Kramer and G.J. den Ottolander (University of Leiden), G.J. Ossenkoppele (Free University of Amsterdam), J.E. Ploem (Lucas Hospital, Amsterdam) for providing clinical and hematologic data of the patients. We thank K. van de Ham
and R. Heruer for preparation of the photographs. J. Gray, H.J.
Cooke, and H.F. Willard are acknowledged for their kind gifts of
the chromosome-specific libraries and the repetitive probes for
chromosomes 1 and 7, respectively. W. van den Ende, J. den Nijsvan Weert, and F.C.T. Havik-Bogaard are thanked for technical
assistance. J.H.J.M. van Krieken, M.H.H. Kramer and A.K. Raap
are acknowledged for critical reading of the manuscript.
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