Comparison of DNA and Karyotype Aneuploidy
in Malignant Lymphomas
TAINA LAKKALA, M.SC, ANTERO LAASONEN, M.SC, KAARLE O. FRANSSILA, M.D.,
LASSE TEERENHOVI, M.D., AND SAKARI KNUUTILA, PH.D.
Tumors from 48 patients with non-Hodgkin's lymphoma were
examined for flow cytometric DNA ploidy and chromosome constitution to determine the degree of concordance of these two
methods. Histologically, there were 24 low-grade, 19 intermediate, and 5 high-grade lymphomas. Flow cytometry revealed an
aneuploid cell population in 19% of the cases. The mean DNA
index of the aneuploid tumors was 1.58 ± 0.71. The frequency
of DNA aneuploidy was only slightly higher (23%) in intermediate than in low-grade lymphomas (17%). None of the five highgrade lymphomas showed DNA aneuploidy. The chromosome
study was successful in 81% of cases (39 of 48), and clonal chromosome abnormalities were observed in 92% of these (36 of 39).
In most of the chromosomally abnormal clones the chromosome
number was in the diploid range. Most tumors with pseudodiploid
(46 chromosomes), hypodiploid (45-44 chromosomes), or hyperdiploid (47-49 chromosomes) clones were DNA diploid by
flow cytometry. On the other hand, all specimens with a chromosome number exceeding 50 were DNA aneuploid by flow cytometry. Therefore, flow cytometric DNA analysis appears to
be a rather coarse method that will detect aneuploidy only when
there is a major increase in chromosome material. (Key words:
Aneuploidy; Flow cytometry; Chromosome aberrations; Lymphoma) Am J Clin Pathol 1990;94:600-605
DURING RECENT YEARS flow cytometric DNA analysis has emerged as a popular method for determining
the DNA content and proliferative activity of tumor cells.
Until some time ago, DNA aneuploidy in a tumor was
regarded as a sign of malignancy, but recently benign tumors with DNA aneuploidy have also been reported.9
DNA aneuploidy has been found to correlate with poorer
prognosis than that found in DNA diploid malignant tumors. 2 In non-Hodgkin's lymphomas, DNA aneuploidy
has been reported to occur in 30-40% of cases, 4 - 61012141619
which is a lower frequency than that observed in most
other malignant solid tumors. 2 However, the frequency
of clonal chromosome abnormalities found in cytogenetic
analysis of non-Hodgkin's lymphomas is as high as 7595%. 3,23 The relationship between flow cytometric DNA
Department of Medical Genetics, University of Turku, Turku;
Pathology Laboratory of the Department of Radiotherapy
and Oncology, Department of Radiotherapy and Oncology,
and Department of Medical Genetics, University of Helsinki,
Helsinki, Finland
content and chromosome abnormalities in non-Hodgkin's
lymphomas has been studied only to a limited extent.5,22
In this study we performed flow cytometric DNA analysis and karyotype analysis on the same specimens in a
series of 48 patients with non-Hodgkin's lymphoma. The
aim of the study was to determine the kinds of chromosome abnormality resulting in such alteration of the DNA
content as to be detectable by DNA flow cytometry (FCM).
Materials and Methods
Flow cytometric DNA analysis as well as histologic and
chromosome studies were performed on the same involved lymph node from each of 48 patients with nonHodgkin's lymphoma. Only those cases with evaluable
flow cytometric histograms were selected for the study.
Of the patients, 22 were men and 26 women. The age
range was 26-92 years, with a median of 58 years (Table 1).
Histologic Studies
The lymphomas were classified according to the updated Kiel classification20 and the International Working
Formulation. 17 They were further grouped as low-, intermediate-, and high-grade lymphomas according to the
Working Formulation. In addition to routine histologic
examination, frozen-section immunohistochemical analysis was performed on all but one case.
Flow Cytometric Studies
Received November 22, 1989; received revised manuscript and accepted for publication March 19, 1990.
Supported by The Research Science Foundation of Laake-Farmos
OY, The Finnish Cancer Society, and Sigrid Juselius Foundation.
Address reprint requests to Dr. Knuutila: Department of Medical Genetics, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki,
Finland.
Tumor biopsies for FCM were cut into small pieces in
RPMI 1640 (a cell culture media) medium (Gipco UK,
Paisley, Scotland) containing 5% dimethylsulfoxide
(DMSO) and deep frozen (-70 °C) until FCM analysis.
For the FCM analysis, the frozen tissue samples were
600
Vol.94.No.s
COMPARISON OF DNA AND KARYOTYPE ANEUPLOIDY IN MALIGNANT LYMPHOMAS
thawed and disaggregated mechanically, filtered through
a 50-jum nylon mesh, and stained with ethidium bromide
(EB) solution (Sigma, St. Louis, MO) containing RNAse
(Sigma) and detergent as described by Vindelav.21 Human
lymphocytes were used as an external ploidy standard and
chicken and rainbow trout erythrocytes as internal reference standards. Immediately before FCM analysis, the
samples were filtered through a 30-/um nylon mesh.
The nuclear suspensions were analyzed with the use of
an EPICS C® Flow Cytometer (Coulter Electronics, Hialeah, FL) with 488-nm argon ion laser excitation. The
instrument was calibrated with fluorescent beads. At least
15,000 nuclei per sample were analyzed using a flow rate
of 50-150 cells/second. Samples were gated simultaneously on both 90-degree and forward light scatter to
exclude signals from debris. EB fluorescence was measured
at a wavelength longer than 610 nm. The DNA index in
samples with DNA aneuploidy was calculated as the ratio
of the abnormal and normal G0/G1 peak modal channels.
Tumors were classified as diploid if they possessed no
additional DNA stemlines. The coefficient of variation
(half-width method) was determined with the use of
Coulter software and the Statpack® program. The mean
coefficient of variation for the diploid G0/G1 peak was
3.9 ± 0.94. Typical flow cytometric DNA histograms of
two DNA aneuploid tumors are presented in Figure 1.
Cytogenetic Studies
For the cytogenetic studies, fresh tissue was processed
as described previously." The cytogenetic preparations
were stained by the trypsin-Giemsa banding method. The
clonality of abnormalities was determined according to
principles established by the First International Workshop
on Chromosomes in Leukemia (1978).7
Results
Histologic classification of the cases is presented in Table 1. Most of the lymphomas were of low or intermediate
grade (24 and 19 cases, respectively); there were only 5
high-grade lymphomas.
Chromosome study was successful in 39 of the lymphomas (81%). Thirty-six of these (92%) had clonal chromosome abnormalities. In 10 cases all the mitoses obtained were chromosomally abnormal, whereas in 26 cases
there were both chromosomally normal and abnormal
mitoses. The karyotypes of the abnormal clones are presented in Table 1. In seven cases there were two different,
albeit related, clones present. In 81% (29 of 36) of the
tumors with chromosomal abnormalities, the chromosome number was close to diploid (i.e., between 44 and
49); in the remaining 7 lymphomas the chromosome
number was higher (for lymphomas with two abnormal
clones, the clone with the higher chromosome number
601
was considered). The chromosome number did not correlate with the histologic grade: in 85% (17 of 20) of lowgrade lymphomas, 75% (9 of 12) of intermediate-grade
lymphomas, and all 4 high-grade lymphomas, the chromosome number in the abnormal clone was 49 or less
(when two clones were present, the clone with the higher
chromosome number was considered).
Flow cytometric DNA aneuploidy was found in nine
of the tumors (19%); the other tumors were DNA diploid.
Multiple clones were found in two tumors (Table 1). Five
of the 12 aneuploid clones were close to diploid, with a
DNA index of 1.12-1.22, whereas 7 showed a DNA index
of 1.57-3.31 (Table 1). The mean DNA index of all the
aneuploid clones was 1.64 ± 0.73 (for lymphomas with
multiple clones, the clone with the highest DNA index
was considered).
There was no clear association between tumor histologic
characteristics and the frequency of DNA aneuploidy;
DNA aneuploidy was observed in 17% (4 of 24) of lowgrade, 26% (5 of 19) of intermediate-grade, and 0% (0 of
5) of high-grade lymphomas.
Figure 2 illustrates the relationship between the chromosome number of the abnormal clone and flow cytometric findings. All seven lymphomas with a chromosome
number higher than 50 in at least one clone showed DNA
aneuploidy by FCM. None of the lymphomas with 4446 or 48-49 chromosomes in the abnormal clone were
DNA aneuploid. However, 2 of the 13 lymphomas with
an abnormal clone of 47 chromosomes (cases 29 and 37,
Table 1) did exhibit DNA aneuploidy. In case 29 there
were two chromosomally abnormal clones, and in FCM
three aneuploid peaks were detected, one of which apparently corresponded to the clone with 47 chromosomes.
In the two cases showing multiple clones in FCM, multiple
clones were also found in chromosome analysis.
The nine tumors with a normal karyotype were diploid
by FCM. One of the nine tumors from which no analyzable mitoses were obtained was DNA aneuploid.
Discussion
In our series of 48 cases of non-Hodgkin's lymphoma,
flow cytometric DNA aneuploidy was detected in 19% of
cases, a frequency somewhat lower than those reported
by others (30_40%). 4 - 6101214 ' 619 The low frequency of
DNA aneuploidy in our series may result, at least partly,
from the fact that half our cases were histologically lowgrade lymphomas. For such tumors, others have reported
DNA aneuploidy in less than 20% of cases. 6 1 0 1 2 1 4 1 9 The
frequency of DNA aneuploidy in different materials also
depends on the criteria of aneuploidy used.
In our series, about half the DNA aneuploid tumors
had DNA indexes exceeding 1.50, the other half being
602
L A K K A L A ET AL.
AJ.C.P. • November 1990
Table 1. Clinical Data and Histologic, Flow Cytometric, and Cytogenetic Findings in 48 Patients with Lymphoma
Patient
Age
Sex
DNA Index
Karyotype of the Abnormal Clone
I. Low-grade lymphomas
Small lymphocytic (lymphocytic, B-cell)
1
2
3
4
5
73
68
57
62
51
• M
F
M
F
M
1.00
1.00
1.00
1.00
1.00
47,XY,+ 12,14q45,X
No abnormal clone present
No abnormal clone present
Chromosome study failed
Follicular, predominantly i
6
7
58
47
F
M
8
9
10
64
26
43
M
F
F
1.00
1.57
1.94
1.00
1.00
1.00
11
12
13
14
15
37
37
49
39
66
M
F
M
F
F
1.00
1.00
1.00
1.00
1.13
47,XX,+7,t(14;18)/48,XX,+7,+8,t(14;18)
2N/4N,ABN
48,XY,+7,+8,t( 14; 18)/48,XY,+7,+8,t( 14; 18),t( 17; 19)
46,XX,-6,+i(6p),t( 14; 18)/45,XX,-6,-9,-12,+i(6p),+der( 12)
46,XX,-l,-3,-4,-7,-10,+der(l)t(l;?4),+der(3)t(3;?),+der(4),
+der(7),+?der(10)?t(l; 10),t(14; 18)
46,X Y,t( 14; 18)/47,XY,+11,6q-,t( 14; 18)
46,XX,+X,-10,6q-,t(14; 18)
46,XY,t(14;18)
47,XX,-22,+der(l),t(14; 18),+mar
51,XX,+3,+3,+3,+ 14,lq+,?5q-,6q-,+mar
Follicular and diffuse predominantly small cleaved cell (centroblastic/centrocytic, follicular and diffuse)
16
73
17
18
19
20
51
70
83
48
F
F
M
F
37
58
62
52
F
M
M
F
3.31
1.00
1.00
1.22
1.00
84,XX,+2,+5,+5,+5,+7,+ 10,+ 10,+ l 1,+ 11,
+ 14,+ 14,+ 16,+ 18,+ 18,+ 19,+20,+20,+21,+22,+22,
+i(lq),+i(lq),lq-,+der(3),+der(3),6q-,+9p+ > +9p+,
+der( 12),t( 14; 18),t( 14; 18),+11 mar
46,XX,-13,+8,t(14;18)
46,XX,-4,-10,+der(4)t( 1; 4), 10q-,t( 14; 18)
Chromosome study failed
49,XX,+7,+ 12,7q-,t(14; 18),+mar/
49,XX,+7,+ 12,t(14; 18),+mar
Diffuse, small cell, *NOS (diffuse, small cell NOS)
21
22
23
24
1.00
1.00
1.00
1.00
47,XX,12q+,t(14;18),+ 18q49,XY,-2,-4,-8,-13,-18,lq+,6q-,+i(18q),+7mar
46,XY,-18,-18,+21,t(l;?),t(l;6),t(3;?),10p-,+i(18q)
47,XX,+?X,-12,6q-,?9p-,+der( 12), 13q+,t( 14; 18)
II. Intermediate-grade lymphomas
Diffuse, small cleaved cell (centrocytic)
25
26
27
28
29
47
70
53
71
70
M
F
F
M
M
30
55
F
1.00
2.06
1.00
1.00
1.15
2.06
2.36
1.00
Chromosome study failed
4N,±,ABN
No abnormal clone present
47,XY,-C,-21 ,t( 14; 18),+18q-,+2mar
47,XY,-15,3p-,7q+,l lq-,+2mar/
4N-,3p-,7q+
47,XX,-10,6q-,+2mar
Diffuse, predominantly small cleaved cell (centroblastic/centrocytic, diffuse)
31
71
F
1.00
46,XX,-3,-7,-10,-13,+der(3),6q-,+der(7),+der(13),t(14; 18),+mar
Diffuse, mixed small and large cell, peripheral T-cell (peripheral T-cell)
32
33
60
62
F
M
1.00
1.00
Chromosome study failed
Chromosome study failed
Diffuse, large cell, noncleaved (centroblastic, diffuse)
34
35
66
74
M
M
1.00
1.00
46,XY,lq+,t(ll;?)
Chromosome study failed
Vol. 94 • No. 5
COMPARISON OF DNA AND KARYOTYPE ANEUPLOIDY IN MALIGNANT LYMPHOMAS
603
Table 1. (Continued)
Patient
Age
Sex
DNA Index
36
37
53
54
M
F
1.66
1.12
38
40
M
1.19
39
40
41
69
87
92
F
F
F
1.00
1.00
1.00
Karyotype of the Abnormal Clone
70,ABN
47,XX,+der(X)t(3; X),-3,+7,-9,-12,-13,-16,-17,+der(3)t
(3;X),+6q-,+der(12)t(l 1; 12),16q+,+2mar
54,XY,+X,-2,+7,+11,-17,+18,+21 ,t(2; 9),+der(2)t(2; 17),
6q-,+der( 12)t( 1; 12),t( 14; 18),+3mar
48,XXq-,+3,-9,+ 12,lq-,lq+,+mar
Chromosome study failed
47,XX,6q-,t(10;l9),+mar
Diffuse, large ccell, Ki-1 -posit, (large anaplastic, Ki-1+)
42
43
39
43
M
F
1.00
1.00
Chromosome study failed
4 5 , X , - X , - 6 , - 8 , - 8 , - 9 , - 2 2 , + lp+, lq-,+der(6),6q-,
+der(8),+der(8),+der(9)
III. High-grade lymphomas
Large cell, immunoblastic (immunoblastic)
44
77
45
64
1.00
M
1.00
45,XX,-2,-3,-8,-13,-14,-14,-15,-16,-18,-20,-21,
- 2 1 ,-22,inv( 1 ),+del inv( 1), 12p-, 17p-,
+der(2p21q)t(2;21),+der(2q21q)t(2;21),
+der(2p22q)t(2;22),+der(3)t(3;?),+der(18)t(X;18),+der(?)t(14;?),
+der(?)t(14;?),+4mar
47,X,-Y,+3,+3,-12,-13,?2q+,+9p+,+9p+,7q+, 14q+, 14q+, 15q+
Lymphoblastic (lymphoblastic)
46
52
M
1.00
4 4 , X Y , - 1 , - 9 , - 1 3 , - 14,t(ll; 14; 17),+2mar
Small noncleaved, non-Burkitt (centroblastic?, diffuse)
47
48
66
60
M
F
1.00
1.00
Histologic classification according to Working Formulation17; terminology in parentheses according to updated Kiel classification.20
close to diploid, whereas in another series near-diploid
DNA indexes were more frequent. 41618 ' 22
Chromosome abnormalities were identified in 92% of
analyzable lymphomas in our material. This result concords with those of previous studies.3,23 In most of the
tumors (63%), the chromosome number in the abnormal
clone was pseudodiploid or close to diploid. In previous
cytogenetic studies on non-Hodgkin's lymphomas, the
chromosome number has been in this range in more than
70% of tumors. 31315,23 Near-diploid chromosome numbers have been reported to be most frequent in low-grade
lymphomas, whereas higher chromosome numbers appear
to be more characteristic of large-cell lymphomas.3,13,15,23
In the current study, our object was to find out which
karyotype abnormalities appear as aneuploid clones in
FCM and which do not. It has been estimated that the
increase or decrease in the nuclear DNA content has to
be at least 5% to be detectable by FCM.22 Because most
non-Hodgkin's lymphomas are cytogenetically pseudodiploid or close to diploid, it is conceivable that the alteration of the DNA content may often be less than the
Chromosome study failed
4 7 , X X , - 4 , - 5 , - 1 0 , - 16,+3q-,22q-,+4mar
M = male; F = female.
* NOS = not possible to classify further.
limit of resolution of FCM and that only a minority of
specimens thus show flow cytometric DNA aneuploidy.
There appeared to be a fair correlation between the
chromosome number and the flow cytometric DNA index
in our series; all tumors with a chromosome number exceeding 50 were DNA aneuploid and all but two tumors
with a clone with a chromosome number of 49 or less
were DNA diploid. However, although none of the four
lymphomas with 48-49 chromosomes were DNA aneuploid, 2 of the 13 lymphomas with a clone with 47 chromosomes were aneuploid. This apparent discrepancy may
result from the fact that all chromosomes are not of the
same size; for instance, chromosome #1 accounts for
about 4.5%, chromosome #9 for less than 2.5%, and chromosome #21 for less than 1 % of the total length of the
human karyotype.8 Therefore, the chromosome number
does not necessarily correspond to the amount of chromosomal material and to the flow cytometric DNA index.
For instance, the gain of five of the smallest chromosomes
#21 or #22 and loss of one chromosome # 1 will nearly
balance each other and, although chromosome number
LAKKALA ET AL.
604
in this hypothetical case is 49, the alteration in the amount
of chromosome material still remains less than 1%. In
complex karyotypes {e.g., our cases 10, 22, 37, 44, 45,
and 46) it may be very difficult to determine the alteration
of the chromosome material. One of the lymphomas, with
47 chromosomes and DNA aneuploidy (case 37), may
have acquired more than 5% of DNA material, whereas
in the other tumor (case 29) the alteration was probably
less than 5%. In several of the DNA diploid tumors (cases
1,9, 11, 17, 21, 24, and 30), the rearrangements seemed
to balance each other quite well, leaving the amount of
chromosome material in the cells close to normal. To
complicate matters, there were some DNA diploid tumors
in which the alteration of the chromosome material suggested DNA aneuploidy although FCM showed them to
be DNA diploid (e.g., cases 20, 44, and 45). One explanation may be that in fact most of the cells were normal
and the small abnormal clone escaped recognition by
FCM. In such cases it might be possible to enhance the
detection rate of DNA abnormalities by using additional
cellular properties, such as surface antigens, to discrimi-
A.J.C.P. • November 1990
Number of
lymphomas
10-
5-
<46
46
47
48
49
>50
Chromosome number of the abnormal clone*
FIG. 2. Histogram of the flow cytometric findings in 36 lymphomas
with clonal chromosome abnormalities. *In cases with more than one
chromosomally abnormal clone, the clone that deviated most from 46
was considered. • = DNA aneuploid; D = DNA diploid.
nate between reactive normal cells and the neoplastic
cells.14
Flow cytometric DNA analysis is a rapid method that,
unlike chromosome analysis, does not require mitotic
cells. It is, however, a very coarse method that will detect
aneuploidy only when there is a major increase in chromosome material. On the other side, karyotype analysis
is a rather laborious and slow method, but it does provide
specific information about the cells examined. In most
cases it can determine whether a cell population is neoplastic, whether the chromosome changes are consistent
with lymphoma, and whether there are chromosome
changes regarded as typical of certain histologic types of
lymphoma.
4)
•fi
E
B
9
C
Acknowledgments. The authors thank Paivi Laurila and Tuula Sariola
for skillful technical assistance.
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FIG. 1. Flow cytometric DNA histograms of two different lymphomas.
A (upper). A multiploid sample with a large near-tetraploid cell population
(large arrow) and small near-triploid cell population (short arrow). B
(lower). A sample with a small near-diploid cell population (arrow).
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