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Fluorescent In Situ Hybridization and Cytogenetic Studies of Trisomy 1 2
in Chronic Lymphocytic Leukemia
By Susan M. Escudier, Josefina M. Pereira-Leahy, Johannes W. Drach, H.U. Weier, Angela M. Goodacre, M. Ann Cork,
Jose M. Trujillo, Michael J . Keating, and Michael Andreeff
Cytogenetic studies (CG) of 4 7 5 chronic lymphocytic leukemia (CLL) cases showed trisomy 1 2 in 6.1 % or 26% of
patients with abnormal karyotypes. Fluorescence in situ
hybridization (FISH) detected trisomy 1 2 in 35% of 11 7 CLL
patients. Only 34.6% of cases detected by FISH were detected by CG. Twelve patients had low levels of trisomic
cells (4%to 1 1%) relative to clonal B cells (47.5% to 86%).
suggestive of clonal evolution. Untreated patients with trisomy 1 2 were predominantly male (P < .05) and had an
increased incidence of splenomegaly (P < .03). Patients
with trisomy 1 2 were more likely to be previously treated
and had advanced Binet stage compared with those without
trisomy 12. The median survival was shorter in patients
with trisomy 1 2 (7.8 years) and patients with other chro-
mosomal abnormalities without trisomy 1 2 by FISH (5.5
years) than in patients with diploid karyotypes (14.4 years).
The response to fludarabine was similar to that of patients
with diploid karyotypes, but there was a trend for earlier
disease progression. FISH detected residual disease in all
patients with trisomy 1 2 in complete (n = 6) or partial
remission (n = 4). As few as 1 trisomic cell in 5,000 was
detected by performing FISH on fluorescence-activatedcell
sorter-sorted cells. Trisomy 12 was absent in T cells in patients with trisomy 12. We conclude that FISH identifies
trisomy 1 2 approximately 2.6 times more often than CG,
readily identifies minimal residual disease, and predicts for
a shorter median survival.
0 1993 by The American Society of Hematology.
C
confound result^.^.^ Analysis of interphase cells could provide
a more representative assessment of clonal chromosomal abnormalities in CLL.
Fluorescent in situ hybridization (FISH) is a rapid method
for detecting chromosomal abnormalities in nondividing interphase cells or during metaphase. Greater numbers of cells
can be evaluated than by conventional cytogenetic analysis,
and an accurate assessment of the proportion of abnormal
cells present can be made.’,’ When the cytogenetic abnormality is known, the presence of minimal residual disease
can be detected using FISH.9-12
In this study, conventional karyotype analysis was performed in 475 CLL samples. FISH was used in 117 CLL
patients to detect trisomy 12 using a centromeric probe for
chromosome 12. Cytogenetic results were then compared with
FISH, and correlated with clinical characteristics of the
patients. Lineage-restriction was investigated using fluorescence-activated cell sorter (FACS)-separated cells selected by
immunophenotype followed by FISH,I2 and the same combination of techniques was used to study minimal residual
disease in I O patients with known trisomy 12 in remission.
LONAL CHROMOSOMAL abnormalities are found in
approximately 50% of patients with chronic lymphocytic leukemia (CLL) and are reported to be associated with
poorer prognosis as compared with diploid karyotypes. In
particular, the presence of complex chromosomal abnormalities, abnormalities of the long arm of chromosome 14,
or a high percentage of abnormal metaphases has been associated with shorter survival.’-4 Trisomy 12 is the most
common clonal chromosomal abnormality described in CLL
(20% to 62% of patients with known cytogenetic abnormalitie~),’-~
and in some studies has been associated with decreased overall survival and need for earlier t~-eatment.~-~
With the use of B-cell mitogens such as lipopolysaccharide
(LPS), 12-0-tetradecanoylphorbol- 13-acetate (TPA), Staphylococcus aureus Cowan (SAC), and Epstein-Barr virus
(EBV), evaluable metaphases are obtained in 80% to 90% of
bone marrow or blood samples from CLL patients. However,
abnormalities are detected in only about one-half of the patients by conventional cytogenetic analysis and diploid metaphases from nonmalignant B cells and dividing T cells may
MATERIALS AND METHODS
From the University of Texas M.D. Anderson Cancer Center, Experimental Hematology and Leukemia Sections, Department of Hematology- and the Section of Cytogenetics, Division of Laboratory
Medicine, Houston, TX; and the University of California, San Francisco, CA.
Submitted July 9, 1992; accepted December 22, 1992.
Supported in part by Grant No. CA 166 72 .fvom the National Institutes of Health and NICHD Grant No. HD17665.
Presented in part at the American Society ofHematology, Denver,
CO, December 1991, and at the American Society of Clinical Onco/ogy, San Diego, CA, May 1992.
Address reprint requests to Susan M. Escudier, MD, Leukemia
Section, Box 61, The University of Texas M.D. Anderson Cancer
Center, 1515 Holcombe Blvd, Houston, TX 77030.
The publication costs ofthis article were de@ayed in part by page
charge payment. This article must therdore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/81 IO-0006$3.00/0
2702
Patient Population
Heparinized peripheral blood and/or bone marrow samples were
obtained from CLL patients at the M.D. Anderson Cancer Center
Leukemia Clinic from January 1991 to September 1992 (n = 61).
Fixed bone marrow cells were obtained from archived material prepared from January 1987 to January 1991 ( n = 56). Most samples
were collected at the patient’s first M.D. Anderson presentation.
Controls were ( I ) the buffy coat cells of normal volunteers (n = 13)
and (2) archived material from CLL patients with abnormalities other
than trisomy 12 (n = 17).
Approval was obtained from the Institutional Review Board for
these studies. Patients and volunteers were informed that blood and/
or bone marrow samples were obtained for research purposes, at the
same time as routine samples were obtained, and that their privacy
would be protected.
FISH
The DNA probe. The hybridization probe was a biotinylated asatellite DNA fragment of approximately 175 bp that binds specifically
Blood. Vol 8 1, No 10 (May 15), 1993: pp 2702-2707
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2703
FISH AND CG STUDIES OF TRISOMY 12 IN CLL
to repeated DNA sequences in the centromeric region of human
chromosome 12. The probe was originally generated by primer-directed in vitro DNA amplificationusing the polymerase chain reaction
(PCR) from flow-sorted metaphase chromosomes 12 using primers
WAI and WA2 as described el~ewhere.'~,'~
In later experiments, we
used a recombinant DNA template containing the 173-bp Hind111
fragment in phosphate-buffered saline (PBS; Stratagene, San Diego,
CA). Total human alphoid blocking DNA was generated using human
genomic DNA and primers WAl and WA2 as described previously.14J5
Cellpreparation. Bufi coat, bone marrow aspirates, and, in some
cases, mononuclear cells separated over a Ficoll-Hypaque density
gradient (Lymphocyte Separation Medium; Organon Teknika, Durham, NC) were fixed in 3: 1 methanokacetic acid and stored at -20°C.
The cell suspensions were dropped on glass slides and air-dried.
Hybridization. The slides were washed sequentially in 0.1 N HCL
with 0.1% Triton X-100, 2X saline sodium citrate (SSC), PBS, PBS
with 1% formaldehyde, PBS, and then 2X SSC. The slides were then
denatured in 70% formamide 2 X SSC solution at 70°C for 4 to 6
minutes. These were dehydrated for 1 minute each in 70%, 85%, and
100%ethanol, and then air-dried. One microliter of the probe was
diluted in 9 pL of hybridization mixture (7 pL master mix [50%
formamide in 2X SSC and glycerin with 1 fiL of salmon sperm DNA
per 10 pL], 1 pL of distilled water, and I pL of a-satellite-blocking
DNA), denatured at 70°C for 5 minutes, and then placed in ice. The
hybridization mixture ( I O pL) was dropped on the slide, cover slipped,
sealed with rubber cement, and incubated at 37°C overnight.
Detection. The slides were washed in 42°C 50% formamide in
2X SSC for 20 minutes, which was followed by two 15-minute washes
with phosphate neutral buffer (PN) at 37°C. The slides were incubated
with PN containing 5% nonfat dry milk (PNM) for 5 minutes, then
incubated with 20 pL avidin-fluorescein conjugate (Vector Laboratories, Burlingame, CA) ( 5 pg/mL PNM) for 20 minutes at room
temperature (RT), and washed in PN twice. If the hybridization signal
was not detectable at this point, it was amplified with biotinylated
anti-avidin (Vector Laboratories) ( 5 pg/mL PNM) incubated for 20
minutes at RT, followed by another layer of avidin-fluorescein isothiocyanate (FITC). Up to two amplifications were performed. The
nuclei were counterstained with propidium iodide (0.25 pg/mL antifade). Hybridization signals were counted in 200 to 500 cells with
a fluorescencemicroscope (Nikon Labophot 2,60X planar objective)
with a 5 I O nm filter (Nikon, Tokyo, Japan).
blood cultures. Two karyotypes were made for each type of metaphase
found and the chromosomes arranged according to ISCN.I6
Cell Sorting
The mononuclear cells were incubated with either Leu-4-FITC
(CD3) alone, Leu-4-FITC and Leu-l2-phycoerythrin (CD19), or LeuI-FITC (CD5) and Leu- 12-phycoerythrin (Becton Dickinson, San
Jose, CA) for 30 minutes at 0°C for each antibody, followed by two
washes with PBS. The cells were then sorted with a FACstar Plus
Cell Sorter (Becton Dickinson) into single-antigen-positive (CD3+,
CD5+, and CD I9+) and double-antigen-positive populations
(CD5+19+).The cells were fixed in methanokacetic acid 3: 1. In some
cases, cells were sorted directly on slides coated with FCS. For FISH,
FACS-sorted single-antigen-negative (CD3-) or single-antigen-positive cells (CD5+CD19-) were used as controls.
Data Analysis
For comparison of distinct variables between two groups, data were
analyzed using the x2test. SuMvd curves were calculated with the method
of Kaplan and Meier" and their significance determined by using logrank analysis.18Time to progression was calculated from the initiation
of treatment. Actuarial survival was measured from the time of diagnosis.
RESULTS
Cytogenetics
Conventional cytogenetic analysis by G-banding was performed in 475 C L L patient samples (Table 1). Evaluable
metaphases were present in 78%. Abnormal karyotypes were
identified in 3 1% of cases with evaluable metaphases. Of those,
29 (7.8% of evaluable, 6.1% of all patients studied) had trisomy 12 alone in one or more metaphases (12.3% ofabnormal
karyotypes) o r in combination with other abnormalities
( 13.2% of abnormal karyotypes) a n d 6.2% (I .9% of evaluable)
had structural abnormalities of chromosome 12. Other common chromosomal abnormalities identified were 1 1q- (1 7%
of abnormal karyotypes), 14q+ (8%), 13q- (7%), t(l1;14)
(6%), 6q- (4%), -17 (4%), +/-19 (4%), +/-18 (4%), +/-21
(3%), -16 (3%), and i(17q) (2%).
Cytogenetics
Both peripheral blood and bone marrow samples were used. Cultures were initiated and samples incubated for 24, 48, and 72 hours
at 37°C. Among the cultures were one peripheral blood culture stimulated with phytohemagglutinin (PHA; Burroughs-Wellcome,
Greenville, NC) and one bone marrow sample stimulated with LPS
(GIBCO, Grand Island, NY) for 72 hours. Ham's FIO tissue culture
medium (GIBCO) supplemented with 10% fetal calf serum (FCS;
Intergen, Purchase, NY) was used.
After the designated culture periods, the samples were harvested
using established cytogenetic techniques, which included a 30-minute
hypotonic treatment (0.057 mol/L KCI) and a 30-minute fixation in
3: 1 methano1:aceticacid fixative. The cell preparations were air-dried
and the metaphases G-banded using a trypsin (Intergen, Purchase,
NY) pretreatment and Gurr's Giemsa stain (Biomedical Specialties,
Santa Monica, CA).
The slide preparations from all cultures were scored for analyzable
metaphases. A maximum of 25 G-banded metaphases from the unstimulated and LPS-stimulated bone marrow and/or from the unstimulated peripheral blood cultures were analyzed. An additional
10 to 25 metaphases were analyzed from PHA-sbmulated peripheral
Table 1. Cytogenetics in CLL (M.D. Anderson Cancer Center)
Karyotype
% Evaluable
% Abnormal
Diploid
+I2
12 structural
1 Iq14q+
t(1 1;14)
13q-
69
8
2
5
2
-
6q-17
+/-19
+/-I8
+/-2 1
-16
i(l7ql
2
2
1
1
1
1
1
1
1
26
6
17
8
6
7
4
4
4
4
3
3
2
Karyotype of CLL patients by usual cytogenetic analysis. Four hundred
seventy-five patients were tested, 78% had evaluable metaphases, and
31 % of those were abnormal.
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2704
ESCUDIER ET AL
Table 2. Comparison of FISH and Metaphase Analysis
Karyotype
Insufficient metaphases
Diploid
Trisomy 12 in 2 1 metaphase
Chromosomal abnormalities other than
trisomy 12
Not studied
All Patients
(n = 11 7)
Trisomy 12 (FISH)
(n = 41)
21
32
11
8
4
10
21
32
4
15
CLL patients analyzed with FISH for trisomy 12. Results of FISH are
compared with those of conventional cytogenetic analysis.
FISH
was 38.7% (range, 4% to 89%). There was no correlation between the percentage of CD19+ or CD20' cells in the samples
and the percentage of cells positive for trisomy 12. Low levels
of trisomic cells (4% to 11%) were found in 12 patients with
47.5% to 86% circulating CD19' or CD20+ cells. In one patient with l l%trisomic cells, these cells were not detectable
8 and 9 months later during therapy, but this clone re-emerged
with 5% trisomy 12 at 16 months. Follow-up studies on two
patients with low-level trisomy at 3 months and 9 months did
not show significant change. The Rai and Binet stages of these
patients were similar to those of the other patients with trisomy
12.
Comparison qf Cytogenetics and FISH
One hundred seventeen patients were evaluated using
FISH. Normal control cells (n = 13) had one hybridization
signal in 3%, two signals in 94.6%, and three signals in 2.2%
of the cells (range, 0.8% to 4.0%; 99% confidence intervals,
1.4% to 3.0%). Archived patient samples with cytogenetic
abnormalities other than trisomy 12 had an average of 2.5%
of cells with one signal, 92.0% with two signals, and 3.5%
with three signals (99% confidence intervals, 1.7%to 6.7%).
Fresh samples were therefore considered positive for trisomy
12 with 24% cells with three signals and stored samples at
27%. The samples were considered evaluable if hybridization
was present in 290% of cells or less than 20% of cells had
zero or one signal. All of the fresh samples were interpretable;
however, approximately one-third of the archived samples
were not interpretable based on these criteria (primarily samples 2 2 years old). The confidence intervals for controls were
identical for both of the two probes used.
Trisomy 12 was present in 41 of 117 patients (35%). The
median percentage of cells with three hybridization signals
Standard cytogenetic analyses were available for 83 of the
patients evaluated with FISH. Trisomy 12 was present in at
least one metaphase in 1 1 patients ( 1 3.3%) and in 2 2 metaphases in 5 patients. Trisomy 12 identified with FISH was
not identified by cytogenetic analysis in 16 of 26 or 6 1.5%
of samples. Diploid karyotypes were present in 4 patients
with trisomy 12 by FISH and other chromosomal abnormalities in 4 patients. Insufficient metaphases precluded
analysis in 8 patients (Table 2). Trisomy 12 was not detected
by cytogenetics in any case with less than 50% trisomic cells
by FISH (0 of lo), compared with those with greater than
50% trisomic cells (9 of 15) (P< .02). Karyotypes were available on 4 patients with trisomy I2 less than 1 1%; 1 was diploid
and 3 had complex karyotypes without trisomy 12. Cytogenetic analysis showed trisomy 12 in one case ( 1 of 20 metaphases) in which FISH did not. This may represent the random gain of a chromosome in a metaphase spread or a level
of trisomy 12 below the level of detection by FISH.
Table 3. Minimal Residual Disease in CLL Patients With Trisomy 12
+12 (%)
CD19/5
Lymphocytes
Case
Date
W BC
(%I
CD19 1%)
1. CH
7/90
319 1
6/92
9/92
10/90
1/92
4/92
7/92
2/90
719 1
10/91
12/90
919 1
1/88
319 1
1/90
9/92
29.5
4.2
4.8
6.1
29.3
3.3
2.9
4.5
120.0
3.7
6.0
11.3
5.6
29.6
6.5
5.2
2.4
89
19
15
15
82
17
29
17
94
33
14
75
15
81
25
72
14
88
ND
ND
ND
93
0.2
ND
ND
93
ND
ND
21
ND
89
34
43
ND
I%)
All
ND
ND
2.3
8.4
ND
ND
ND
1.3
ND
ND
8.2
ND
ND
ND
ND
ND
2.5
76
8
8
14
59
ND
6
4
60
6
ND
24
6
ND
65
ND
7
~~
2. M M
3. JF
4. MB
5.
cc
6. AD
CD19 or CD1915
Status
ND
ND
63
ND
ND
Dx
CR
CR
CR
Dx
CR
CR
CR
Dx
CR
CR
Dx
CR
Dx
CR
Dx
CR
~~
7
ND
29
ND
ND
55
ND
ND
ND
ND
ND
52
Serial studies of patients with trisomy 12 in complete remission.
Abbreviations: WBC. white blood cell count (X 1 03/dL); CDI 9, lymphocytes expressing CD19; CD19/5, lymphocytes coexpressingC D l 9 and CD5;
+ 12. percentage of cells with trisomy 12 for peripheral blood buffy coat or mononuclear cells (All) or CDI 9/5-sorted cells; Dx, diagnosis; CR, complete
remission.
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FISH AND CG STUDIES OF TRISOMY 12 IN CLL
2705
1.o
Residual Leukemia and Restriction to B-Cell Lineage
In 6 patients in complete remission by National Cancer
Institute (NCI) criteria (hemoglobin > 11 g/dL, lymphocytes
<4,OOO/pL, neutrophils > 1,5OO/pL, and platelets > 150,000/
pL), trisomy 12 was still detectable (Table 3). Blood or bone
marrow was tested on nine occasions, with a median of 6.5%
of mononuclear cells with trisomy 12 (range, 4.3% to 65%).
In four of these patients, malignant cells were enriched by
sorting CD 19+or CD 19+5+cells. Trisomy 12 was present in
a median of 52% of cells (range, 7% to 62.5%). With this
technique, in one case 7% trisomy was detected in 0.2% of
CD 19' cells, or less than 1 cell in 5,000 was detected (Table
3). Five patients with partial remissions also had detectable
trisomy 12 by FISH on unseparated blood or bone marrow
or in CD19' or CD19'5' cells. Two patients underwent autologous bone marrow harvests in remission. The unmanipulated marrows had 6% and 8% trisomy 12. After the samples
were immunopurged with an anti-CD I9 antibody, trisomy
12 was not detected above background levels.
T cells from 10 samples from 9 patients with trisomy 12 were
sorted (CDS'CD19- or CD3+CD19-) and did not display trisomy 12 (median 3.8% in T cells v mean 7 1% in B cells).
Clinical Correlation
Clinical characteristics of patients with trisomy 12 identified by FISH were compared with those of patients without
Table 4. Clinical Characteristics of CLL Patients With
Trisomv 12. Didoid KanrtotvDe. and Other Abnormal Karvotvoes
Trisomy
12
Present
-
Trisomy
12
Absent
Diploid
Other
~
WBC (x1O ~ / ~ L )
36.8
36.8
36.7
53.3
Absolute lymphocytes
30.5
35.0
31.9
50.1
(x1O ~ / ~ L )
Hemoglobin (g/dL)
12.2
12.9
13.5
10.4
03/pL)
Platelets (XI
169
163
186
157
Rai stage (%)
0
12.2
26.3
35.7
17.6
1
26.3
25.0
31.7
5.9
11.8
2
14.5
17.8
14.6
3
14.5
10.7
23.5
12.2
4
18.9
10.7
41.2
29.3
Binet stage (96)
A
29.4
39.0
52.6
67.9
B
17.6
24.4
21.1
21.4
C
26.3
10.7
52.9
36.6
Response to fludarabine (%) (n = 29) (n = 44) (n = 15) (n = 12)
CR
16.6
37.9
38.6
53.3
PR
16.6
31.0
11.4
13.3
NR
66.7
27.5
43.2
20.0
ED
0
3.4
6.8
13.3
Treatment
No prior treatment
47.2
58.1
29.4
78.6
Prior treatment
70.6
52.8
41.9
21.4
Comparison of clinical characteristics of patients with trisomy 12 by
FISH, patients with diploid karyotypes without trisomy 12 by FISH, and
patients with other chromosomal abnormalities without trisomy 12 by
FISH.
Abbreviations: WBC, white blood cell count; CR, complete remission;
PR, partial remission; NR, no response; ED, early death.
-
Trisomy 12
0.8
.->0
3
0.6
c
.=
5
e
0.4
a
...
0.2
i
In = 17)
I
0.0
0
36
72
108
144
180
216
252
288
324
360
Months
Fig 1. Survival of CLL patients with trisomy 1 2 detected by FISH,
diploid karyotype, or other abnormal karyotypes (n = 86).
trisomy 12, patients with diploid karyotypes (n = 28), and
patients with abnormal karyotypes other than trisomy 12 (n
= 17) (Table 4). Advanced Binet stage was seen more frequently in patients with trisomy 12 than in diploid patients
(P < .02). There was a trend for trisomy 12 patients to have
lower hemoglobin levels than diploid patients (P < .1). Trisomy I2 patients were more likely to have had prior treatment
than patients with diploid karyotypes (18 of 41 v 6 of 28) (P
< .02). The overall response rate to fludarabine was comparable to that found in previous studies." The complete
and partial remission rate was greater in patients with trisomy
12 than in those with other abnormalities (20 of 29 v 4 of
12) (P < .05), but was equivalent to that in patients with
diploid karyotypes. When only patients who had prior treatment were considered, patients with trisomy 12 had a higher
complete and partial remission rate than those without trisomy 12 (1 1 of 18 v 5 of 22) (P < .02). Previously untreated
patients with trisomy 12 had an increased incidence of
splenomegaly and were predominantly male ( I 5 of 18 v 24
of 44) (P < .04).
Median survival from the time of diagnosis was significantly shorter in patients with trisomy 12 by FISH (7.8 years)
and in patients with other abnormal karyotypes without trisomy 12 by FISH (5.5 years) than in patients with diploid
karyotypes without trisomy 12 by FISH (14.4 years). The
median follow-up was 4 years (Fig I). When patients with
trisomy 12 were compared with all other patients, there was
no difference in survival (Fig 2). Median time to progression
after treatment with fludarabine-based regimens tended to
be shorter in patients with other abnormal karyotypes (21.5
months) than in trisomy 12 patients (not reached) and diploid
patients (not reached) (P = . I ) (Fig 3).
DISCUSSION
The median overall survival for CLL patients is 9 years."
The presence of clonal chromosomal abnormalities predicts
for a shorter overall survival time. Specifically, complex abnormalities, increased percentages of abnormal metaphases,
abnormalities of the long arm of chromosome 14, and trisomy
12 have been associated with shortened survival. Although
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ESCUDIER ET AL
2706
1 .o
0.8
u
9
0.6
0.2
0
33
66
99
132
165 198
Months
231
264
297
330
Fig 2. Survival of CLL patients with and without trisomy 12 by
FISH (n = 1 1 7).
cytogenetic abnormalities are prognostically important in
CLL, approximately 50% to 70% of patients have either a
diploid karyotype or insufficient metaphases for analysis. In
recent studies of CLL, FISH detected a higher incidence of
trisomy 12 than conventional cytogenetic^.^'-^^ A decreased
overall survival was suggested in the study of Anastasi et a1,22
but was not statistically significant.
With the use of FISH, we were able to detect 2.6 times as
many cases of trisomy I2 as were detected with conventional
cytogenetic analysis in the 83 samples studied with both
techniques. This was primarily due to the detection of trisomy
12 in samples that were classified as diploid or had insufficient
metaphases for conventional analysis. We identified 4 1 patients with trisomy 12. Of l l patients found to have trisomy
12 in one or more metaphases with conventional cytogenetics,
10 were confirmed with FISH. In cases oftrisomy 12 identified
with FISH that were originally classified as cytogenetically
diploid or as having insufficient metaphases, dividing normal
T cells may have led to misleading result^.^.^ In 4 patients
with trisomy 12 by FISH and additional abnormal karyotypes,
3 had low numbers of trisomic cells (7.3%, 8.5%, and 1 I%),
most likely representing clonal evolution. About one-fourth
of patients with trisomy 12 had a low percentage of trisomic
cells, suggesting clonal evolution. This finding may also suggest that trisomy 12 is a secondary event in at least a proportion of the patients. If trisomy 12 was present in more
than 50% of cells by FISH, it was more likely to also be
detected with conventional cytogenetics, but in none of the
samples with less than 50% trisomic cells was cytogenetic
analysis successful.
Untreated patients with trisomy 12 tended to present with
more splenomegaly than patients without trisomy 12. This
is consistent with the more advanced stage seen in the trisomy
12 patients. Patients with trisomy 12 were also more likely
to be previously treated. This may be due to the more advanced stage requiring treatment sooner, as reported by othe r ~or,
, ~alternatively, that this is a secondary event associated
with disease progression. It is less likely that trisomy 12 develops secondary to chemotherapy.
In 1984, Han et a12 reported trisomy 12 in 63% of CLL
patients with abnormal karyotypes (23.6% of evaluable pa-
tients). Eighty-six percent of the patients with a normal
karyotype, but only 57% of those with an abnormal karyotype
survived I O years. Survival of patients with trisomy I2 alone
was not significantly different from that of patients with diploid karyotypes.' Juliusson3 reported trisomy 12 in 32.6% of
52 patients with evaluable metaphases (28.3% of all patients).
Trisomy 12 was associated with a shorter therapy-free survival, but not with overall ~ u r v i v a l A
. ~ cooperative study reported cytogenetic analyses on 433 CLL patients. Trisomy
12 was present in 3 1% of abnormal karyotypes ( 17% of evaluable patients, 15.5% of all patients). In univariate analysis,
trisomy 12 was significantly associated with a poorer survival
rate (P < ,005). The median survival of patients with any
clonal abnormalities was 7.7 years and greater than 15 years
in patients with normal karyotype^.^ This study has recently
been updated with similar results in 662 patients. The median
survival of patients with trisomy 12 alone was approximately
7.7 years.25 We have confirmed these results with a shorter
median survival in patients with trisomy 12 (7.8 years) compared with patients with diploid karyotypes (14.4 years).
When these patients were treated, the response rate was similar to that of diploid patients and of patients without trisomy
12, but there was a trend for these patients to relapse sooner
(Fig 3). These findings suggest a higher proliferative rate in
patients with cytogenetic abnormalities. In addition, patients
in clinical remission all had residual disease, which could
also contribute to the poorer survival. However, the absence
of an equally sensitive marker for residual disease in patients
with diploid karyotypes precludes knowledge of the level of
residual disease in these patients.
We have confirmed the restriction of trisomy 12 to B cells
and its absence in T cells using the combined FISH/FACS
approach. Knuutila et a15 and Autio et aI6 have previously
reported similar results using a combination of immunophenotyping and cytogenetic analysis. Our results obtained
in 200 to 500 cells in each sample rules out even low levels
of T cells with this abnormality.
Minimal residual disease was readily detected with FISH
in 6 CLL patients in complete remission and in 4 patients
in partial remission. Using FACS to separate CDI9'5' cells
enhanced our ability to detect residual disease up to eightfold.
'5a
0.6
I
.E
~
-b
0.4
E
0.2
.-6
: In = 4)
...........
-----
n
-.*.a
"."
~
0
6
12
18
24
30
36
Months
Trisomy 12
Dlplold
Other abnormal
Karyotype
~
42
48
54
60
Fig 3. Time to progression after complete remission in CLL patients with and without trisomy 12 by FISH (n = l 17).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
FISH AND CG STUDIES OF TRISOMY 12 IN CLL
At last follow-up, one of the patients in CR had relapsed.
Robertson et alZ6reported a significantly decreased remission
duration in patients with residual disease detected with
Southern blotting for gene rearrangements, persistent interstitial or nodular lymphocyte infiltrates in the bone marrow,
or greater than 5% CD19+5+cells in the blood or bone marrow. Approximately three-fourths of patients in complete remission by NCI criteria had residual lymphocyte infiltrates
in the bone marrow. Forty-three percent of these patients
had residual disease by flow cytometry.26By sorting CD 19+5+
cells, we were able to detect much lower levels of residual
disease (1 in 5,000). This approach, recently described for
patients with acute myeloid leukemia and myelodysplastic
syndrome," will be useful in monitoring intensive therapies
such as bone marrow transplantation and assessing the efficacy of bone marrow purging.
In conclusion, FISH greatly enhanced detection of trisomy
12 in CLL, predicted for a shorter survival, and readily detected residual disease in patients in clinical complete remission. With the further development of probes for FISH
for prognostically important cytogenetic abnormalities, such
as t( 1 1;14) and 14q translocations, specific abnormalities will
be identified in cytogenetically "diploid" cases and better
prognostic models may be developed leading to more selective
therapies.
ACKNOWLEDGMENT
The authors thank Sherry Pierce, RN, for expert data management
and statistical assistance.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1993 81: 2702-2707
Fluorescent in situ hybridization and cytogenetic studies of trisomy 12
in chronic lymphocytic leukemia
SM Escudier, JM Pereira-Leahy, JW Drach, HU Weier, AM Goodacre, MA Cork, JM Trujillo, MJ
Keating and M Andreeff
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