Supplemental Material
Methods in detail
Metaphase Cytogenetics
For metaphase induction, 107 peripheral blood or bone marrow cells were cultured in RPMI
1640 medium (Gibco, Gaithersburg, MD, USA) with 20% fetal calf serum in the presence of
the immunostimulatory CpG-oligonucleotide DSP30 (2 µM) (TIBMolBiol, Berlin, Germany)
and interleukin 2 (IL-2) (200 U/ml) (PreproTech Inc., Rocky Hill, USA) as described.1 After
48h of culture at 37°C, colcemid (Sigma, Munich, Germany) at a concentration of 0.15µg/ml
was added for another 24h before chromosome preparation. In addition a parallel culture
with 12-O-tetradecanoylphorbol-13-acetate (TPA) was set. After 72h, colcemid at a
concentration of 0.15µg/ml was added to the TPA stimulated culture for another 2h before
chromosome preparation. Chromosome preparation and staining was done according to
standard protocols as described elsewhere.2 In each case of complex aberrations results of
G-banding analysis were confirmed by 24-color FISH.3 Chromosomes were classified
according to the International System for Human Cytogenetic Nomenclature (ISCN 1995).4
Scoring system for counting abnormalities
Only clonal abnormalities according to ISCN definition are reported in this study. 4 Balanced
aberrations as well as a gain or loss of a whole chromosome were counted as one
abnormality, respectively, whereas unbalanced structural aberrations leading to imbalances
of two chromosomal regions were counted as two abnormalities. In detail additional copies of
a rearranged chromosome, changes in ploidy, ringchromosomes consisting of material of
one chromosome, and balanced insertions were counted as one abnormality, whereas
inverted duplications and triplications were counted as two abnormalities. The count for
complex derivative chromosomes was calculated from the number of imbalances that
occurred due to the rearrangements.
Fluorescence in situ hybridization
Interphase FISH was done as described previously.2,5 The FISH panel which was used in all
patients included probes for the detection of trisomy 12, IGH-rearrangements and deletions
of 6q21, 11q22.3 (ATM), 13q14 (D13S25 and D13S319) and 17p13 (TP53). Interphase FISH
for the detection of t(11;14)(q13;q32) was routinely done on all samples. Samples with
t(14;18)(q32;q21) in metaphase cytogenetics were confirmed by FISH analysis. The
hierarchical model to define genetic subgroups in FISH analysis was applied according to
Döhner.6 The major categories are patients with del(17p), patients with del(11q) but not
2
del(17p), patients with trisomy 12 but not del(11q) and not del(17p), patients with del(13q) as
sole abnormality, and normal karyotype.
Additional FISH probes for NMYC (2p24), BCL6 (3q27), MYC (8q24) were used in a
subgroup of patients to confirm abnormalities detected in chromosome banding analysis on
interphases of uncultivated cells using peripheral blood or bone marrow smears.
To determine the rate of false positive interphases (cut off level) 5 bone marrow smears and
5 cytogenetic preparations obtained of normal healthy donors were analyzed. 500 interphase
nuclei were scored on each slide. The cut off level was determined as mean + 2 standard
deviations and was between 3 and 4% for probes for deection of deletions, 0.9% for gains
and 0.2% for translocations. For the detection of translocations (t(11;14) and t(14;18)) double
fusion probes were used.
Analysis of the IgVH status
IgVH status was analyzed as previously described.1 In brief, monoclonal immunoglobulin
rearrangements were PCR amplified from cDNA using 6 different consensus framework
region 1 (FR1) VH forward primers and 1 consensus joining region JH reverse primer. PCR
products were agarose gel purified and analyzed by direct sequencing. Sequences were
aligned to immunoglobulin sequences in the V-BASE (http://vbase.mrc-cpe.cam.ac.uk/) and
IMGT (http://imgt.cines.fr/) databases und evaluated according to ERIC recommendations.7
Immunophenotyping
Immunophenotyping was performed as described elsewhere applying four-color-staining.8
The antibodies anti- (Dako, Hamburg, Germany), anti- (Dako), anti-CD19 (Immunotech,
Marseilles, France), and anti CD38 (Immunotech) were conjugated to the fluorochromes
fluorescein isothiocyanate, phycoerythrin, ECD, and phycoerythrin-cyanin 5.1, respectively.
The antibodies 1E7.2 (detection of ZAP-70 Caltag, Hamburg, Germany), UCHT1 (detection
of CD3, Immunotech), and HM47 (detection of CD79a, Immunotech) were conjugated to the
fluorochromes phycoerythrin, ECD, and phycoerythrin-cyanin 5.1, respectively. For
intracellular analysis Fix & Perm (Caltag) was used for fixation and permeabilization of cells.
The respective combinations of antibodies were added to 106 mononuclear cells (volume,
100 µl) and incubated for 10 minutes. After addition of 2 ml lysing solution (ammonium
chloride–based; prepared by Carl Roth GmbH, Karlsruhe, Germany), the samples were
incubated for an additional 10 minutes and were then washed twice in phosphate-buffered
saline (PBS) and resuspended in 0.5 mL PBS. Multiparameter flow cytometry analysis was
performed using a FC500 flow cytometer (Beckman Coulter, Krefeld, Germany). 20 000
3
events were acquired. Analysis of list mode files was performed using CXP Software
(Beckman Coulter). A sample was considered CD38 positive if 30% or more cells were
positive as compared to isotype-stained cell used as negative controls.9 A sample was
considered ZAP-70 positive if 20% or more cells were positive as compared to T-cell used as
positive controls.10
4
Results in detail
In addition to translocations involving an immunoglobulin locus 38 reciprocal translocations
were observed that involved 13q14 (7.6%). The breakpoints on the partner chromosomes
were 1p36, 1q32, 1q42 (n=2), 3q21, 3q29, 4p16, 4q21, 5q13 (n=2), 5q15 (n=2), 5q31, 6p23
(n=3), 6p21, 6q11, 6q23 (n=2), 6q25, 7p21, 8p21, 8q22, 9p22, 10q24, 11p15, 11q13, 11q21,
11q23, 12q21, 12q24, 13q34, 15q25, 16q24 (n=2), 17q11, and 20q13, respectively (figure
2). Interestingly, interphase FISH demonstrated a 13q14 deletion in 36 of these cases.
Therefore, metaphase FISH was performed with a whole chromosome painting probe for
chromosome 13 and a probe for the locus D13S25 or D13S319, respectively, on
metaphases. These analyses confirmed a loss of 13q material within the breakpoint region in
all 36 cases (figure 2). In 17 cases the 13q14-translocation was the sole abnormality. 15
cases showed one, 4 cases two additional abnormalities. In one case each 3, 4, and 5
additional abnormalities were detected, respectively. Recurring additional aberrations were
deletions of the long arm of the homologous chromosome 13 (n=12), deletions 11q (n=5),
deletions 2q (n=3), and an isochromosome 2p (n=2).
In addition, in the total cohort 30 other reciprocal translocations were observed. However,
none of these was recurring.
Numerical aberrations
Trisomy 12 was the most frequent numerical abnormality and was observed in 68 cases
(13.6%). Further recurrent trisomies were of chromosomes 3 (n=3), 18 (n=6), and 19 (n=11).
Gains of chromosomes X, 7, 21, and 22 were observed in one case each, doubling of the Y
chromosome in two cases.
Losses of whole chromosomes were observed in 42 cases. The most frequent
chromosomes lost were X (n=9) and Y (n=13). Recurrent losses were found also for
chromosomes 4 (n=2), 8 (n=2), 15 (n=5), 17 (n=4), and 18 (n=2). Losses of chromosomes 3,
10, 12, 13, 21 and 22 were only detected once each.
Structural unbalanced abnormalities leading to gain and/or loss of genetic material
The most frequent structural aberrations leading to loss of genetic material were a partial
loss of the long arm of chromosome 13 (n=274) and of the long arm of chromosome 11
(n=60). Further recurring losses affected 1p (n=2), 2p (n=3), 2q (n=16), 3p (n=5), 3q (n=5),
4p (n=6), 4q (n=3), 5p (n=3), 5q (n=7), 6p (n=6), 6q (n=26), 7q (n=5), 8p (n=12), 8q (n=3), 9p
(n=4), 9q (n=4), 11p (n=2), 12p (n=4), 12q (n=2), 14q (n=14), 15q (n=7), 16p (n=5), 17p
(n=37), 18p (n=7), 18q (n=3), 19p (n=3), 19q (n=3), 20p (n=2), 20q (n=4), and 21q (n=2).
5
Recurrently gained regions by structural abnormalities were 1p (n=2), 1q (n=3), 2p (n=13), 2q
(n=4), 3p (n=2), 3q (n=7), 4q (n=5), 5p (n=5), 5q (n=11),6p (n=3), 6q (n=6), 7p (n=6), 7q
(n=8), 8p (n=3), 8q (n=14), 9p (n=2), 9q (n=3), 10p (n=2), 10q (n=2), 11p (n=5), 11q (n=7),
12q (n=6), 13q (n=17), 14q (n=3), 15q (n=9), 17p (n=3), 17q (n=14), 18q (n=3), and 21q
(n=2).
Frequency of genetic aberrations with respect to disease state
378 cases were analyzed at diagnosis of CLL, while in 90 cases the analysis was performed
during the course of the disease. No data with respect to the timepoint of diagnosis was
available in 32 cases. A comparison of the frequency of genetic abnormalities as determined
by FISH and cytogenetic classes as well as the number of aberrations revealed no significant
differences between cases at diagnosis and cases analyzed during the course of the disease
with exception of 6q deletions which were less frequently observed in the cases analyzed at
diagnosis (3.2% vs. 9.0%, p=0.007).
Clonal evolution
Clonal evolution was detected in 125 cases. Recurring abnormalities during clonal evolutions
were: deletions of 13q (37x), 11q (9x), 2p (12x), 6q (8x), 7q (2x), 8p (2x), 14q (2x). Gains of
whole chromosome were observed: chromosome 12 (6x), 19 (5x), 3 (2x), 18 (2x). Recurrent
losses of whole chromosomes were loss of chromosome X (3x) and Y (2x). Twenty-one nonrecurrent reciprocal translocation and 63 unbalanced translocations were observed. Gains
and losses found in the primary clone are are compared to gains and losses due to changes
during clonal evolution in figure S5.
References
1 Dicker F, Schnittger S, Haferlach T, Kern W, Schoch C. Immunostimulatory
oligonucleotide-induced metaphase cytogenetics detect chromosomal aberrations in
80% of CLL patients: A study of 132 CLL cases with correlation to FISH, IgVH status,
and CD38 expression. Blood 2006; 108: 3152-3160.
2 Schoch C, Schnittger S, Bursch S, Gerstner D, Hochhaus A, Berger U et al.
Comparison of chromosome banding analysis, interphase- and hypermetaphaseFISH, qualitative and quantitative PCR for diagnosis and for follow-up in chronic
myeloid leukemia: A study on 350 cases. Leukemia 2002; 16: 53-59.
3 Schoch C, Haferlach T, Bursch S, Gerstner D, Schnittger S, Dugas M et al. Loss of
genetic material is more common than gain in acute myeloid leukemia with complex
aberrant karyotype: A detailed analysis of 125 cases using conventional chromosome
analysis and Fluorescence in situ hybridization including 24-color FISH. Genes,
Chromosomes & Cancer 2002; 35: 20-29.
6
4 ISCN. In: Mitelman F, ed. ISCN 1995, Guidelines for Cancer Cytogenetics,
Supplement to: An International System for Human Cytogenetic Nomenclature. S.
Karger; Basel, 1995; 1-110.
5 Buhmann R, Kurzeder C, Rehklau J, Westhaus D, Bursch S, Hiddemann W et al.
CD40L stimulation enhances the ability of conventional metaphase cytogenetics to
detect chromosome aberrations in B-cell chronic lymphocytic leukaemia cells. Br J
Haematol 2002; 118: 968-975.
6 Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L et al. Genomic
aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343:
1910-1916.
7 Ghia P, Stamatopoulos K, Belessi C, Moreno C, Stilgenbauer S, Stevenson F et al.
ERIC recommendations on IGHV gene mutational status analysis in chronic
lymphocytic leukemia. Leukemia 2007; 21: 1-3.
8 Kern W, Haferlach T, Schnittger S, Schoch C. Detection of t(14;18)(q32;q21) in B-cell
chronic lymphocytic leukemia. Arch Pathol Lab Med 2005; 129: 410-411.
9 Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL et al. Ig V gene mutation
status and CD38 expression as novel prognostic indicators in chronic lymphocytic
leukemia. Blood 1999; 94: 1840-1847.
10 Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M. et al. ZAP-70
expression as a surrogate for immunoglobulin-variable-region mutations in chronic
lymphocytic leukemia. N Engl J Med 2003; 348: 1764-1775.
7
Table S1: Clinical stage according to Binet
N
Binet A
Binet B
Binet A/B*
Binet C
no data
53
25
266
78
78
* Binet A or B according to haemoglobin concentration an platelet count, no data on lymph
node status available to differentiate between Binet A and B
8
Table S2: Karyotypes of cases with cytogenetically independent subclones in 10 patients
with CLL
Patient-No. Karyotype
1
46,XX,del(2)(q33q37) [4]
46,XX,del(13)(q12),del(14)(q11q32),der(15)ins(15;13)(q13;q?q?),
der(16)t(13;16)(q?;p11) [2]
46,XX [4]
2
46,XX,del(6)(q15q25) [3],
46,XX,der(1)del(1)(p32)del(1)(q32),der(9)t(1;9)(?;q34),del(17)(p11) [14]
3
46,XX,dup(15)(q26q11),i(17)(q10) [8]
46,XX,inv(1)(p11q44),der(3)t(3;4)(q21;?)t(4;5),der(4)t(4;5)(p11;?)t(3;5),
del(5)(q15q33),del(6)(q15) [cp5]
46,XX [7]
4
46,XX,del(11)(q13q23) [5]
46,XX,t(1;13)(q32;q14) [3]
46,XX [13]
5
46,XX,del(11)(q14q25) [11]
46,XX,t(5;13)(q13;q14) [2]
46,XX [7]
6
47,XX,t(7;8)(p13;p23),+12 [6]
46,XX.ish del(13)(q14q14)(D13S319-) [5]
46,XX [14]
7
46,XY,del(13)(q14q21) [6]
45,X,-Y [9]
46,XY [10]
8
46,XY,del(6)(q15q23) [3]
45,X,-Y,del(6)(q15q23) [11]
46,XY,add(4)(q35) [2]
46,XY [4]
9
46,XY,del(13)(q14q21).ish del(13)(q14q21)(D13S25-),
del(13)(q14q14)(D13S25-) [13]
46,XY,t(5;13)(q15;q14),del(11)(q21q25),del(13)(q14q21).ish
der(5)t(5;13)(q15;q14)(D13S25-),del(13)(q14q21)(D13S25-),
der(13)t(5;13)(q15;q14)(D13S25-) [3]
47,XY,+12 [3]
46,XY [2]
10
46,XY,t(4;13)(q21;q14) [8]
47,XY,+12 [2]
46,XY [10]
9
Figure Legends
Figure S1-S3:
Chromosomal gains and losses as determined by chromosome banding analysis in CLL. Losses are
shown in red to the left side and gains in green to the right of the affected chromosomal region
(created by using CyDAS (http://www.cydas.org/OnlineAnalysis/)). The thickness of the bars
represents the number of cases showing the respective gain or loss.
S1. cases with a translocation involving one of the immunoglobulin loci (14q32, 2p11, 22q11) (n=31)
S2. cases without a translocation involving an immunoglobulin locus and without a complex aberrant
karyotype (n=387)
S3. cases with a complex aberrant karyotype defined as showing ≥3 abnormalities (n=82)
Figure S4:
Localization of the breakpoints on the partner chromosomes involved in 38 cases showing reciprocal
13q14-translocations
Figure S5:
In 125 cases clonal evolution was observed. Gains and losses found in the primary clone are shown
on the left and are compared to gains and losses due to changes during clonal evolution shown on the
right. Losses are shown in red to the left side and gains in green to the right of the affected
chromosomal region (created by using CyDAS (http://www.cydas.org/OnlineAnalysis/)). The thickness
of the bars represents the number of cases showing the respective gain or loss.
10
Figure S1
11
Figure S2
12
Figure S3
13
Figure S4
14
Figure S5