Hereditas 97: 221-225 (1982)

Chromosome break points in clonal and nonclonal chromosome changes in human chronic lymphocytic leukemia KIRSI AUTIO and JIM SCHRODER

Folkhiilsan Institute of Genetics, Helsinki, Finland

AUTIO, K. and SCHRODER, J. 1982. Chromosome break points in clonal and nonclonal chromosome changes in human chronic lymphocytic leukemia. - Hereditas 97: 221-225. Lund, Sweden. ISSN 0018-0661. Received April 2, 1982

The break points in clonal and nonclonal chromosome changes in human chronic lymphocytic leukemia, CLL, are described. In nonclonal changes the areas most often affected were those near the telomerer (28.6 %) and centromeres (19.0 %), chromosomes 1 , 2 and X being involved more frequently than others. In clonal changes chromosomes 6 and 14 were more often involved in aberrations than the other chromosomes.

Kirsi Autio, Folkhiilsan Institute of Genetics, P . B . 819, SF-WlOl Helsinki 10, Finland

As data accumulate on chromosome changes in various malignant conditions, it is becoming cru- cially important to pinpoint the exact sites of chromosome breakage. To date, chronic mye- locytic leukemia (CML) is the only human malignant disease in which a characteristic and consistent karyotypic finding, i.e. the Philadelphia chromosome (Ph'), has been established. How- ever, the 14q+ anomaly in lymphoproliferative disorders (e.g., Burkitt lymphoma, lymphosar- coma, etc.) is slowly emerging as a specific karyo- typic anomaly comparable to the Ph' in CML (ZECH et al. 1976; KAISER-MCCAW et al. 1977; MANOLOVA et al. 1979).

The karyotypes of the malignant cells in chronic lymphocytic leukemia, CLL, have until recently been poorly known. However, during the past 2 years we (AUTIO et al. 1979; SCHRODER et al. 1981) and others (GAHRTON et al. 1980a and b; MORITA et al. 1981) have shown that this disease is character- ized by specific chromosome changes. In the present paper we report the chromosomal break points in clonal and nonclonal chromosome changes in 27 patients with CLL.

Patients

Blood was obtained from 22 randomly selected patients with nonclonal chromosome abnormali- ties, and from all 5 patients with clonal chromo- some abnormalities among 100 consecutively studied patients with CLL. Eleven patients were

males and 16 females. Their ages ranged from 49 to 91 years, the average age being 72. White blood cell counts ranged from 6 to 364~10~11 (mean 42x 109/1), 60-99 % being lymphocytes. Studies of surface Ig on these lymphocytes and their ability to rosette with sheep red blood cells (SRBC) showed that all the patients had B-cell CLL.

Methods

Cell culture and preparation

Lymphocyte suspensions were obtained from peripheral blood by centrifugation through a Ficoll-Paque gradient (Pharmacia Fine Chemicals, Sweden). The mitogens used were leukoagglutinin (LA) (IOpglml), protein A (PA) from Staphylo- coccus aureus, Cowan I , Lot 2414 (100pg/rnl) (both from Pharmacia Fine Chemicals, Sweden), pokeweed mitogen (PWM) (Grand Island Biolog- ical Company, U.S.A) at a dilution of I : 100 and lipopolysaccharide B (LPS) from Escherichia coli 055:B5 (Difco Laboratories, U. S. A.) (40pg/ml). In order to obtain mitoses from spontaneously dividing lymphoblasts, cells were also cultured without mitogens.

Lymphocytes were cultured in closed 10 ml dis- posable culture flasks with a total of 2-4x1O6 lymphocytes in 2 ml of culture medium (RPMI 1640 supplemented with 10 % fetal calf serum, 32 IU/ml penicillin and 32 pg/ml streptomycin, all from Gibco Bio-Cult, Glasgow, Scotland). The cultures were harvested after 2 (no mitogen, LPS),

222 K. A U T ~ O AND J . SCHRODER Hereditas 97 (1982)

4 P

g. 1

U

4

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18

3 1 2 =' y& *

3 3 2 -- *

19 20 21 22 Y X

Fig. 1. Diagram of the human chromosomes indicating break points of nonclonal (small arrows) and clonal (large arrows) chromosome changes.

Hereditas 97 (1982) HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA 223

19 20 21 22 Y X Fig. 3. Karyotype of a cultured human lymphocyte: 46,XY, t(14;15) (nonclonal). Fig. 2. Karyotype of LPS-cultured human lymphocyte:

46,XY, t(6;13), t ( l l ; l4 ) , del (6) (clonal).

4 (LA), 5 (PA), 6 (no mitogen, LPS) and 7 (PWM) locations in LPS cultured cells and in 10 % days. of PWM cultured cells; t(6;7)(q27;q22), t(7; 13)(q22;

One hour before harvesting, 0.5 p g of desace- q12), t(11;14)(ql l;q32) and t(17;?)(ql I ; ? ) . The tylmethylcolchicine (Cokemid@, Ciba. Switzer- third patient had del (6) (q 16) and two different land) was added to each culture flask. Chromo- translocations in LPS' cultures; t( 11;14)(q21; some preparations were made by conventional q32) and t(6;13)(p23;q14) (Fig. 2). One of the re- methods. maining two patient had the following transloca-

tion in all LPS-cultured cells t(6;20)(q 16;p13),

G-banding and chromosome analysis

Five to 10 days after preparation the slides were incubated for 1 h at 60°C in 2xSSC (salinelsodium citrate) and rinsed several times in distilled water. They were then treated with 0.1 % trypsin in phosphate-buffered saline (PBS) for 1 min, washed three times in PBS and stained with Giemsa for 12 min. When possible, karyotype analysis was based on a minimum of 10 cells from

while the fifth patient had trisomy 12 and a dele- tion of chromosome 14 (14q-)(q22) in LPS cul- tures and in control cultures.

Four breaks could be found in chromosome 6 (p23, q16, q16, q27), one break in chromosome 7 (q22), two in chromosomes 11 (q l l , q21) and 13 (q12, q14), three in chromosome 14 (q22, q32, q32), and one each in chromosomes 17 (q l l ) and 20 (p13).

The nonclonal rearrangements found in 22 pa- each culture of each patient. Chromosome break tients can be seen in Fig. l and 3. The total number points were determined in all five cases with of nonclonal breakages was 63 and the total sites clonal chromosome changes, and 22 cases with of breakages 50; in 13 cases a break occurred at nonclonal chromosome changes. the same site twice. A relatively high proportion of

the break points were close to the telomeres (28.6 %) and centromeres (19.0 %), and only 7.9 % of the break points were at identical sites in both clonal and nonclonal abnormalities.

Eight of all nonclonal breaks (12.7 %) had oc- The distribution and number of breaks in the dif- curred at six sites of chromosome 1, and two ferent chromosomes can be seen in Fig. 1. Clonal breaks had occurred in the bands q25 and p12. chromosome changes were found in five patients. Seven breaks ( I 1. I %) had occurred at six sites of One patient had trisoniy 12 in 10 % of his LA and chromosome 2 (twice in the centromere region). PA cultured cells, another had four different trans- Three breaks (4.8 %) had occurred in chromo-

Results

224 K. AUTIO AND J . SCHRODER Hereditas 97 (1982)

some 3, two at the telomere and one at p21. Two breaks (3.2 %) had occurred in chromosome 4, both at the telomere. In chromosomes 6 and 7 four break points (6.4 9%) were found: two of those in chromosome 7 were in band p22. Chromosomes 8 and 9 each had two breaks (3.2 %), and chromo- somes 1 1 and 12 four breaks (6.4 %), two of those in chromosome 12 being in band q 13. Two breaks (3.2 %) were found in chromosome 14 and four breaks (6.4 %) at three sites of chromosome 15, two of these four at the telomere. One break (1.6 %) was found at the telomere of chromosome 16, two breaks (3.2 %) at the telomere of chromo- some 17 and three breaks (4.8 % ) in two bands of chromosome 18, two of these being in band q21. One break ( I .6 9%) had occurred in the centromere region on chromosome 20 and three breaks (4.8 9%) in two different bands of chromosome 21. Seven breaks ( 1 1 . 1 %) had occurred in the X chromosome in five different bands, two were in the centromere region, and two others in band q24. No breaks were found in chromosomes 5, 10, 19, 22 and Y.

Discussion Two types of chromosome change seem to occur in human CLL. Nonclonal chromosomal translo- cations can be found in both T- and B-cell mitogen cultures (CROSSEN 1974; FLEISCHMAN and PRIC~GINA 1977), while the clonal abnormalities seem to be restricted to B lymphocytes (GAHRTON et al. 1980a and b: SCHRODER et al. 1981). The nonclonal changes involve different chromosomes and chromosome sites in different cells, but certain chromosomes and chromosome regions are in- volved more often than others.

The chromosomes most often involved in non- clonal changes are numbers 1 (8 breaks out of 63, 12.7 %), 2 (7 breaks, 11 .1 %), X (7 breaks, 1 1 . 1 %), 6, 7, I 1 , I2 and 15 (all with four breaks, 6.4 %). The regions most often involved in break- ages were the telomeres, or regions close to the telomeres (28.6 9i of all breaks) in chromosomes 2, 3, 4, 7, 1 1 , 15, 16, 17, 18, 21 and X , and in the centromere regions (19.0 96 of all breaks) in chro- mosomes 2 , 6 , 8, 9, I I , 12, 20, 21 and X. A break- age had occurred twice in thirteen sites, the chro- mosomes involved being 1, 2, 3, 4, 7, 12, 15, 17, 18, 21 and X .

The most common clonal chromosome change in C L L reported by us (AUTIO et al. 1979; SCHRO-

DER e t al. 1981) and others (GAHRTON et al. 1980a and b; M ~ R I T A et al. 1981) is trisomy 12. Chromo- somes 14 and 17 are also often involved in aberra- tions in C L L (AUTIO et al. 1979; GAHRTON et al. 1980b: MORITA et al. 1981: SCHRODER et al. 1981). MORITA e t al. (1981) have reported marker 14q+ in two patients, with break points a t q32. We have reported here two cases of t(11;14) with break points a t q l l;q32 and q21;q32. A similar or identi- cal translocation has also been reported by GAHR- TON e t al. (1980b) with break points q13:q32 (two cases). Thus, this telomeric band in chromosome 14 (q32) seems to be vulnerable to breakage. In one patient we also found a deletion 14 with the break point in band q22. Translocations involving chromosome 14 and chromosomes 8, 14 and 18 have also been reported in C L L (KAISER-MCCAW et al. 1975: FLEISCHMAN and F’RIGOGINA 1977: MITEL- MAN and LEVAN 1981).

Abnormalities of chromosome 17 have been seen as a clonal change in some cases of C L L (FINAN et al. 1978: MORITA et al. 1981). Two cases with isochromosome 17q have been described by FINAN et al. (1978) and MORITA et al. (1981). We found a translocation between chromosome 17 and an unidentifiable chromosome in one patient. GAHRTON et al. (1980b) reported three deletions involving chromosome 6, with break points in re- gions q15, q21 and p12. Chromosome 6 was in- volved in three clonal translocations in our mate- rial, the translocations being t(6;7)(q27;q22), t(6:20)(q16:p13) and t(6: 13)(p23;q14) and in one deletion q 16.

The break points occurring in nonclonal and clonal chromosome abnormalities seem to be clustered in different chromosomes (clonal 6, 14, 17, and nonclonal in I , 2, X) and also in different chromosome regions. The mechanisms for this are unclear, but we suggest that the clonal changes are the primary event involved in malignant prolifera- tion whereas the nonclonal abnormalities are a secondary phenomenon caused by treatment of the disease or by other factors.

Acknowledgments. - This work was supported by the Finnish National Research Council for Natural Sciences, the Sigrid Juselius Foundation and the Emil Aaltonen Foundation. We wish to thank all the clinicians who helped us to obtain blood samples from the patients.

Literature cited AUTIO, K . , TURUNEN, O., PENTTILA, O., ERAMAA, E., DE LA

CHAPELLE, A. and SCHRODER, J . 1979. Human chronic

Hereditus 97 (1982) HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA 225

lymphocytic leukemia: Karyotypes in different lymphocyte populations. - Cancer Genet. Cytogenet. I : 147-155

CROSSEN, P. E. 1974. Giemsa banding patterns in chronic lymphocytic leukaemia. - Humangenetik 27: 151-156

FINAN, J . , DANIELE, R., ROWLANDS, D. Jr . and NOWELL, P. 1978. Cytogenetics of chronic T cell leukemia including two patients with a 14qf translocation. - Virchows Arch. E Cell Pathol. 29: 191-127

FLEISCHMAN, E. W. and PRIGOGINA, E. L. 1977. Karyotype peculiarities of malignant lymphomas. - Hum. Genet. 35: 269-279

GAHRTON, G., ROBERT, K.-H., FRIBERG, K., ZECH, L. and BIRD, A. G. 1980a. Extra chromosome 12 in chronic lymphocytic leukemia. -Lancet (I): 146147

GAHRTON, G., ROBERT, K.-H., FRIBERG, K., ZECH, L. and BIRD, A. G. 1980b. Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealed by polyclonal B- cell-mitogen stimulation. -Blood 56: 640-647

KAISER-MCCAW, B., HECHT, F., HARNDEN, D. G. and TEPLITZ, R. L. 1975. Somatic rearrangement of chromosome 14 in human lymphocytes. - Proc. Nut. Acad. Sci. 72: 207 1-2075

KAISER-MCCAW, B., EPSTEIN, A. L., KAPLAN, H. S. and HECHT, F. 1977. Chromosome 14 translocation in African and North American Burkitt's lymphoma. - Int. J. Cancer 19: 482486

MANOLOVA, Y., MANOLOV, G . , KIELER, J., LEVAN, A. and KLEIN, G. 1979. Genesis of the 14q+ marker in Burkitt's lymphoma. - Hereditas 90: 5-10

MITELMAN, F. and LEVAN, G. 1981. Clustering of aberrations to specific chromosomes in human neoplasms IV. A survey of 1,871 cases. - Hereditas 95: 79-139

MORITA, M., MINOWADA, J . and SANDBERG, A. A. 1981. Chromosomes and causation of human cancer and leukemia. XLV. Chromosome patterns in stimulated lymphocytes of chronic lymphocytic leukemia. - Cancer Genet. Cytogenet. 3: 293-306

SCHRODER, J . , VUOPIO, P. and AUTIO, K. 1981. Chromosome changes in human chronic lymphocytic leukemia. - Cancer Genet. Cytogenet. 4 : 11-21

ZECH, L., HAGLUND, U . , NILSSON, K. and KLEIN, G. 1976. Characteristic chromosomal abnormalities in biopsies and lymphoid cell lines from patients with Burkitt and non-Burkitt lymphomas. -Znt. .I. Cancer 17: 47-56