99
Cancer Letters, 49 (1989) 99- 105 Elsevier Scientific Publishers Ireland Ltd.
Clonal chromosomal leukemia B.N.
Nayak,
J. Sokal
and
changes
in chronic lymphocytic
M. Ray
Departments of Pediatrics and Child Health, Human Genetics, Uniuersity of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, (Canada) (Received 7 July 1989) (Revision received 13 August 1989) (Accepted 5 September 1989)
Summary Cytogenetic analysis was carried out in 28 B-CLL patients (21 males and 7 females, 3885 years old, with median age 64 years, disease stage O-IV). Peripheral nominator cells (1 x 10’) or isolated B-lymphocytes were incubated in oitro for 5 - 7 days. The cells were stimulated by pokeweed mitogen (PWM), or phorbol myristate-acetate (PMA), with or without 10% conditioned medium (CM) derived from a T cell leukemia line or 10% B-cell growth factor (BCGF). Twenty-two patients (79%) responded to PWM + CM; 5 out of 5 patients responded to PWM + BCGF. The average mitotic index 1+ S.E.M.) for PWM, PMA, PWM + CM, PMA + CM, PWM + BCGFwere0.13 f 0.01, 0.24 + 0.13, 0.51 & 0.11, 0.14 + 0.06 and 0.63 -I- 0.15, respectively. Cytogenetic analysis revealed the presence of abnormal karyotypes in 22 patients. Fourteen patients (50%) had clonal chromosome aberrations which included: monosomy 1, 9, 17, 18, 21, and X chromoCorrespondence to: M. Ray, Cytogenetics Laboratory, Health Sciences Centre, Room FE 028, Community Services Building, 685 William Avenue, Winnipeg, Manitoba, R3E 022, Canada.
0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland
some, and trisomy of chromosomes 7, 9, 20, 21 and 22. The clonal structural aberrations del(5) were i(6q), inv(l2) (q15q24), (p13pl5), del(l0) (q24). No homogeneously staining regions (HSR) were observed. Four patients with resistance to anti-neoplastic drugs showed the presence of double minute chromosomes (dmin) ranging in frequency from 5 to50%.
Keywords: chronic lymphocytic leukemia; clonal chromosome aberrations; pokeweed mitogen; phorbol-myristate acetate; B-cell growth factor; double minute chromosomes. Introduction Chronic lymphocytic leukemia (CLL) is the commonest form of leukemia in North America and Europe, accounting for 30% of all cases, and is characterized by the accumulation of relatively mature-appearing lymphocytes in the peripheral blood, marrow, lymph nodes, spleen and liver [l]. Approximately 95% of patients have a clonal expansion of Blymphocytes with < 5% being of T-cell origin [2]. The clinical presentation and rate of
lreland Ltd.
100
disease progression are highly variable and prolsnosis can be best predicted by the clinical . _ stage of the disease, using the Rai classification [3,4]. However, analysis of chromosomal changes in clonal B-cells from chronic B-cell lymphocytic leukemia (B-CLL) patients has been difficult due to a low spontaneous mitotic index [5-71, and lack of effective universal Bcell monoclonal mitogens. Until recently, there has been variable success in obtaining analysable metaphase chromosomes from peripheral lymphocytes of patients with B-CLL, using a number of mitogens, such as PWM [8- 121, lipopolysaccharides (LPS) [ 111, leucoagglutinin (LA) [ 131, tetradecanoyl-o-phorbol-13acetate (TPA) [lo], PMA [9], and cytochalasin-B [8]. Results of these studies show that approximately 50% of CLL patients present with karyotypic abnormalities and 25% patients have trisomy 12, a characteristic chromosome anomaly. It has been suggested that patients with trisomy 12 may require treatment sooner after diagnosis than those with normal karyotypes, or other abnormalities [6], while multiple aberrations within the same cell carry a poorer prognosis than trisomy 12 [6,7,14,15]. These studies would thus suggest that an increasing complexity of clonal chromosomal abnormalities may be associated with an increased rate of disease progression. Evidence in the literature indicates that mitogen derived from either plant or bacterial sources yield different frequencies of trisomy 12 as compared with mitogen of viral origin [6,8--131. However, a viral mitogen such as Epstein-Barr virus, caused chromosomal abnormality in cells from normal individuals [5]. Thus there is a need for a standardized protocol and characterization of the clonal aberrations in B-CLL patients. The present investigation was undertaken to determine the mitogen response and clonal chromosome aberrations and their relationship to the stage of CLL. Materials and methods Patients
Twenty-eight
patients
with B-cell CLL were
studied; 21 patients were male and 7 were female. Their ages ranged from 38 to 85 years (median 64 years) with Rai stage O-IV. Cell culture and chromosome
analysis
Peripheral mononuclear cells (approximately 98% lymphocytes) were isolated from heparinized venous blood by centrifugation over Ficoll400-diatrizoate sodium (Pharmacia) and 1 x lo7 cells/ml were incubated for 4-5 days in 10 ml of RPM1 1640 culture medium (GIBCO) with 10% fetal bovine serum (Bocknek) at 37 OC in an atmosphere of 95 % air and 5% CO,. PWM (GIBCO), (l:lOO), alone or with 10% BCGF (Cellular Products Inc.), or in combination with 10% of medium prepared from a T-cell supernatant Rex leukemia cell line (cell line was provided by Dr. J. Pawly, Dept. of Molecular Immunology, Roswell Park Memorial Institute, Buffalo, N.Y.) or PMA (GIBCO), 1 pg/ml were used to stimulate the cells. Phytohaemagglutinin M form (PHA-M, GIBCO) was used as a polyclonal T-cell activator at a concentration of 100 pg/ml. Colcemid (GIBCO), 0.05 pg/ml, was added 2-2.5 h before harvesting. Chromosome analyses were performed by conventional Giemsa staining (4%), to determine the gross morphological changes in chromosomes such pulverization, dmin and as gaps, breaks, homogeneously staining regions (HSR) and by a modified G-banding technique [16] to determine the specific chromosome aberrations. B and T-cell separation
Peripheral mononuclear cells from three patients were isolated as described above. Monocyte removal was performed by suspending the mononuclear cells in 10 ml of RPM1 in a plastic flask and incubating at 37OC for 1 h, after which 1.0 x lo7 of the nonadherent lymphocytes were mixed with 100 ~1 of packed 2-aminoethylisothiouronium bromide-treated sheep red blood cells (SRBC) and incubated for 1 h at 4OC 117,181. The cells were then separated by centrifugation over two consecutive Ficoll gradients. SRBC were lysed with 10 mM Tris/140 mM ammonium chlor-
101
ide, pH 7.4 and populations were (Cellular Products combination with cells. Successful carried out only in
the enriched lymphocyte cultured as above. BCGF Inc.), was used alone and in PWM to stimulate the Bchromosome analysis was 1 (G. W .) out of 3 patients.
Analysis Fifteen to 20 G-banded metaphases per patient were analyzed in order to identify the chromosome(s) involved in numerical and structural changes. Approximately 1000 cells were counted to estimate the mitotic index (MI) for each mitogen or mitogen combinations. A clonal chromosomal abnormality was defined by one of the following criteria: (i) Gain of specific chromosome in at least two cells; (ii) Loss of specific chromosome in at least three cells, or (iii) identical structural aberrations in at least two cells [ 191. Results The MI f S.E.M. values for PWM, PWM + CM, PWM + BCGF, PMA, PMA + CM, PHA and PHA + CM were 0.13 f 0.01, 0.51 + 0.11, 0.63 +- 0.15, 0.24 f 0.13, 0.14 f 0.06,0.36 + 0.39and0.52 -+ 0.11, respectively (Table 1). In 79% of patients analysable metaphases were obtained when the peripheral lymphocytes were stimulated by PWM in combination with CM. Addition of condition medium or growth factor along with PMA yielded a lower number of metaphases
Table 1.
compared to PMA alone. Cells which were not stimulated with mitogens had no metaphases. Cells treated with PHA gave MI value of 0.36, PHA along with PWM and CM yielded considerably higher MI value (0.52). Twenty-two of the 28 patients (79%) had abnormal karyotypes. Fifteen of the 21 male patients (71%) and all 7 female patients showed karyotypic abnormalities. Fifty per cent of the patients showed clonal chromosome aberrations (Table 2). Nine out of 21 males (43%) and 5 out of 7 females (71%) had clonal chromosomal aberrations. The number of clones per patient ranged from 1 to 4. The numerical clonal chromosome aberrations included trisomy of chromosomes 7, 9, 20, 21 and 22, and monosomy for chromosome 1,9, 17, 18,21 and X. The clonal structural rearrangements observed were: de1 (5) (p13p15), i(6q), del(l0) (q24), inv(l2) (q15q24). Two patients showed the presence of a clonal marker, chromosome of apparent identical morphology. The nonclonal structural aberrations encountered in these patients include del(5) (q31), del(6) (p22), del(8) (q22q24), del(8) (q24), del(l1) (q24), del(12) (q22), del(15) (q24), t(1;13) (p36;q34), t(l1; 11) (q14;q24), t(13;18) (pll,qll), t(X;12) (q22;q21). Four patients out of 28 showed dmin with a frequency ranging between 5 and 50% (Fig. 1). Patients with dmin were found to have developed resistance to a number of antineoplastic drugs. Six out of 28 patients belonging to different stages of CLL had apparent normal karyotypes.
Mitotic index value. PWM
PWM + CM
PMA
PMA + CM
PWM + BCGF
of patients Patients
25
28
18
8
5
responded Patients not
15 (60%)
22 (79%)
9 (50%)
5 (62.5%)
(100%)
responded
10 (40%)
6 (21%)
9 (50%)
3 (37.5%)
0 (0%)
0.51 + 0.11
0.24 + 0.13
0.14 + 0.06
0.63 + 0.15
Total no.
Mean MI
0.13 + 0.01
102
Table 2.
Clonal chromosome aberrations in CLL patients.
Patients
Sex
Stage
Mitogens
No. of cells analyzed
CIonaI abberations
AB
M
I
PWM + CM
20
45,XY, - 18
TB
F
IV
PWM PWM + CM
20 15
BCGF
15
45,x, -x 45,xx, - 9 45,xx, - 17 45,x, -x
PWM
20
PWM + CM
20
LC
M
IV
45,XY, - 21 46,XY,inv(12) 47,XY,inv(12) 47,XY, + 9
(q15q24) (q15q24),
+ mar
JD
F
0
PWM + CM
20
45,Xx,
AGO
M
III
PWM
20
46,XY,2p + ,del(5) (~13~15) 45,XY, - 1
- 8, - 9, + mar
46,XY, (6q) AJ
F
II
PWM + CM
20
46,XX,21p
ML
F
IV
PMA
25
47,xx,
APL
M
IV
PWM
20
47,XY, + 2
GR
M
I
PWM
20
45,XY, - 17
us
M
IV
PWM + CM
20
47,XY, + 20
LS
M
I
PWM + CM
20
45,XY, - 17 47,XY, + 21
AS
F
II
PWM + CM PWM + PMA
20 20
47,Xx, + mar 45,x, -x 47,xx, + 7 47,Xx, + mar
GV
M
IV
PWM + CM
20
45,x, -Y 45,XY, - 10
GW
M
II
PWM
15
PWM + CM
20
PWM + BCGF
20
46,XY,deI(5) (~13~15) 47,XY, + 4,deI(lO) (q33) 46,XY,del(5) (~13~15) 46,XY,del(lO) (q24) 46,XY,del(lO) (q24)
PWM, Pokeweed mitogen; PMA, Phorbol-12-myristate-13-acetate; T-cell leukemia cell line growth medium.
+
+ 22
BCGF, B-cell growth factor; CM, supernatant of
103
Fig. 1.
Double minute chromosomes
(dmin) in CLL.
Discussion
High frequencies of clonal and non-clonal chromosome abnormalities are a prominent feature of the lymphocytes in CLL [13,14,20]. Some of the clonal abnormalities constitute the primary karyotypic change in the proliferation of the neoplastic B-cells, while non-clonal a secondary abnormalities are usually phenomenon, possibly related to treatment [5,13,21,23]. A high frequency of cytogenetitally abnormal cells, and multiple chromosome abnormalities, has previously been associated with poor prognosis [6,7,14,22]. The results of this study show that 50% of the patients had clonal chromosome abnormonosomy of included which mality chromosome 1, 9, 17, 18, 21 and X chromosome and trisomy of chromosomes 7, 9, 20, 21 and 22. The clonal structural chromosomal aberrations observed in these patients were del(5) (p13p15), i (6q), de1 (10) (q24) and inv (12) (q15 q24). According to the other studies, trisomy 12 has been shown as a common abnormality in CLL patients [8,11,12,23], although other aberrations such as de1 (6s)
(8,231, trisomy3 [11,23,24], trisomy8,9, 16, 18, 20 and 21 [ll], monosomy 8, 10, 16 (24), x (12) and 149 + [23] have also been reported. Sequential chromosome study [ 1 l] suggests that in some B-CLL patients two distinct clones, one of which is a major clone with trisomy 12 and another with various other abnormalities with lower frequencies might co-exist in vivo for relatively long periods. These studies however, indicate the extent of chromosomal changes in CLL patients and complexity in the clonal evolution and selection of neoplastic cells. Four of 28 patients showed dmin. These paired chromatin bodies have been found in a number of human tumors of neurogenic origin and in certain leukemias [25,26]. Those patients showing dmin demonstrated a resistance to a number of antineoplastic drugs. Although the role of dmin is not known, it is believed that dmins are extra-mitotically transferred between cells and may contain amplified human proto-oncogenes [27] and can be produced from precursors derived from a chromosomal deletion [28]. The presence of multiple clones in B-CLL patients would indicate that there were more than one subgroup in the leukemic lymphocyte population of the patients. It is difficult to conclude whether the chromosomal aberrations observed in CLL patients represent the population of malignant B-cells or both B- and T-cells. Some of the polyclonal activators, such as PWM, PMA may stimulate B- and Tcells [29-331. However, when chromosomal analysis was performed on immunologically phenotyped cells, PWM and TPA have been shown to stimulate clonal B-cells with either Lambda or Kappa immunoglobulin light chains with 47, + 12 karyotype [lo]. In the present study clonal trisomy 12 or 14q + were not observed. Whether this is due to particular mitogens used or other factors that might be unique to the patients, or the population under study, are not known. Analysis of some reported results [5,6,8-10,13,34] show that trisomy 12 was observed in 42% of cases of B-
104
CLL when cells stimulated by Epstein Barr Virus (EBV) compared to 9%) 7% and 3% for LPS, PWM and PA, respectively. EBV confounded the chromosomal analysis since it caused chromosomal abnormality including tetraploidy in 7-25% of cells in normal individuals [5]. If trisomy 12 is considered as a characteristic aberration of B-leukemic cells in B-CLL patients, then the choice of mitogens is perhaps important in studying the biochemical and clinical significance of specific chromosoma1 changes in these patients. In B-CLL, a great number of cells are B-cell types, a small proportion of cells are (< 5%) T-cells [2,35381. When mitogens such as PWM and PMA are employed, it is likely that some T-cells would be stimulated since these mitogens are believed to be B- and T-cell mitogens. Recently, it has been shown that inversion of chromosomes 14 of the segment qllq32 has been associated with T-cell disease [39]. Another aberration involving interstitial deletion or translocation of chromosome 13 at band position q14 has been shown in cells stimulated by PMA [9], a tumor-promoting agent that was shown to induce differentiation of human leukemic cells [33,40]. Further studies are required to study the cell specificity and clinical significance of these aberrations. It is important however, to point out that the lack of effective monoclonal B-cell activators limits the evaluation of intrinsic chromosomal changes in patients with B-CLL, although involvement of T-cells in B-CLL disease complex cannot be overlooked.
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Acknowledgements 12
The authors would like to express their sincere thanks to Dr. L.G. Israels for reviewing the manuscript and providing the facilities and to Dr. J.B. Johnston for kindly providing the blood samples. We gratefully acknowledge the financial assistance from the Cancer Treatment and Research Foundation of Manitoba and the Childrens Hospital of Winnipeg Research Foundation Inc.
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Perri R.T. and Kay N.E. (1986) Abnormal T cell function in early stage chronic lymphocytic leukemia (CLL) patients. Am. J. Hematol., 22,55-61. Callen D.F. and Ford J.H. (1983) Chromosome abnormalities in chronic lymphocytic leukemia revealed by TPA as a mitogen. Cancer Genet. Cytogenet., 10.87-93. Miller R.A. and Gralow J. (1984) The induction of Leu-1 antigen expression in human malignant and normal B cells by phorbol myristate acetate (PMA). J. Immunol., 133, 3408-3414. Nadler L., Ritz J., Bates M.P., Park E.K.. Anderson K.C., Sallan S.E. and Schlossman S.F. (1982) Induction of human B-cell antigen in non-T cell acute lymphoblastic leukemia. J. Clin. Invest., 70,433. LeBien T.W., Bollum F.J., Yasmineh W.G. and Kersey J.H. (1982). Phorbol ester-induced differentiation of a non-T, non-B leukemia cell line: model for human lymphoid progenitor cell development. J. immunol., 128, 1316-1320. Gahrton G., Robert K.H., Friberg K., Zech L. and Bird A.G. (1980) Extra chromosome 12 in chronic lymphocytic leukemic. Lancet, i, 146-147. Brouet J.C. and Seligmann, M. (1981) T derived chronic lymphocytic leukemia. Pathol. Res. Pratt., 171, 262267. Foa R., Catovsky D., Brozovic M., Marsh G., Ooyirilang kumaran T.. Cherchi M.. Galton D.A.G. (1979) Clinical staging and immunological findings in chronic lymphocytic leukemia. Cancer, 44,483. Kay N.E.. Johnson J.D., Stanek R., Douglas S.D. (1979) T-cell subpopulations in chronic lymphocytic leukemia: Abnormalities in distribution and in vitro receptor maturation. Blood, 54,540. Catovsky D., Laurta F., Matutes E., Foa R., Mantovani V., Tura S., Galton D.A.G. (1981) Increase in T lymphocytes in B-cell chronic lymphocytic leukemia II. Correlation with clinical stage and findings in B-prolymphocytic leukemia. Br. J. Haematol., 49,539. Zech L., Gahrton G., Hammarstrom L., Juliusson G., Mellstedt H. and Robert K.H. (1984) Inversion of chromosome 14 marks human T cell chronic lymphocytic leukemia. Nature, 308,858-860. Nadler L., Ritz J., Bates M.P., Park E.K., Anderson K.C., Sallan S.E. and Schlossman S.F. (1982) Induction of human B-cell antigen in non-T-cell acute lymphoblastic leukemia. J. Clin. Invest., 70,433-442.
Cancer Letters, 49 (1989) 99- 105 Elsevier Scientific Publishers Ireland Ltd.
Clonal chromosomal leukemia B.N.
Nayak,
J. Sokal
and
changes
in chronic lymphocytic
M. Ray
Departments of Pediatrics and Child Health, Human Genetics, Uniuersity of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, (Canada) (Received 7 July 1989) (Revision received 13 August 1989) (Accepted 5 September 1989)
Summary Cytogenetic analysis was carried out in 28 B-CLL patients (21 males and 7 females, 3885 years old, with median age 64 years, disease stage O-IV). Peripheral nominator cells (1 x 10’) or isolated B-lymphocytes were incubated in oitro for 5 - 7 days. The cells were stimulated by pokeweed mitogen (PWM), or phorbol myristate-acetate (PMA), with or without 10% conditioned medium (CM) derived from a T cell leukemia line or 10% B-cell growth factor (BCGF). Twenty-two patients (79%) responded to PWM + CM; 5 out of 5 patients responded to PWM + BCGF. The average mitotic index 1+ S.E.M.) for PWM, PMA, PWM + CM, PMA + CM, PWM + BCGFwere0.13 f 0.01, 0.24 + 0.13, 0.51 & 0.11, 0.14 + 0.06 and 0.63 -I- 0.15, respectively. Cytogenetic analysis revealed the presence of abnormal karyotypes in 22 patients. Fourteen patients (50%) had clonal chromosome aberrations which included: monosomy 1, 9, 17, 18, 21, and X chromoCorrespondence to: M. Ray, Cytogenetics Laboratory, Health Sciences Centre, Room FE 028, Community Services Building, 685 William Avenue, Winnipeg, Manitoba, R3E 022, Canada.
0 1990 Elsevier Scientific Publishers 0304-3835/90/$03.50 Published and Printed in Ireland
some, and trisomy of chromosomes 7, 9, 20, 21 and 22. The clonal structural aberrations del(5) were i(6q), inv(l2) (q15q24), (p13pl5), del(l0) (q24). No homogeneously staining regions (HSR) were observed. Four patients with resistance to anti-neoplastic drugs showed the presence of double minute chromosomes (dmin) ranging in frequency from 5 to50%.
Keywords: chronic lymphocytic leukemia; clonal chromosome aberrations; pokeweed mitogen; phorbol-myristate acetate; B-cell growth factor; double minute chromosomes. Introduction Chronic lymphocytic leukemia (CLL) is the commonest form of leukemia in North America and Europe, accounting for 30% of all cases, and is characterized by the accumulation of relatively mature-appearing lymphocytes in the peripheral blood, marrow, lymph nodes, spleen and liver [l]. Approximately 95% of patients have a clonal expansion of Blymphocytes with < 5% being of T-cell origin [2]. The clinical presentation and rate of
lreland Ltd.
100
disease progression are highly variable and prolsnosis can be best predicted by the clinical . _ stage of the disease, using the Rai classification [3,4]. However, analysis of chromosomal changes in clonal B-cells from chronic B-cell lymphocytic leukemia (B-CLL) patients has been difficult due to a low spontaneous mitotic index [5-71, and lack of effective universal Bcell monoclonal mitogens. Until recently, there has been variable success in obtaining analysable metaphase chromosomes from peripheral lymphocytes of patients with B-CLL, using a number of mitogens, such as PWM [8- 121, lipopolysaccharides (LPS) [ 111, leucoagglutinin (LA) [ 131, tetradecanoyl-o-phorbol-13acetate (TPA) [lo], PMA [9], and cytochalasin-B [8]. Results of these studies show that approximately 50% of CLL patients present with karyotypic abnormalities and 25% patients have trisomy 12, a characteristic chromosome anomaly. It has been suggested that patients with trisomy 12 may require treatment sooner after diagnosis than those with normal karyotypes, or other abnormalities [6], while multiple aberrations within the same cell carry a poorer prognosis than trisomy 12 [6,7,14,15]. These studies would thus suggest that an increasing complexity of clonal chromosomal abnormalities may be associated with an increased rate of disease progression. Evidence in the literature indicates that mitogen derived from either plant or bacterial sources yield different frequencies of trisomy 12 as compared with mitogen of viral origin [6,8--131. However, a viral mitogen such as Epstein-Barr virus, caused chromosomal abnormality in cells from normal individuals [5]. Thus there is a need for a standardized protocol and characterization of the clonal aberrations in B-CLL patients. The present investigation was undertaken to determine the mitogen response and clonal chromosome aberrations and their relationship to the stage of CLL. Materials and methods Patients
Twenty-eight
patients
with B-cell CLL were
studied; 21 patients were male and 7 were female. Their ages ranged from 38 to 85 years (median 64 years) with Rai stage O-IV. Cell culture and chromosome
analysis
Peripheral mononuclear cells (approximately 98% lymphocytes) were isolated from heparinized venous blood by centrifugation over Ficoll400-diatrizoate sodium (Pharmacia) and 1 x lo7 cells/ml were incubated for 4-5 days in 10 ml of RPM1 1640 culture medium (GIBCO) with 10% fetal bovine serum (Bocknek) at 37 OC in an atmosphere of 95 % air and 5% CO,. PWM (GIBCO), (l:lOO), alone or with 10% BCGF (Cellular Products Inc.), or in combination with 10% of medium prepared from a T-cell supernatant Rex leukemia cell line (cell line was provided by Dr. J. Pawly, Dept. of Molecular Immunology, Roswell Park Memorial Institute, Buffalo, N.Y.) or PMA (GIBCO), 1 pg/ml were used to stimulate the cells. Phytohaemagglutinin M form (PHA-M, GIBCO) was used as a polyclonal T-cell activator at a concentration of 100 pg/ml. Colcemid (GIBCO), 0.05 pg/ml, was added 2-2.5 h before harvesting. Chromosome analyses were performed by conventional Giemsa staining (4%), to determine the gross morphological changes in chromosomes such pulverization, dmin and as gaps, breaks, homogeneously staining regions (HSR) and by a modified G-banding technique [16] to determine the specific chromosome aberrations. B and T-cell separation
Peripheral mononuclear cells from three patients were isolated as described above. Monocyte removal was performed by suspending the mononuclear cells in 10 ml of RPM1 in a plastic flask and incubating at 37OC for 1 h, after which 1.0 x lo7 of the nonadherent lymphocytes were mixed with 100 ~1 of packed 2-aminoethylisothiouronium bromide-treated sheep red blood cells (SRBC) and incubated for 1 h at 4OC 117,181. The cells were then separated by centrifugation over two consecutive Ficoll gradients. SRBC were lysed with 10 mM Tris/140 mM ammonium chlor-
101
ide, pH 7.4 and populations were (Cellular Products combination with cells. Successful carried out only in
the enriched lymphocyte cultured as above. BCGF Inc.), was used alone and in PWM to stimulate the Bchromosome analysis was 1 (G. W .) out of 3 patients.
Analysis Fifteen to 20 G-banded metaphases per patient were analyzed in order to identify the chromosome(s) involved in numerical and structural changes. Approximately 1000 cells were counted to estimate the mitotic index (MI) for each mitogen or mitogen combinations. A clonal chromosomal abnormality was defined by one of the following criteria: (i) Gain of specific chromosome in at least two cells; (ii) Loss of specific chromosome in at least three cells, or (iii) identical structural aberrations in at least two cells [ 191. Results The MI f S.E.M. values for PWM, PWM + CM, PWM + BCGF, PMA, PMA + CM, PHA and PHA + CM were 0.13 f 0.01, 0.51 + 0.11, 0.63 +- 0.15, 0.24 f 0.13, 0.14 f 0.06,0.36 + 0.39and0.52 -+ 0.11, respectively (Table 1). In 79% of patients analysable metaphases were obtained when the peripheral lymphocytes were stimulated by PWM in combination with CM. Addition of condition medium or growth factor along with PMA yielded a lower number of metaphases
Table 1.
compared to PMA alone. Cells which were not stimulated with mitogens had no metaphases. Cells treated with PHA gave MI value of 0.36, PHA along with PWM and CM yielded considerably higher MI value (0.52). Twenty-two of the 28 patients (79%) had abnormal karyotypes. Fifteen of the 21 male patients (71%) and all 7 female patients showed karyotypic abnormalities. Fifty per cent of the patients showed clonal chromosome aberrations (Table 2). Nine out of 21 males (43%) and 5 out of 7 females (71%) had clonal chromosomal aberrations. The number of clones per patient ranged from 1 to 4. The numerical clonal chromosome aberrations included trisomy of chromosomes 7, 9, 20, 21 and 22, and monosomy for chromosome 1,9, 17, 18,21 and X. The clonal structural rearrangements observed were: de1 (5) (p13p15), i(6q), del(l0) (q24), inv(l2) (q15q24). Two patients showed the presence of a clonal marker, chromosome of apparent identical morphology. The nonclonal structural aberrations encountered in these patients include del(5) (q31), del(6) (p22), del(8) (q22q24), del(8) (q24), del(l1) (q24), del(12) (q22), del(15) (q24), t(1;13) (p36;q34), t(l1; 11) (q14;q24), t(13;18) (pll,qll), t(X;12) (q22;q21). Four patients out of 28 showed dmin with a frequency ranging between 5 and 50% (Fig. 1). Patients with dmin were found to have developed resistance to a number of antineoplastic drugs. Six out of 28 patients belonging to different stages of CLL had apparent normal karyotypes.
Mitotic index value. PWM
PWM + CM
PMA
PMA + CM
PWM + BCGF
of patients Patients
25
28
18
8
5
responded Patients not
15 (60%)
22 (79%)
9 (50%)
5 (62.5%)
(100%)
responded
10 (40%)
6 (21%)
9 (50%)
3 (37.5%)
0 (0%)
0.51 + 0.11
0.24 + 0.13
0.14 + 0.06
0.63 + 0.15
Total no.
Mean MI
0.13 + 0.01
102
Table 2.
Clonal chromosome aberrations in CLL patients.
Patients
Sex
Stage
Mitogens
No. of cells analyzed
CIonaI abberations
AB
M
I
PWM + CM
20
45,XY, - 18
TB
F
IV
PWM PWM + CM
20 15
BCGF
15
45,x, -x 45,xx, - 9 45,xx, - 17 45,x, -x
PWM
20
PWM + CM
20
LC
M
IV
45,XY, - 21 46,XY,inv(12) 47,XY,inv(12) 47,XY, + 9
(q15q24) (q15q24),
+ mar
JD
F
0
PWM + CM
20
45,Xx,
AGO
M
III
PWM
20
46,XY,2p + ,del(5) (~13~15) 45,XY, - 1
- 8, - 9, + mar
46,XY, (6q) AJ
F
II
PWM + CM
20
46,XX,21p
ML
F
IV
PMA
25
47,xx,
APL
M
IV
PWM
20
47,XY, + 2
GR
M
I
PWM
20
45,XY, - 17
us
M
IV
PWM + CM
20
47,XY, + 20
LS
M
I
PWM + CM
20
45,XY, - 17 47,XY, + 21
AS
F
II
PWM + CM PWM + PMA
20 20
47,Xx, + mar 45,x, -x 47,xx, + 7 47,Xx, + mar
GV
M
IV
PWM + CM
20
45,x, -Y 45,XY, - 10
GW
M
II
PWM
15
PWM + CM
20
PWM + BCGF
20
46,XY,deI(5) (~13~15) 47,XY, + 4,deI(lO) (q33) 46,XY,del(5) (~13~15) 46,XY,del(lO) (q24) 46,XY,del(lO) (q24)
PWM, Pokeweed mitogen; PMA, Phorbol-12-myristate-13-acetate; T-cell leukemia cell line growth medium.
+
+ 22
BCGF, B-cell growth factor; CM, supernatant of
103
Fig. 1.
Double minute chromosomes
(dmin) in CLL.
Discussion
High frequencies of clonal and non-clonal chromosome abnormalities are a prominent feature of the lymphocytes in CLL [13,14,20]. Some of the clonal abnormalities constitute the primary karyotypic change in the proliferation of the neoplastic B-cells, while non-clonal a secondary abnormalities are usually phenomenon, possibly related to treatment [5,13,21,23]. A high frequency of cytogenetitally abnormal cells, and multiple chromosome abnormalities, has previously been associated with poor prognosis [6,7,14,22]. The results of this study show that 50% of the patients had clonal chromosome abnormonosomy of included which mality chromosome 1, 9, 17, 18, 21 and X chromosome and trisomy of chromosomes 7, 9, 20, 21 and 22. The clonal structural chromosomal aberrations observed in these patients were del(5) (p13p15), i (6q), de1 (10) (q24) and inv (12) (q15 q24). According to the other studies, trisomy 12 has been shown as a common abnormality in CLL patients [8,11,12,23], although other aberrations such as de1 (6s)
(8,231, trisomy3 [11,23,24], trisomy8,9, 16, 18, 20 and 21 [ll], monosomy 8, 10, 16 (24), x (12) and 149 + [23] have also been reported. Sequential chromosome study [ 1 l] suggests that in some B-CLL patients two distinct clones, one of which is a major clone with trisomy 12 and another with various other abnormalities with lower frequencies might co-exist in vivo for relatively long periods. These studies however, indicate the extent of chromosomal changes in CLL patients and complexity in the clonal evolution and selection of neoplastic cells. Four of 28 patients showed dmin. These paired chromatin bodies have been found in a number of human tumors of neurogenic origin and in certain leukemias [25,26]. Those patients showing dmin demonstrated a resistance to a number of antineoplastic drugs. Although the role of dmin is not known, it is believed that dmins are extra-mitotically transferred between cells and may contain amplified human proto-oncogenes [27] and can be produced from precursors derived from a chromosomal deletion [28]. The presence of multiple clones in B-CLL patients would indicate that there were more than one subgroup in the leukemic lymphocyte population of the patients. It is difficult to conclude whether the chromosomal aberrations observed in CLL patients represent the population of malignant B-cells or both B- and T-cells. Some of the polyclonal activators, such as PWM, PMA may stimulate B- and Tcells [29-331. However, when chromosomal analysis was performed on immunologically phenotyped cells, PWM and TPA have been shown to stimulate clonal B-cells with either Lambda or Kappa immunoglobulin light chains with 47, + 12 karyotype [lo]. In the present study clonal trisomy 12 or 14q + were not observed. Whether this is due to particular mitogens used or other factors that might be unique to the patients, or the population under study, are not known. Analysis of some reported results [5,6,8-10,13,34] show that trisomy 12 was observed in 42% of cases of B-
104
CLL when cells stimulated by Epstein Barr Virus (EBV) compared to 9%) 7% and 3% for LPS, PWM and PA, respectively. EBV confounded the chromosomal analysis since it caused chromosomal abnormality including tetraploidy in 7-25% of cells in normal individuals [5]. If trisomy 12 is considered as a characteristic aberration of B-leukemic cells in B-CLL patients, then the choice of mitogens is perhaps important in studying the biochemical and clinical significance of specific chromosoma1 changes in these patients. In B-CLL, a great number of cells are B-cell types, a small proportion of cells are (< 5%) T-cells [2,35381. When mitogens such as PWM and PMA are employed, it is likely that some T-cells would be stimulated since these mitogens are believed to be B- and T-cell mitogens. Recently, it has been shown that inversion of chromosomes 14 of the segment qllq32 has been associated with T-cell disease [39]. Another aberration involving interstitial deletion or translocation of chromosome 13 at band position q14 has been shown in cells stimulated by PMA [9], a tumor-promoting agent that was shown to induce differentiation of human leukemic cells [33,40]. Further studies are required to study the cell specificity and clinical significance of these aberrations. It is important however, to point out that the lack of effective monoclonal B-cell activators limits the evaluation of intrinsic chromosomal changes in patients with B-CLL, although involvement of T-cells in B-CLL disease complex cannot be overlooked.
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