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Chromosomal abnormalities in chronic lymphocytic leukemia
Chromosomal Abnormalities in Chronic Lymphocytic Leukemia Kong-Oo Goh
ABSTRACT: Peripheral blood lymphocytes from five chronic lymphocytic leukemia (CLL) patients were cultured with PHA for 3-6 days. Chromosomal analysis with G-banding showed 25% of the diploid metaphoses were pseudodiploid as a result of either a chromosomal deletion or a translocation. Abnormal clones with 1 8 p - were seen in two patients, and two other patients had 18p- metaphases, but with other inconsistent abnormal chromosomes. None of the patients had trisomy 12, which has been thought by some to carry a poor prognosis. Although no conclusion can be reached regarding the significance of the chromosomal abnormalities in CLL, the type of chromosomal abnormalities in these patients suggests that they are the result of chromosomal breakage and abnormal repair. INTRODUCTION The cytogenetics of chronic l y m p h o c y t i c l e u k e m i a (CLL) has not been s t u d i e d as extensively as that of chronic myelocytic leukemia has, where a specific abnormal chromosome (Ph) is seen. Furthermore, it is difficult to obtain adequate metaphases in the direct bone marrow or the standard PHA-stimulated peripheral b l o o d lymphocyte cultures of CLL patients. Several investigators have reported normal chromosomal patterns in this disease. We found abnormalities in PHA-stimulated peripheral blood l y m p h o c y t e cultures of CLL patients after 3 - 6 days [1]. Although this observation was confirmed by Ducos and Colombies [2], Crossen [3] d i d not find chromosomal abnormalities in 20 CLL patients. Because PHA stimulates T cells, Crossen believed that the metaphases analyzed from the PHA-stimulated cultures m a y have been the normal T cells of the B-cell CLL patients. Using various mitogens to stimulate different l y m p h o c y t e populations, several investigators [4-7] have recently confirmed our original observations [1]. This article reports additional cytogenetic studies in CLL patients using a G-banding technique. MATERIALS AND METHODS Heparinized peripheral blood l y m p h o c y t e s from five patients (Table 1) and four normal controls were cultured with PHA according to a m e t h o d described previously [1]. The cultures were terminated 3 - 6 days after in vitro incubation at 37°C. The metaphases for cytogenetic studies were prepared according to a standard technique, and the slides were treated with trypsin and stained with Giemsa [8]. The From the Monroe Community Hospital, University of Rochester School of Medicine and Dentistry, Rochester, NY. Address requests for reprints to Dr. Kong-Oo Gob, Monroe Community Hospital; University of Rochester School of Medicine and Dentistry, Rochester, NY 14603. Received October 24, 1983; accepted June 18, 1984. 103 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderhilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics16, 103-107 (1985) 0165-4608/85/$03.30
104
K.-O. Goh Table 1
Clinical s u m m a r y of five chronic l y m p h o cy t i c leukemia patients
Patient
1
Age Sex Lymphadenopathy Splenomegaly Hematocrit (%) White blood cells ( x 109/L} Lymphocytes (%) B cells (%) Platelet count ( x 109/L) Previous treatment
2
87 M + 0 45 24 83 93 240 None
3
82 F + + 37 90 98 70 49 Cyclophosphamide and prednisone
73 M + 0 35 42 90 57 200 None
4
5
78 F + + 43 30 76 59 260 None
51 M + 0 45 93 93 74 120 None
ch r o m o s o m a l n u m b e r was determined from direct microscopic ex am i n at i o n at 1455 x , and 10-50 r a n d o m l y selected, normal-appearing metaphases from each patient were photographed and karyotyped in detail. The standard international classification [9] for Giemsa-trypsin banded chromosomes was used.
RESULTS The results of the c h r o m o s o m a l analyses are s u m m a r i z e d in Table 2. The m o d al diploid c h r o m o s o m a l n u m b e r was 46 in each of the patients, representing 72% of the metaphases analyzed. The frequency of diploid metaphases in each of the patients varied b e tw e e n 57% and 79%. In four patients, the diploid metaphases were seen in 7 0 % - 7 9 % of the metaphases analyzed. In patient 4, however, only 57% of the metaphases had a normal modal c hr o m o so m al number; the rest were h y p o d i p loid. There was no consistent c h r o m o so m al pattern in the h y p o d i p l o i d or hyperdiploid metaphases in these patients. Twenty-five percent of the diploid metaphases in these patients were pseudodiploid. This abnormality was the result of ch r o m o so m al deletion or translocation (Fig. 1). An abnormal clone of 1 8 p - was seen in patients 1 and 3 (Fig. 2). A l t h o u g h four metaphases with 1 8 p - were seen in two other patients (patients 2 and 5), these metaphases had other inconsistent abnormal chromosomes. No marker chrom o s o m e was seen in patient 4, w h o had m a n y aneuploidies. Trisomy 12 was not seen in any of the metaphases analyzed in these patients.
Table 2
S u m m a r y of c h r o m o s o m a l analyses of five chronic lymphocytic leukemias Chromosomal number -<44
Patient 1 2 3 4 5 Total
23 18 15 20 60 136
45 46 47 (No. of metaphases) 20 24 30 l0 36 120
147(79)b 180(77) 148(74) 40(57) 264(70) 779(72)
2 5 3 0 6 16
->48 3 8 5 O 12 28
aNormal chromosomal number but abnormal karyotype. ~rhe numbers inside the parentheses are the percentages. %Vith other inconsistent abnormal chromosomes.
Total
Number pseudodiploid a (%)
Abnormal clones
195 235 201 70 378 1079
39(27) 45(25) 47(31) 6(15) 56(21) 193(25)
18p 18p - c 18p 18p - c
Chromosomal Abnormalities in CLL
105
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Figure 1 Two karyotypes from patient 3 showing pseudodiploidies. The karyotype in the left has: (a) a missing chromosome #4 and #10 (arrow), (b) a deletion of the short arms (p} of a chromosome #18, {c) two marker chromosomes (M1 and M2) and (d) a small fragment (F). The karyotype in the right shows: (a} a missing chromosome #2 (arrow), {b) a deletion of the short arms of a chromosome #20, (c) a marker chromosome (M3} and (d} acentric fragments (F}, probably the short arms of chromosome #20.
The m o d a l chromosomal n u m b e r in the four controls was 46, w h i c h was seen in 92% of the 200 metaphases analyzed. There were six p s e u d o d i p l o i d metaphases seen in this group, and these were characterized by metaphases with a r a n d o m chromosomal loss. No consistent chromosomal pattern was seen in these metaphases. DISCUSSION Autio et al. [10] analyzed the chromosomal patterns of p e r i p h e r a l blood cultures of ten CLL patients stimulated with various mitogens. They found that leukoagglutinin (LA} and protein A (PA) stimulated the l y m p h o c y t e s of all of their patients, but p o k e w e e d mitogen (PWM} stimulated only five and l i p o p o l y s a c c h a r i d e B (LPS) only three patients. A l t h o u g h the majority of the metaphases from the LA-, PA-, and PWM-stimulated cultures had normal karyotypes, 26% s h o w e d c h r o m o s o m a l abnormalities. Translocations were seen in several of their patients, but only one had a trisomy 12 clone. The trisomy stem cell line in this patient was observed over a p e r i o d of 4 years, and it was seen in the metaphases obtained from the cultures stimulated with LA, a T-cell stimulator, and PA, a B- and T-cell stimulator. This suggests that this abnormal stem cell line m a y have been present in both the B and T cells of a B-cell CLL patients. Although trisomy 12 has been frequently reported in CLL patients, Nowell et al. [5] d i d not find it in any of six patients. Neither d i d we find trisomy 12 in our present group of patients. Recently, Robert et al. [11] suggested that the disease of patients w i t h trisomy 12 m a y be more aggressive than that of patients w h o have a normal karyotype or w h o have abnormalities other than trisomy 12. However, this view is not s u p p o r t e d by the observations of Han et al. [12], nor do our present cytogenetic findings resolve it.
106
K.-O. Goh
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Figure 2 Four karyotypes showing 1 8 p - . The top two karyotypes are from patient 3, and the bottom two are from patient 1.
The discovery by Autio et al. [10] of chromosomal abnormalities in 26% of cells is very similar to our present observation at 25% of the metaphases of our patients are p s e u d o d i p l o i d . Gahrton et al. [13] observed chromosomal abnormalities in either B- or T-cell-stimulated cultures of CLL patients. They believed that their observation was a result of a T-cell-induced B-cell activator factor(s), as suggested by Robert [14]. The factor(s) can cause B cells to undergo DNA synthesis. The aberrations they saw in the T-cell-stimulated cultures m a y actually be B cells, rather than T cells, w h i c h were stimulated to undergo mitosis by the factor(s). Perhaps the chromosomal abnormalities found in our PHA-stimulated cultures, reported previously [1] and e x t e n d e d here with a G-banding technique, were the abnormal B cells stimulated to undergo mitosis, as e x p l a i n e d by Gahrton et al. [13]. Two of our patients had an 1 8 p - clone of cells. Two other patients had two metaphases with 1 8 p - each. In addition, the metaphases from the latter two pa-
Chromosomal Abnormalities i n CLL
10 7
tients had other inconsistent abnormal chromosomes. Because the cytogenetic studies in four of our patients were carried out before the patients were treated, and because PHA is not k n o w n to cause any chromosomal aberrations (as Epstein-Barr virus does [4]), the abnormal metaphases found in our patients are associated with their primary disease. This is further supported by the observation that there are preferential chromosomal abnormalities in CLL involving chromosomes #2, #14, and #18 [6], and chromosome # 1 8 was involved in four of our patients. The abnormalities found in our patients were the results of chromosomal breakage and abnormal repair. This may imply that these processes occur frequently i n CLL. Marie A. Herrmann and Laura Hansen gave technical assistance. Marge Laley and Theresa Tong provided secretarial service.
REFERENCES 1. Goh KO (1967): Pseudodiploid chromosomal patterns in chronic lymphocytic leukemia. J Lab Clin Med 69:938-949. 2. Ducos, J, Colombies P (1968): Chromosomes in chronic lymphocytic leukemia. Lancet 1:1308. 3. Crossen PE (1975): Giemsa banding patterns in chronic lymphocytic leukaemia. Humangenetik 27:151-156. 4. Morita M, Minowada J, Sandberg AA (1981): Chromosomes and causation of cancer and leukemia. XLV. Chromosome patterns in stimulated lymphocytes of chronic lymphocytic leukemia. Cancer Genet Cytogenet 3:293-306. 5. Nowell P, Shankey TV, Finan J, Guerry D, Besa E (1981): Proliferation, differentiation and cytogenetics of chronic leukemic B lymphocytes cultured with Mitomycin-treated normal cells. Blood 57:444-451. 6. Schroder J, Vuopio P, Autio K (1981): Chromosome changes in human chronic lymphocytic leukemia. Cancer Genet Cytogenet 4:11-21. 7. Nowell P, Daniele R, Rowlands D, Finan J (1980): Cytogenetics of chronic B-cell and Tcell leukemia. Cancer Genet Cytogenet 1:273-280. 8. Goh KO, Herrmann MA (1984): Abnormal chromosomes in histocytic lymphoma. Am J Med Sci 288:48-52. 9. ISCN (1978): An international system for human cytogenetics nomenclature (1978). Cytogenet Cell Genet 21:309-404. I0. Autio K, Turunen O, Penttila O, Eramaa E, de la Chapelle A, Schroder J (1979): Human chronic lymphocytic leukemia: Karyotypes in different lymphocyte population. Cancer Genet Cytogenet 1:147-I 55. 11. Robert K-H, Gahrton G, Friberg K, Zech L, Nilsson B (1982): Extra chromosome 12 and prognosis in chronic lymphocytic leukemia. Scand J Haematol 28:163-168. 12. Hart T, Ozer H, Sadamori N, Gomez GA, Henderson ML, Bloom L, Emrich L], Reese PA, Sandberg AA (1983): Prognostic significance of cytogenetic changes in patients with chronic lymphocytic leukemia. Blood 62:173a, 1983 (abstr). 13. Gahrton G, Robert K-H, Friberg K, Zech L, Bird AG (1980): Nonrandom chromosome aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell mitogen stimulation. Blood 56:640-647. 14. Robert K-H, Miller E, Gahrton G, Erikson H, Nilsson B (1978): B-cell activation of peripheral blood lymphocytes from patients with chronic lymphocytic leukemia. Clin Exp Immunol 33:302-308.
ABSTRACT: Peripheral blood lymphocytes from five chronic lymphocytic leukemia (CLL) patients were cultured with PHA for 3-6 days. Chromosomal analysis with G-banding showed 25% of the diploid metaphoses were pseudodiploid as a result of either a chromosomal deletion or a translocation. Abnormal clones with 1 8 p - were seen in two patients, and two other patients had 18p- metaphases, but with other inconsistent abnormal chromosomes. None of the patients had trisomy 12, which has been thought by some to carry a poor prognosis. Although no conclusion can be reached regarding the significance of the chromosomal abnormalities in CLL, the type of chromosomal abnormalities in these patients suggests that they are the result of chromosomal breakage and abnormal repair. INTRODUCTION The cytogenetics of chronic l y m p h o c y t i c l e u k e m i a (CLL) has not been s t u d i e d as extensively as that of chronic myelocytic leukemia has, where a specific abnormal chromosome (Ph) is seen. Furthermore, it is difficult to obtain adequate metaphases in the direct bone marrow or the standard PHA-stimulated peripheral b l o o d lymphocyte cultures of CLL patients. Several investigators have reported normal chromosomal patterns in this disease. We found abnormalities in PHA-stimulated peripheral blood l y m p h o c y t e cultures of CLL patients after 3 - 6 days [1]. Although this observation was confirmed by Ducos and Colombies [2], Crossen [3] d i d not find chromosomal abnormalities in 20 CLL patients. Because PHA stimulates T cells, Crossen believed that the metaphases analyzed from the PHA-stimulated cultures m a y have been the normal T cells of the B-cell CLL patients. Using various mitogens to stimulate different l y m p h o c y t e populations, several investigators [4-7] have recently confirmed our original observations [1]. This article reports additional cytogenetic studies in CLL patients using a G-banding technique. MATERIALS AND METHODS Heparinized peripheral blood l y m p h o c y t e s from five patients (Table 1) and four normal controls were cultured with PHA according to a m e t h o d described previously [1]. The cultures were terminated 3 - 6 days after in vitro incubation at 37°C. The metaphases for cytogenetic studies were prepared according to a standard technique, and the slides were treated with trypsin and stained with Giemsa [8]. The From the Monroe Community Hospital, University of Rochester School of Medicine and Dentistry, Rochester, NY. Address requests for reprints to Dr. Kong-Oo Gob, Monroe Community Hospital; University of Rochester School of Medicine and Dentistry, Rochester, NY 14603. Received October 24, 1983; accepted June 18, 1984. 103 © 1985 Elsevier Science Publishing Co., Inc. 52 Vanderhilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics16, 103-107 (1985) 0165-4608/85/$03.30
104
K.-O. Goh Table 1
Clinical s u m m a r y of five chronic l y m p h o cy t i c leukemia patients
Patient
1
Age Sex Lymphadenopathy Splenomegaly Hematocrit (%) White blood cells ( x 109/L} Lymphocytes (%) B cells (%) Platelet count ( x 109/L) Previous treatment
2
87 M + 0 45 24 83 93 240 None
3
82 F + + 37 90 98 70 49 Cyclophosphamide and prednisone
73 M + 0 35 42 90 57 200 None
4
5
78 F + + 43 30 76 59 260 None
51 M + 0 45 93 93 74 120 None
ch r o m o s o m a l n u m b e r was determined from direct microscopic ex am i n at i o n at 1455 x , and 10-50 r a n d o m l y selected, normal-appearing metaphases from each patient were photographed and karyotyped in detail. The standard international classification [9] for Giemsa-trypsin banded chromosomes was used.
RESULTS The results of the c h r o m o s o m a l analyses are s u m m a r i z e d in Table 2. The m o d al diploid c h r o m o s o m a l n u m b e r was 46 in each of the patients, representing 72% of the metaphases analyzed. The frequency of diploid metaphases in each of the patients varied b e tw e e n 57% and 79%. In four patients, the diploid metaphases were seen in 7 0 % - 7 9 % of the metaphases analyzed. In patient 4, however, only 57% of the metaphases had a normal modal c hr o m o so m al number; the rest were h y p o d i p loid. There was no consistent c h r o m o so m al pattern in the h y p o d i p l o i d or hyperdiploid metaphases in these patients. Twenty-five percent of the diploid metaphases in these patients were pseudodiploid. This abnormality was the result of ch r o m o so m al deletion or translocation (Fig. 1). An abnormal clone of 1 8 p - was seen in patients 1 and 3 (Fig. 2). A l t h o u g h four metaphases with 1 8 p - were seen in two other patients (patients 2 and 5), these metaphases had other inconsistent abnormal chromosomes. No marker chrom o s o m e was seen in patient 4, w h o had m a n y aneuploidies. Trisomy 12 was not seen in any of the metaphases analyzed in these patients.
Table 2
S u m m a r y of c h r o m o s o m a l analyses of five chronic lymphocytic leukemias Chromosomal number -<44
Patient 1 2 3 4 5 Total
23 18 15 20 60 136
45 46 47 (No. of metaphases) 20 24 30 l0 36 120
147(79)b 180(77) 148(74) 40(57) 264(70) 779(72)
2 5 3 0 6 16
->48 3 8 5 O 12 28
aNormal chromosomal number but abnormal karyotype. ~rhe numbers inside the parentheses are the percentages. %Vith other inconsistent abnormal chromosomes.
Total
Number pseudodiploid a (%)
Abnormal clones
195 235 201 70 378 1079
39(27) 45(25) 47(31) 6(15) 56(21) 193(25)
18p 18p - c 18p 18p - c
Chromosomal Abnormalities in CLL
105
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Figure 1 Two karyotypes from patient 3 showing pseudodiploidies. The karyotype in the left has: (a) a missing chromosome #4 and #10 (arrow), (b) a deletion of the short arms (p} of a chromosome #18, {c) two marker chromosomes (M1 and M2) and (d) a small fragment (F). The karyotype in the right shows: (a} a missing chromosome #2 (arrow), {b) a deletion of the short arms of a chromosome #20, (c) a marker chromosome (M3} and (d} acentric fragments (F}, probably the short arms of chromosome #20.
The m o d a l chromosomal n u m b e r in the four controls was 46, w h i c h was seen in 92% of the 200 metaphases analyzed. There were six p s e u d o d i p l o i d metaphases seen in this group, and these were characterized by metaphases with a r a n d o m chromosomal loss. No consistent chromosomal pattern was seen in these metaphases. DISCUSSION Autio et al. [10] analyzed the chromosomal patterns of p e r i p h e r a l blood cultures of ten CLL patients stimulated with various mitogens. They found that leukoagglutinin (LA} and protein A (PA) stimulated the l y m p h o c y t e s of all of their patients, but p o k e w e e d mitogen (PWM} stimulated only five and l i p o p o l y s a c c h a r i d e B (LPS) only three patients. A l t h o u g h the majority of the metaphases from the LA-, PA-, and PWM-stimulated cultures had normal karyotypes, 26% s h o w e d c h r o m o s o m a l abnormalities. Translocations were seen in several of their patients, but only one had a trisomy 12 clone. The trisomy stem cell line in this patient was observed over a p e r i o d of 4 years, and it was seen in the metaphases obtained from the cultures stimulated with LA, a T-cell stimulator, and PA, a B- and T-cell stimulator. This suggests that this abnormal stem cell line m a y have been present in both the B and T cells of a B-cell CLL patients. Although trisomy 12 has been frequently reported in CLL patients, Nowell et al. [5] d i d not find it in any of six patients. Neither d i d we find trisomy 12 in our present group of patients. Recently, Robert et al. [11] suggested that the disease of patients w i t h trisomy 12 m a y be more aggressive than that of patients w h o have a normal karyotype or w h o have abnormalities other than trisomy 12. However, this view is not s u p p o r t e d by the observations of Han et al. [12], nor do our present cytogenetic findings resolve it.
106
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Figure 2 Four karyotypes showing 1 8 p - . The top two karyotypes are from patient 3, and the bottom two are from patient 1.
The discovery by Autio et al. [10] of chromosomal abnormalities in 26% of cells is very similar to our present observation at 25% of the metaphases of our patients are p s e u d o d i p l o i d . Gahrton et al. [13] observed chromosomal abnormalities in either B- or T-cell-stimulated cultures of CLL patients. They believed that their observation was a result of a T-cell-induced B-cell activator factor(s), as suggested by Robert [14]. The factor(s) can cause B cells to undergo DNA synthesis. The aberrations they saw in the T-cell-stimulated cultures m a y actually be B cells, rather than T cells, w h i c h were stimulated to undergo mitosis by the factor(s). Perhaps the chromosomal abnormalities found in our PHA-stimulated cultures, reported previously [1] and e x t e n d e d here with a G-banding technique, were the abnormal B cells stimulated to undergo mitosis, as e x p l a i n e d by Gahrton et al. [13]. Two of our patients had an 1 8 p - clone of cells. Two other patients had two metaphases with 1 8 p - each. In addition, the metaphases from the latter two pa-
Chromosomal Abnormalities i n CLL
10 7
tients had other inconsistent abnormal chromosomes. Because the cytogenetic studies in four of our patients were carried out before the patients were treated, and because PHA is not k n o w n to cause any chromosomal aberrations (as Epstein-Barr virus does [4]), the abnormal metaphases found in our patients are associated with their primary disease. This is further supported by the observation that there are preferential chromosomal abnormalities in CLL involving chromosomes #2, #14, and #18 [6], and chromosome # 1 8 was involved in four of our patients. The abnormalities found in our patients were the results of chromosomal breakage and abnormal repair. This may imply that these processes occur frequently i n CLL. Marie A. Herrmann and Laura Hansen gave technical assistance. Marge Laley and Theresa Tong provided secretarial service.
REFERENCES 1. Goh KO (1967): Pseudodiploid chromosomal patterns in chronic lymphocytic leukemia. J Lab Clin Med 69:938-949. 2. Ducos, J, Colombies P (1968): Chromosomes in chronic lymphocytic leukemia. Lancet 1:1308. 3. Crossen PE (1975): Giemsa banding patterns in chronic lymphocytic leukaemia. Humangenetik 27:151-156. 4. Morita M, Minowada J, Sandberg AA (1981): Chromosomes and causation of cancer and leukemia. XLV. Chromosome patterns in stimulated lymphocytes of chronic lymphocytic leukemia. Cancer Genet Cytogenet 3:293-306. 5. Nowell P, Shankey TV, Finan J, Guerry D, Besa E (1981): Proliferation, differentiation and cytogenetics of chronic leukemic B lymphocytes cultured with Mitomycin-treated normal cells. Blood 57:444-451. 6. Schroder J, Vuopio P, Autio K (1981): Chromosome changes in human chronic lymphocytic leukemia. Cancer Genet Cytogenet 4:11-21. 7. Nowell P, Daniele R, Rowlands D, Finan J (1980): Cytogenetics of chronic B-cell and Tcell leukemia. Cancer Genet Cytogenet 1:273-280. 8. Goh KO, Herrmann MA (1984): Abnormal chromosomes in histocytic lymphoma. Am J Med Sci 288:48-52. 9. ISCN (1978): An international system for human cytogenetics nomenclature (1978). Cytogenet Cell Genet 21:309-404. I0. Autio K, Turunen O, Penttila O, Eramaa E, de la Chapelle A, Schroder J (1979): Human chronic lymphocytic leukemia: Karyotypes in different lymphocyte population. Cancer Genet Cytogenet 1:147-I 55. 11. Robert K-H, Gahrton G, Friberg K, Zech L, Nilsson B (1982): Extra chromosome 12 and prognosis in chronic lymphocytic leukemia. Scand J Haematol 28:163-168. 12. Hart T, Ozer H, Sadamori N, Gomez GA, Henderson ML, Bloom L, Emrich L], Reese PA, Sandberg AA (1983): Prognostic significance of cytogenetic changes in patients with chronic lymphocytic leukemia. Blood 62:173a, 1983 (abstr). 13. Gahrton G, Robert K-H, Friberg K, Zech L, Bird AG (1980): Nonrandom chromosome aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell mitogen stimulation. Blood 56:640-647. 14. Robert K-H, Miller E, Gahrton G, Erikson H, Nilsson B (1978): B-cell activation of peripheral blood lymphocytes from patients with chronic lymphocytic leukemia. Clin Exp Immunol 33:302-308.