Trisomy 12 in Epstein-Barr Virus-Transformed Lymphoblastoid Cell Lines of Normal Individuals and Patients with Nonhematologic Malignancies Semyon Risin, Vicki L. Hopwood, and Sen Pathak
ABSTRACT: Karyotypes of 36 lymphoblastoid cell lines established by Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) of eight n o r m a l individuals and 28 patients with various nonhematologic malignancies were analyzed. In seven lines (19.4%), cells with trisomy 12 were noted, with clonality in two of these lines. In two of 11 metaphases with such trisomy, chromosome 12 was involved in structural rearrangements [t(8;12)(q12;p12) and t(12;12)(q11;q24)]. No cells with trisomy 12 were observed in phytohemagglutinin (PHA)-stimulated PBL cultures of these individuals. In 250 individuals (normal and with nonhematologic malignancies) examined in our laboratory in the last 5 years, extra copies of chromosome 12 in PHA-stimulated PBL cultures were observed in only five of 23,216 cells (0.02%). There were no cases of clonality in these samples. The frequency of an extra chromosome 12 was comparable to that of the other chromosomes except 21 and X, whose frequency of occurrence was 0.08% and 0.09%, respectively. These findings should be considered random events in PHA-stimulated PBL. On the contrary, in lymphoblastoid cell lines established by EBV transformation, trisomy of chromosome 12 was the most frequent numerical abnormality. It was observed in 64.7% of all cases with chromosome gains and therefore could not be considered a random occurrence. The specificity of this phenomenon for EBV transformation is supported by the results of cytogenetic analysis of eight lymphoblastoid cell lines established by an alternative procedure in our laboratory [1]. In 400 cells analyzed not a single cell with trisomy 12 was observed. We suggest that EBV transformation might either randomly induce formation of such cells in immortalized B-cell populations or show potentially blastomogenic cells or proneness to their formation in certain individuals who could be predisposed to develop lymphoproliferative diseases, especially chronic lymphocytic leukemia (CLL) in which trisomy of chromosome 12 is the most common alteration.
INTRODUCTION Trisomy 12 is the most c o m m o n chromosomal aberration in patients with B-cell chronic l y m p h o c y t i c leukemia (CLL). It was detected in studies in w h i c h B-cell activators had been used for c h r o m o s o m e analysis [2, 3] and later was well d o c u m e n t e d in m a n y other investigations [4-11]. It was also shown in some cases of B-cell p r o l y m p h o c y t i c leukemia (PLL) [12, 13], hairy cell leukemia [14], and in l y m p h o cytic l y m p h o m a [15], as well as in some l y m p h o b l a s t o i d cell lines derived from these patients [16-20]. In CLL patients, this clonal a b n o r m a l i t y was observed in more than one third of patients who s h o w e d different cytogenetic changes [21, 22]. Clinical implications of these findings have been discussed extensively [8, 9, 21-23]. Reports
show that trisomy 12 m a y be an i m p o r t a n t prognostic factor. Patients with single n u m e r i c a l abnormalities, such as trisomy 12, t e n d e d to have poorer survival than patients with other c h r o m o s o m a l aberrations [21, 22], but the nature of the cells with extra copies of c h r o m o s o m e 12 as well as the significance of this a b n o r m a l i t y in blastomogenesis remain unclear. We report our observations of trisonly 12 in Epstein-Barr virus (EBV) transformed l y m p h o b l a s t o i d cell lines derived from peripheral blood l y m p h o c y t e s (PBL) of normal individuals and patients with n o n h e m a t o l o g i c malignancies. Our observations raise further questions about the nature and true significance of trisomy 12 in h u m a n malignancies in general and in certain l e u k e m i a s in particular. MATERIALS AND METHODS
From the Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. Address reprint requests to: Dr. S. Pathak, Cellular Genetics Laboratory, Box 181, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Received October 14, 1991; accepted January 8, 1992. 164 Cancer Genet Cytogenet 60:164 169 (1992) 0165-4608/92/$05.O0
Patients Blood samples were p r o c u r e d from n o r m a l i n d i v i d u a l s and from patients with various nonhematologic malignancies (Table 1). A n informed written consent was obtained from each participant before their blood was aspirated.
© 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Trisomy 12 in Lymphoblastoid Cell Lines
165
Table 1 Distribution of analyzed samples according to diagnosis No. of samples EBV-transformed lymphoblastoid cell lines
PHA-stimulated 72-hour cultures Diagnosis Normal individuals (asymptomatic family members of breast cancer and colon cancer families) Breast cancer Colon cancer Precancerous (adenomatous) colon polyps Other solid tumors Total Abbreviations:
Analyzed
With Trisomy12
Analyzed
With Trisomy12
88
2
8
1
68 41 19 34 250
2 0 1 0 5
7 12 3 6 36
3 1 1 1 7
PHA.phytohemagglutin;EBV,Epstein-Barrvirus.
Peripheral Blood Lymphocyte Culture Total heparinized blood (0.5 ml) was mixed for cultivation with 4.5 ml RPMI 1640 (JRM Biosciences, Lenexa, KS), supplemented with 20% fetal bovine serum (FBS, Sigma, St. Louis, MO), 2 mM L-glutamine, penicillin (50 U/ml), streptomycin (100 tzg/ml), and 1.3% phytohemagglutinin (PHA, Wellcome Research Laboratories, Research Triangle Park, NC). Blood cultures were incubated at 37°C for 72 hours.
Establishment of Lymphoblastoid Cell Lines To establish lymphoblastoid cell lines, the EBV transformation procedure was used [24]. Mononuclear cells (MNC) from PB were isolated using the Sepracell-MN continuous density gradient as recommended by the manufacturer (Sepracell, Oklahoma City, OK). The isolated MNC were resuspended in RPMI 1640 medium supplemented with 15% FBS, mixed in a 1 : 1 ratio with the supernatant of an EBV-infected marmoset cell line culture (B95-8), used as a source for EBV [251, and cultivated at 37°C in a humidified atmosphere with 5% CO 2. The cell lines were analyzed cytogenetically in early passages (
ABSTRACT: Karyotypes of 36 lymphoblastoid cell lines established by Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) of eight n o r m a l individuals and 28 patients with various nonhematologic malignancies were analyzed. In seven lines (19.4%), cells with trisomy 12 were noted, with clonality in two of these lines. In two of 11 metaphases with such trisomy, chromosome 12 was involved in structural rearrangements [t(8;12)(q12;p12) and t(12;12)(q11;q24)]. No cells with trisomy 12 were observed in phytohemagglutinin (PHA)-stimulated PBL cultures of these individuals. In 250 individuals (normal and with nonhematologic malignancies) examined in our laboratory in the last 5 years, extra copies of chromosome 12 in PHA-stimulated PBL cultures were observed in only five of 23,216 cells (0.02%). There were no cases of clonality in these samples. The frequency of an extra chromosome 12 was comparable to that of the other chromosomes except 21 and X, whose frequency of occurrence was 0.08% and 0.09%, respectively. These findings should be considered random events in PHA-stimulated PBL. On the contrary, in lymphoblastoid cell lines established by EBV transformation, trisomy of chromosome 12 was the most frequent numerical abnormality. It was observed in 64.7% of all cases with chromosome gains and therefore could not be considered a random occurrence. The specificity of this phenomenon for EBV transformation is supported by the results of cytogenetic analysis of eight lymphoblastoid cell lines established by an alternative procedure in our laboratory [1]. In 400 cells analyzed not a single cell with trisomy 12 was observed. We suggest that EBV transformation might either randomly induce formation of such cells in immortalized B-cell populations or show potentially blastomogenic cells or proneness to their formation in certain individuals who could be predisposed to develop lymphoproliferative diseases, especially chronic lymphocytic leukemia (CLL) in which trisomy of chromosome 12 is the most common alteration.
INTRODUCTION Trisomy 12 is the most c o m m o n chromosomal aberration in patients with B-cell chronic l y m p h o c y t i c leukemia (CLL). It was detected in studies in w h i c h B-cell activators had been used for c h r o m o s o m e analysis [2, 3] and later was well d o c u m e n t e d in m a n y other investigations [4-11]. It was also shown in some cases of B-cell p r o l y m p h o c y t i c leukemia (PLL) [12, 13], hairy cell leukemia [14], and in l y m p h o cytic l y m p h o m a [15], as well as in some l y m p h o b l a s t o i d cell lines derived from these patients [16-20]. In CLL patients, this clonal a b n o r m a l i t y was observed in more than one third of patients who s h o w e d different cytogenetic changes [21, 22]. Clinical implications of these findings have been discussed extensively [8, 9, 21-23]. Reports
show that trisomy 12 m a y be an i m p o r t a n t prognostic factor. Patients with single n u m e r i c a l abnormalities, such as trisomy 12, t e n d e d to have poorer survival than patients with other c h r o m o s o m a l aberrations [21, 22], but the nature of the cells with extra copies of c h r o m o s o m e 12 as well as the significance of this a b n o r m a l i t y in blastomogenesis remain unclear. We report our observations of trisonly 12 in Epstein-Barr virus (EBV) transformed l y m p h o b l a s t o i d cell lines derived from peripheral blood l y m p h o c y t e s (PBL) of normal individuals and patients with n o n h e m a t o l o g i c malignancies. Our observations raise further questions about the nature and true significance of trisomy 12 in h u m a n malignancies in general and in certain l e u k e m i a s in particular. MATERIALS AND METHODS
From the Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. Address reprint requests to: Dr. S. Pathak, Cellular Genetics Laboratory, Box 181, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Received October 14, 1991; accepted January 8, 1992. 164 Cancer Genet Cytogenet 60:164 169 (1992) 0165-4608/92/$05.O0
Patients Blood samples were p r o c u r e d from n o r m a l i n d i v i d u a l s and from patients with various nonhematologic malignancies (Table 1). A n informed written consent was obtained from each participant before their blood was aspirated.
© 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Trisomy 12 in Lymphoblastoid Cell Lines
165
Table 1 Distribution of analyzed samples according to diagnosis No. of samples EBV-transformed lymphoblastoid cell lines
PHA-stimulated 72-hour cultures Diagnosis Normal individuals (asymptomatic family members of breast cancer and colon cancer families) Breast cancer Colon cancer Precancerous (adenomatous) colon polyps Other solid tumors Total Abbreviations:
Analyzed
With Trisomy12
Analyzed
With Trisomy12
88
2
8
1
68 41 19 34 250
2 0 1 0 5
7 12 3 6 36
3 1 1 1 7
PHA.phytohemagglutin;EBV,Epstein-Barrvirus.
Peripheral Blood Lymphocyte Culture Total heparinized blood (0.5 ml) was mixed for cultivation with 4.5 ml RPMI 1640 (JRM Biosciences, Lenexa, KS), supplemented with 20% fetal bovine serum (FBS, Sigma, St. Louis, MO), 2 mM L-glutamine, penicillin (50 U/ml), streptomycin (100 tzg/ml), and 1.3% phytohemagglutinin (PHA, Wellcome Research Laboratories, Research Triangle Park, NC). Blood cultures were incubated at 37°C for 72 hours.
Establishment of Lymphoblastoid Cell Lines To establish lymphoblastoid cell lines, the EBV transformation procedure was used [24]. Mononuclear cells (MNC) from PB were isolated using the Sepracell-MN continuous density gradient as recommended by the manufacturer (Sepracell, Oklahoma City, OK). The isolated MNC were resuspended in RPMI 1640 medium supplemented with 15% FBS, mixed in a 1 : 1 ratio with the supernatant of an EBV-infected marmoset cell line culture (B95-8), used as a source for EBV [251, and cultivated at 37°C in a humidified atmosphere with 5% CO 2. The cell lines were analyzed cytogenetically in early passages (
