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Trisomy 3 in cold agglutinin disease
Trisomy 3 in Cold Agglutinin Disease Judith Gordon, Leslie Silberstein, Lisa Moreau, and Peter C. Nowell
ABSTRACT: Cold agglutinin disease (CAD) is a form of autoimmune hemolytic anemia generally considered to result from an antibody-producing clonal expansion of B lymphocytes. We studied 16 patients with CAD and demonstrated a ehromosomally abnormal clone in the peripheral blood (PB) of six. Trisomy 3 was the only abnormality in two patients, trisomy 12 the only abnormality in one, and both trisomy 3 and trisomy 12 were present in three patients. A lymphoma was subsequently diagnosed in two of these six patients and in none of the patients without a karyotypic abnormality. The results suggest that trisomy 3 confers a slight growth advantage in the B-cell lineage, particularly at a relatively late stage of differentiation. The resultant clone may present clinically as CAD and, in some cases, progress to a clinical lymphoma.
INTRODUCTION Trisomy for chromosome 3 occurs relatively commonly in a variety of neoplasms, usually as part of a complex, highly aneuploid karyotype. In hematopoietic disorders, it is more frequent in lymphoid than in myeloid tumors, particularly advanced lymphomas [1]. As a single abnormality, or in association with only one or two additional karyotypic alterations, trisomy 3 is quite rare and has been described most commonly in low-grade preneoplastic or neoplastic clonal proliferations of T cells. These have included such disorders as lymphogranulomatosus X, angioimmunoblastic lympadenopathy, and Lennert's l y m p h o m a [2, 3]. We recently described several cases of cold agglutinin disease (CAD), a form of autoimmune hemolytic anemia, in which a chromosomally abnormal B-cell clone was identified in the peripheral blood (PB) with trisomy 3 alone or trisomy 3 in combination with trisomy 12 [4]. We have extended this study to include additional cases and to obtain clinical follow-up data on CAD patients with and without a demonstrable cytogenetic abnormality.
MATERIALS AND METHODS Sixteen patients with CAD have now been studied cytogenetically. The diagnosis was established on the basis of a hemolytic cold autoantibody, and additional clinical details on some of the patients were reported previously [4]. The duration of the From the Department of Pathologyand LaboratoryMedicine, Universityof Pennsylvan/aSchoolof Medicine, Philadelph/a. Address reprint requests to: Dr. Peter C. Nowell, Depatment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082. Received July 24, 1989; accepted September 13, 1989.
89 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 46:89-92 (1990)
01654608/90/$03.50
90
J. Gordon et al.
Table 1
Six CAD patients with karyotypic abnormalities
Patient no.
Abnormal karyotype
Survival after study (too)
Lymphoma
1 2 3 4 5 6
51,XX, + 3,+4, + 9,+ 12, + 13, + 18 47,XY, + 3 47,XX, + 3 47,XX, + 12; 48,XX, + 3, + 12 47,X, - X, + 3, + 12 47,XX, + 12
60 a 20 50a 20 19a 6a
Yes No Yes No Nob No
Patients 1-4 are the same as those similarly numbered in reference 4. a Patient alive. bPatient has "mature lymphocytosis of bone marrow." patients' a u t o i m m u n e disorder ranged from 6 weeks to 10 years before study, and none had an established diagnosis of lymphoma. The patients ranged in age from 37 to 78 years, although all but one were aged more than 60 years.
Cytogenetic Studies Peripheral blood (PB) m o n o n u c l e a r cells from each patient were cultured for 7 2 - 9 6 hours w i t h a combination of p o k e w e e d mitogen (PWM) and a phorbol ester (phorbol12-myristate-13-acetate, TPA) s u p p l e m e n t e d with a crude preparation of interleukin2 (IL-2) that contained B-cell growth factor activity [5]. Standard trypsin-Giemsa b a n d e d c h r o m o s o m e preparations were made [5]. At least 20 counts and three karyotype analyses were performed for each study, with additional counts and analyses as required to characterize any karyotypically abnormal clone. RESULTS A c h r o m o s o m a l l y abnormal clone, constituting 15% to 65% of the metaphases examined, was identified in the PB of six of the 16 CAD patients studied (Table 1). In two patients the only karyotypic abnormality was trisomy 3, three patients also had trisomy 12, and in one patient the only abnormality was trisomy 12. Cells from the k a r y o t y p i c a l l y abnormal clone of two patients (Patients I and 5) have been established as Epstein-Barr virus (EBV)-transformed cultures and shown to be the source of the patients' pathological a u t o a n t i b o d y [4] ( u n p u b l i s h e d observations, L. Silberstein). Three of these six patients also had a B-cell clone in the PB shown by i m m u n o p h e n o typing [4].
Clinical Follow-up A l l six of the patients w i t h a karyotypically abnormal clone and seven of the other 10 CAD patients have been followed for a period of 6 - 6 0 months. The diagnosis of l y m p h o m a has been made in two of the six patients with a chromosome alteration (Table 1) and in none of those w i t h a normal karyotype. A splenectomy was performed on patient I eight weeks after the original cytogenetic study, and a well-differentiated l y m p h o m a was identified in the spleen and adjacent nodes. She is alive after 5 years, with her l y m p h o m a partially controlled by therapy. A l y m p h node b i o p s y after 47 months s h o w e d evidence of karyotypic evolution, recently associated with more aggressive clinical disease. In patient 3, a well-differentiated l y m p h o m a was diagnosed on BM b i o p s y 6 weeks after c h r o m o s o m e study, and 4 years after therapy she remains in remission. Patients
Trisomy 3 in Cold Agglutinin Disease
91
2 and 4 both died 20 months after chromosome study without progression to lymphoma, and patients 5 and 6 are clinically unchanged after 19 months and 6 months, respectively. Patient 5 had "mature lymphocytosis of the bone marrow" when first studied, but a definite diagnosis of lymphoma has not been established. Among the 10 patients without a karyotypically abnormal clone demonstrable in the blood, one died of complications of her autoimmune disease 3 months after study, six have been followed for 15-51 months (median = 40 months) without evidence of a lymphocytic neoplasm, and three were lost to follow-up. In three cases, a repeat chromosome study after 2-5 months again showed no karyotypic abnormality.
DISCUSSION
Our present findings, which appear to be the only cytogenetic data on a series of patients with CAD, confirm our previous impression that when a chromosomally abnormal clone is demonstrable in this disorder, trisomy 3 or trisomy 12 or both are typically observed, often without additional alterations. The presence of trisomy 12, singly or in combination, clearly links these low-grade lymphoid dyscrasias to chronic B-cell leukemia, in which an extra chromosome 12 is the most common finding and in which trisomy 3 occasionally occurs as an additional alteration [1, 6]. Cold agglutinin disease, however, represents the first instance of trisomy 3, without other extensive cytogenetic changes, being frequently associated with a particular B-cell disorder. Contrafatto [7], in an early banding study, identified the single large extra chromosome often observed in WaldenstrOm's macroglobulinemia as a 3 p - in two cases, and further suggested that "anomalies of chromosome 3 occur particularly often in monoclonal gammopathies." Trisomy 3 alone has been most frequently reported in low-grade T-cell tumors (lymphogranulomatosus X, angioimmunoblastic lymphadenopathy, Lennert's lymphoma) [2, 3, 8]. Very few patients with high-grade tumors have only trisomy 3 [9-11], and Yunis et al. [10] reported a diffuse large-cell lymphoma that had 48 chromosomes with trisomy 3, trisomy 12, and the t(14;18) translocation often associated with lowgrade B-cell neoplasms. These limited data from both B-cell and T-cell tumors suggest that extra dosage of chromosome 3 confers a slight growth advantage on lymphocytes in either lineage, in much the same way that trisomy 12 appears to act only within the B-cell phenotype. This effect of trisomy 3 also appears to involve a relatively late stage of differentiation, resulting, with B cells, in a clone that actively secretes immunoglobulin. Some evidence shows that rearrangements involving the c-myc gene may also contribute to this low-grade clonal expansion [12], and additional somatic genetic changes, either visible or submicroscopic, may be necessary for progression to more aggressive disease. This conclusion appears to be supported by the initial follow-up data on our patients. Although patient 3, with trisomy 3 alone, was diagnosed as having lymphoma shortly after our study, her disease has been very indolent; the one other patient with a lymphoma, and a somewhat more aggressive course, also had the most extensive karyotypic changes in addition to trisomy 3 (patient 1). Although two of the other four patients died in less than 2 years, of unrelated causes, our present data indicate that the presence of a karyotypically abnormal clone in CAD does not necessarily predict prompt progression to frank neoplasia. The present results also suggest, however, that CAD patients with a cytogenetically aberrant clone may be at greater risk of developing lymphoma than those with a normal karyotype. Whether such information will be of clinical value in the prognosis and management of the clinically heterogeneous group of patients who have this disease remains to be determined by additional studies and longer follow-up.
92
J. Gordon et al. This work was supported in part by Grants No. CA42232, CA15822, and DK39065 from the National Institutes of Health, Bethesda, Maryland. The authors thank Drs. E. C. Besa, A. J. Solan, K. Sethi, R. B. Sklaroff, S. Shattil, R. Steingart, R. M. Brontz, and C. N. Liedman for their cooperation.
REFERENCES 1. Helm S, Mitelman F (1987): Cancer Cytogenetics. Alan R. Liss, New York. 2. Godde-Salz E, Feller AC, Lennert K (1986): Cytogenetic and immunohistochemicalanalysis of lymphoepithelioid cell lymphoma (Lennert's ]ymphoma): Further substantiation of its T-cell nature. Leuk Res 10:313-323. 3. Godde-Salz E, Feller AC, Lennert K (1987): Chromosomal abnormalities in lymphogranulomatosis X (Lg.X) and angioimmunoblasticlymphadenopathy (AILD). Leuk Res 11:181-190. 4. Silberstein LE, Robertson GA, Hannam-Harris AC, Moreau L, Besa E, Nowell PC (1986): Etiologic aspects of cold agglutinin disease: evidence for cytogenetically defined clones of lymphoid cells and the demonstration that an anti-Pr cold antibody is derived from a chromosomally aberrant B-cell clone. Blood 67:1705-1709. 5. Nowell PC, Vonderheid EC, Besa E, Hoxie JA, Moreau L, Finan JB (1986): The most common chromosome change in 85 chronic B cell or T cell tumors: A 14q32 translocation. Cancer Genet Cytogenet 19:219-227. 6. Juliusson G, Oscier DG, Ross FM, Castoldi GL, Elonen E, Knuutila S, Gahrton G (1989) InternationalWorkshop on Chromosomes in Chronic Lymphocytic Leukemia: First compilation of cytogenetic data on 427 patients from Scandinavia, United Kingdom, and Italy [Abstract]. Proceedings of Xth Congress of the InternationalSociety of Hematology (in press). 7. Contrafatto G (1977): Marker chromosome of macroglobulinemia identified by G-banding. Cytogenet Cell Genet 18:370-373. 8. Kaneko Y, Larson RA, Variakojis D, et al. (1982): Non-random chromosome abnormalities in angioimmunoblastic lymphadenopathy. Blood 60:877. 9. Mark J, Dahlenfors R, Ekedahl C (1979): Recurrent chromosomal aberrations in non-Hodgkin's and non-Burkitt lymphomas. Cancer Genet Cytogenet 1:39-56. 10. Yunis JJ, Oken MM, Theologides A, Howe RB, Kaplan ME (1984): Recurrent chromosomal defects are found in most patients with non-Hodgkin's lymphoma. Cancer Genet Cytogenet 13:17-28. 11. Cabanillas F, Pathak S, Trujillo J, Manning J, Katz R, McLaughlin P, Velasquez WS, Hagemeister FB, Goodacre A, Cork A, Butler JJ, Freireich EJ (1988): Frequent nonrandom chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma. Cancer Res 48:5557-5564. 12. Perl A, Wang N, Williams JM, Hunt MJ, Rosenfeld SI, Condemi JJ, Packman CH, Abraham GN (1987): Aberrant immunoglobulin and c-myc gone rearrangements in patients with nonmalignant monoclonal cryoglobulinemia. J Immunol 139:3512-3520.
ABSTRACT: Cold agglutinin disease (CAD) is a form of autoimmune hemolytic anemia generally considered to result from an antibody-producing clonal expansion of B lymphocytes. We studied 16 patients with CAD and demonstrated a ehromosomally abnormal clone in the peripheral blood (PB) of six. Trisomy 3 was the only abnormality in two patients, trisomy 12 the only abnormality in one, and both trisomy 3 and trisomy 12 were present in three patients. A lymphoma was subsequently diagnosed in two of these six patients and in none of the patients without a karyotypic abnormality. The results suggest that trisomy 3 confers a slight growth advantage in the B-cell lineage, particularly at a relatively late stage of differentiation. The resultant clone may present clinically as CAD and, in some cases, progress to a clinical lymphoma.
INTRODUCTION Trisomy for chromosome 3 occurs relatively commonly in a variety of neoplasms, usually as part of a complex, highly aneuploid karyotype. In hematopoietic disorders, it is more frequent in lymphoid than in myeloid tumors, particularly advanced lymphomas [1]. As a single abnormality, or in association with only one or two additional karyotypic alterations, trisomy 3 is quite rare and has been described most commonly in low-grade preneoplastic or neoplastic clonal proliferations of T cells. These have included such disorders as lymphogranulomatosus X, angioimmunoblastic lympadenopathy, and Lennert's l y m p h o m a [2, 3]. We recently described several cases of cold agglutinin disease (CAD), a form of autoimmune hemolytic anemia, in which a chromosomally abnormal B-cell clone was identified in the peripheral blood (PB) with trisomy 3 alone or trisomy 3 in combination with trisomy 12 [4]. We have extended this study to include additional cases and to obtain clinical follow-up data on CAD patients with and without a demonstrable cytogenetic abnormality.
MATERIALS AND METHODS Sixteen patients with CAD have now been studied cytogenetically. The diagnosis was established on the basis of a hemolytic cold autoantibody, and additional clinical details on some of the patients were reported previously [4]. The duration of the From the Department of Pathologyand LaboratoryMedicine, Universityof Pennsylvan/aSchoolof Medicine, Philadelph/a. Address reprint requests to: Dr. Peter C. Nowell, Depatment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082. Received July 24, 1989; accepted September 13, 1989.
89 © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010
Cancer Genet Cytogenet 46:89-92 (1990)
01654608/90/$03.50
90
J. Gordon et al.
Table 1
Six CAD patients with karyotypic abnormalities
Patient no.
Abnormal karyotype
Survival after study (too)
Lymphoma
1 2 3 4 5 6
51,XX, + 3,+4, + 9,+ 12, + 13, + 18 47,XY, + 3 47,XX, + 3 47,XX, + 12; 48,XX, + 3, + 12 47,X, - X, + 3, + 12 47,XX, + 12
60 a 20 50a 20 19a 6a
Yes No Yes No Nob No
Patients 1-4 are the same as those similarly numbered in reference 4. a Patient alive. bPatient has "mature lymphocytosis of bone marrow." patients' a u t o i m m u n e disorder ranged from 6 weeks to 10 years before study, and none had an established diagnosis of lymphoma. The patients ranged in age from 37 to 78 years, although all but one were aged more than 60 years.
Cytogenetic Studies Peripheral blood (PB) m o n o n u c l e a r cells from each patient were cultured for 7 2 - 9 6 hours w i t h a combination of p o k e w e e d mitogen (PWM) and a phorbol ester (phorbol12-myristate-13-acetate, TPA) s u p p l e m e n t e d with a crude preparation of interleukin2 (IL-2) that contained B-cell growth factor activity [5]. Standard trypsin-Giemsa b a n d e d c h r o m o s o m e preparations were made [5]. At least 20 counts and three karyotype analyses were performed for each study, with additional counts and analyses as required to characterize any karyotypically abnormal clone. RESULTS A c h r o m o s o m a l l y abnormal clone, constituting 15% to 65% of the metaphases examined, was identified in the PB of six of the 16 CAD patients studied (Table 1). In two patients the only karyotypic abnormality was trisomy 3, three patients also had trisomy 12, and in one patient the only abnormality was trisomy 12. Cells from the k a r y o t y p i c a l l y abnormal clone of two patients (Patients I and 5) have been established as Epstein-Barr virus (EBV)-transformed cultures and shown to be the source of the patients' pathological a u t o a n t i b o d y [4] ( u n p u b l i s h e d observations, L. Silberstein). Three of these six patients also had a B-cell clone in the PB shown by i m m u n o p h e n o typing [4].
Clinical Follow-up A l l six of the patients w i t h a karyotypically abnormal clone and seven of the other 10 CAD patients have been followed for a period of 6 - 6 0 months. The diagnosis of l y m p h o m a has been made in two of the six patients with a chromosome alteration (Table 1) and in none of those w i t h a normal karyotype. A splenectomy was performed on patient I eight weeks after the original cytogenetic study, and a well-differentiated l y m p h o m a was identified in the spleen and adjacent nodes. She is alive after 5 years, with her l y m p h o m a partially controlled by therapy. A l y m p h node b i o p s y after 47 months s h o w e d evidence of karyotypic evolution, recently associated with more aggressive clinical disease. In patient 3, a well-differentiated l y m p h o m a was diagnosed on BM b i o p s y 6 weeks after c h r o m o s o m e study, and 4 years after therapy she remains in remission. Patients
Trisomy 3 in Cold Agglutinin Disease
91
2 and 4 both died 20 months after chromosome study without progression to lymphoma, and patients 5 and 6 are clinically unchanged after 19 months and 6 months, respectively. Patient 5 had "mature lymphocytosis of the bone marrow" when first studied, but a definite diagnosis of lymphoma has not been established. Among the 10 patients without a karyotypically abnormal clone demonstrable in the blood, one died of complications of her autoimmune disease 3 months after study, six have been followed for 15-51 months (median = 40 months) without evidence of a lymphocytic neoplasm, and three were lost to follow-up. In three cases, a repeat chromosome study after 2-5 months again showed no karyotypic abnormality.
DISCUSSION
Our present findings, which appear to be the only cytogenetic data on a series of patients with CAD, confirm our previous impression that when a chromosomally abnormal clone is demonstrable in this disorder, trisomy 3 or trisomy 12 or both are typically observed, often without additional alterations. The presence of trisomy 12, singly or in combination, clearly links these low-grade lymphoid dyscrasias to chronic B-cell leukemia, in which an extra chromosome 12 is the most common finding and in which trisomy 3 occasionally occurs as an additional alteration [1, 6]. Cold agglutinin disease, however, represents the first instance of trisomy 3, without other extensive cytogenetic changes, being frequently associated with a particular B-cell disorder. Contrafatto [7], in an early banding study, identified the single large extra chromosome often observed in WaldenstrOm's macroglobulinemia as a 3 p - in two cases, and further suggested that "anomalies of chromosome 3 occur particularly often in monoclonal gammopathies." Trisomy 3 alone has been most frequently reported in low-grade T-cell tumors (lymphogranulomatosus X, angioimmunoblastic lymphadenopathy, Lennert's lymphoma) [2, 3, 8]. Very few patients with high-grade tumors have only trisomy 3 [9-11], and Yunis et al. [10] reported a diffuse large-cell lymphoma that had 48 chromosomes with trisomy 3, trisomy 12, and the t(14;18) translocation often associated with lowgrade B-cell neoplasms. These limited data from both B-cell and T-cell tumors suggest that extra dosage of chromosome 3 confers a slight growth advantage on lymphocytes in either lineage, in much the same way that trisomy 12 appears to act only within the B-cell phenotype. This effect of trisomy 3 also appears to involve a relatively late stage of differentiation, resulting, with B cells, in a clone that actively secretes immunoglobulin. Some evidence shows that rearrangements involving the c-myc gene may also contribute to this low-grade clonal expansion [12], and additional somatic genetic changes, either visible or submicroscopic, may be necessary for progression to more aggressive disease. This conclusion appears to be supported by the initial follow-up data on our patients. Although patient 3, with trisomy 3 alone, was diagnosed as having lymphoma shortly after our study, her disease has been very indolent; the one other patient with a lymphoma, and a somewhat more aggressive course, also had the most extensive karyotypic changes in addition to trisomy 3 (patient 1). Although two of the other four patients died in less than 2 years, of unrelated causes, our present data indicate that the presence of a karyotypically abnormal clone in CAD does not necessarily predict prompt progression to frank neoplasia. The present results also suggest, however, that CAD patients with a cytogenetically aberrant clone may be at greater risk of developing lymphoma than those with a normal karyotype. Whether such information will be of clinical value in the prognosis and management of the clinically heterogeneous group of patients who have this disease remains to be determined by additional studies and longer follow-up.
92
J. Gordon et al. This work was supported in part by Grants No. CA42232, CA15822, and DK39065 from the National Institutes of Health, Bethesda, Maryland. The authors thank Drs. E. C. Besa, A. J. Solan, K. Sethi, R. B. Sklaroff, S. Shattil, R. Steingart, R. M. Brontz, and C. N. Liedman for their cooperation.
REFERENCES 1. Helm S, Mitelman F (1987): Cancer Cytogenetics. Alan R. Liss, New York. 2. Godde-Salz E, Feller AC, Lennert K (1986): Cytogenetic and immunohistochemicalanalysis of lymphoepithelioid cell lymphoma (Lennert's ]ymphoma): Further substantiation of its T-cell nature. Leuk Res 10:313-323. 3. Godde-Salz E, Feller AC, Lennert K (1987): Chromosomal abnormalities in lymphogranulomatosis X (Lg.X) and angioimmunoblasticlymphadenopathy (AILD). Leuk Res 11:181-190. 4. Silberstein LE, Robertson GA, Hannam-Harris AC, Moreau L, Besa E, Nowell PC (1986): Etiologic aspects of cold agglutinin disease: evidence for cytogenetically defined clones of lymphoid cells and the demonstration that an anti-Pr cold antibody is derived from a chromosomally aberrant B-cell clone. Blood 67:1705-1709. 5. Nowell PC, Vonderheid EC, Besa E, Hoxie JA, Moreau L, Finan JB (1986): The most common chromosome change in 85 chronic B cell or T cell tumors: A 14q32 translocation. Cancer Genet Cytogenet 19:219-227. 6. Juliusson G, Oscier DG, Ross FM, Castoldi GL, Elonen E, Knuutila S, Gahrton G (1989) InternationalWorkshop on Chromosomes in Chronic Lymphocytic Leukemia: First compilation of cytogenetic data on 427 patients from Scandinavia, United Kingdom, and Italy [Abstract]. Proceedings of Xth Congress of the InternationalSociety of Hematology (in press). 7. Contrafatto G (1977): Marker chromosome of macroglobulinemia identified by G-banding. Cytogenet Cell Genet 18:370-373. 8. Kaneko Y, Larson RA, Variakojis D, et al. (1982): Non-random chromosome abnormalities in angioimmunoblastic lymphadenopathy. Blood 60:877. 9. Mark J, Dahlenfors R, Ekedahl C (1979): Recurrent chromosomal aberrations in non-Hodgkin's and non-Burkitt lymphomas. Cancer Genet Cytogenet 1:39-56. 10. Yunis JJ, Oken MM, Theologides A, Howe RB, Kaplan ME (1984): Recurrent chromosomal defects are found in most patients with non-Hodgkin's lymphoma. Cancer Genet Cytogenet 13:17-28. 11. Cabanillas F, Pathak S, Trujillo J, Manning J, Katz R, McLaughlin P, Velasquez WS, Hagemeister FB, Goodacre A, Cork A, Butler JJ, Freireich EJ (1988): Frequent nonrandom chromosome abnormalities in 27 patients with untreated large cell lymphoma and immunoblastic lymphoma. Cancer Res 48:5557-5564. 12. Perl A, Wang N, Williams JM, Hunt MJ, Rosenfeld SI, Condemi JJ, Packman CH, Abraham GN (1987): Aberrant immunoglobulin and c-myc gone rearrangements in patients with nonmalignant monoclonal cryoglobulinemia. J Immunol 139:3512-3520.