Nonrandom karyotype abnormalities in 36 multiple myeloma patients

ELSEVIER

Nonrandom Karyotype Abnormalities in 36 Multiple Myeloma Patients Ravindran Ankathil, Jayaprakash Madhavan, V.P. Gangadharan, G. Rajasekharan Pillai, and M. Krishnan Nair

ABSTRACT: G-banded analysis performed on pretreated bone marrow samples of 36 multiple myeloma patients allowed the identification of clonal chromosome abnormalities. Abnormalities consisting of trisomies, monosomies, translocations, deletions, and marker chromosomes apparently followed a nonmndom pattern. The chromosomes involved in the production of abnormal karyotypes were numbers 1,2,3,11,12,14,1Z and 18. Even though no specific chromosome pattern has been identified, the involvement of chromosomes 1, 3, and 14 was found to be more frequent. Many of the chromosomes and chromosomal breakpoints involved in these abnormalities correspond to the location of identified oncogenes or tumor suppressor genes. Hence, it is presumed that these chromosome abnormalities may be playing an important role in the genesis of multiple myeloma by altering the structure or function of oncogenes or tumor suppressor genes.

INTRODUCTION

During recent years, many cytogenetic studies have aimed at establishing karyotypic characteristics related to specific malignancies. The plasma cell disorders represent a group of diseases that are biologically different from other hematologic malignancies. Due to low mitotic activity and difficulty in obtaining sufficient metaphase spreads, very few reports are available regarding the cytogenetic aspects of multiple myeloma. Thus, chromosome studies were undertaken in multiple myeloma patients to detect the abnormalities of chromosome constitution in their bone marrow cells. We were specifically interested to see whether or not cytogenetic analysis could provide additional information for the management of individual cases.

establishing the diagnosis and before the start of any kind of therapy. Chromosome preparations were obtained directly or after short-term (24-48 h) culture of bone marrow aspirate in F-10 medium with 20% fetal calf serum. Mitotic cells were accumulated by exposure to Colcemid (0.1 ~g/mL medium) for 30 minutes before harvesting. Cell suspensions were rinsed twice in Hanks balanced salt solution and exposed to hypotonic KC1 (0.75 M) for 30 minutes, fixed in methanol/acetic acid fixative in 3:1 ratio, and air dried. Chromosome preparations were stained with conventional Giemsa and handed with trypsin-Giemsa [3]. Chromosome identification and karyotype preparations were done according to the International System for Human Cytogenetic Nomenclature [4].

MATERIALS AND METHODS Thirty-six multiple myeloma patients (21 males and 15 females) attending the outpatient clinics of the Regional Cancer Centre, Thiruvananthapuram (S. India) were included in the study. They were diagnosed and staged according to Durie and Salmon's classification and criteria [1, 2]. The ages of the patients ranged from 38 to 75 years, with a median age of 58 years. Chromosome studies were carried out using bone marrow samples of the patients during the course of

From the Regional Cancer Centre, Thiruvananthapumm, India. Address reprint requests to: Dr. I~vindran Ankathil, Assistant Professor, Division of Cancer Research, Regional Cancer Centre, Thiruvananthapumm - 695 011, India. Received June 13, 1994; accepted September 1, 1994.

RESULTS The modal chromosome number of the patients ranged from 45 to 47 chromosomes. Seven patients showed a hypodiploid modal number with 45 chromosomes. Pseudodiploidy with 46 chromosomes as the modal population was encountered in 27 patients and two patients presented a hyperdiploid mode with 47 chromosomes. Abnormalities consisting of addition or loss of whole or part of the chromosomes apparently followed a nonrandom pattern. These abnormalities were observed either as single or combined anomalies. The most striking observation was the detection of marker chromosomes in 12 of the 36 patients (33%). In three patients, large metacentric marker chromosomes which in size were larger than the group A chromosomes were observed. These were formed as a result of the

Cancer Genet Cytogenet 83:71-74 (1995)

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R. A n k a t h i l et al.

a d d i t i o n of c h r o m a t i n m a t e r i a l to t h e s h o r t a r m of c h r o m o s o m e 2 (2 p + ). S m a l l m e t a c e n t r i c m a r k e r s of t h e size of g r o u p G chromosomes were observed in four patients and doublem i n u t e s w e r e s e e n i n five. Chromosomes that were involved in producing the abnorm a l k a r y o t y p e s w e r e 1,2,3,11,12,14,17, a n d 18. T h e k a r y o t y p e p a t t e r n s of t h e 36 p a t i e n t s are g i v e n i n T a b l e 1. It c a n b e s e e n t h a t n u m b e r s 1 a n d 14 w e r e t h e m o s t f r e q u e n t l y i n v o l v e d c h r o m o s o m e s ; 14 q + a b n o r m a l i t y w a s d e t e c t e d i n six p a tients. D e l e t i o n of t h e s h o r t a r m of c h r o m o s o m e I (1 p - ) w a s detected in four patients and a translocation between chrom o s o m e s 1 a n d 2, i.e., t(1;2)(q32;q37), w a s d e t e c t e d i n t w o patients. C h r o m o s o m e 3, w h i c h w a s i n v o l v e d e i t h e r i n trisom i e s o r m o n o s o m i e s i n five p a t i e n t s , w a s t h e n e x t f r e q u e n t l y i n v o l v e d c h r o m o s o m e . T h r e e p a t i e n t s s h o w e d d e l e t i o n of t h e l o n g a r m of c h r o m o s o m e 11. M o n o s o m y 12 w a s o b s e r v e d i n f o u r p a t i e n t s , of w h i c h it w a s i n v o l v e d i n p s e u d o d i p l o i d y i n t w o cases. C h r o m o s o m e 17 w a s i n v o l v e d i n f o u r p a t i e n t s ,

w h e r e i n t w o p a t i e n t s s h o w e d m o u o s o m y 17 a n d t h e o t h e r t w o p a t i e n t s s h o w e d d e l e t i o n of t h e s h o r t a r m (17 p - ). M o n o s o m y 18 w a s d e t e c t e d i n t w o p a t i e n t s .

DISCUSSION Chromosome studies utilizing banding techniques were f o u n d to b e s o m e w h a t d i f f i c u l t i n m y e l o m a p a t i e n t s . Low i n v i t r o p r o l i f e r a t i o n of p l a s m a cells, d i f f i c u l t i e s i n o b t a i n i n g g o o d s p r e a d s , b l u r r e d a n d c o n t r a c t e d c h r o m o s o m e s i n dir e c t b o n e m a r r o w p r e p a r a t i o n s , a n d r e s i s t a n c e to G - b a n d i n g c o u l d b e t h e r e a s o n s for this. T h i s m a y b e t h e r e a s o n for t h e s c a r c i t y of p u b l i s h e d d a t a o n t h e c y t o g e n e t i c s of m u l t i p l e m y e l o m a c o m p a r e d to o t h e r h e m a t o l o g i c d i s o r d e r s . A variety of c h r o m o s o m e a b n o r m a l i t i e s s u c h as t r i s o m i e s , monosomies, translocations, deletions, and marker chromosomes have been observed in these multiple myeloma pa-

Table 1

K a r y o t y p e p a t t e r n i n 36 m u l t i p l e m y e l o m a p a t i e n t s

Patient no.

Age

Sex

Stage of disease

No. of metaphases examined

Group

1 2 3 4 5 6 7 8 9 10 11 12

63 60 59 54 58 72 52 49 55 73 61 54

M M F F F F M M M M M F

111B 11A 111A 111A 11A 11B 111B 111 A 111 A 111 B 11A 111 B

20 12 22 13 19 18 15 14 11 9 10 17

AN AN AN AN AN AA AN AN AN AA AA AN

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

55 58 65 42 60 67 65 66 50 61 62 65 47 38 60 52 68 63 43 58 68 49 75 58

F F F M M F M M F M F M M M M M M F M M M F F F

11B 11B 111B 111A 111 A 111 A 11 B 111 A 11B 111 B 11 A 11 B 111 A 111 B 111A 111A 111 B 111 B 111A 11 B 11B 111 B 11A 111 A

8 11 15 20 12 10 17 21 10 15 18 12 22 8 19 16 21 17 14 20 19 12 22 16

AN AA AN AN AA AA AN AA AN AA AN AN AN AA AA AN AN AN AN AN AN AA AN AA

Karyotype 46,XY[8]/45,XY, - 3112] 46,XY[5]/46,XY, l d m i n [ 7] 46,XX[7]/45,XX, - 18115] 46,XX[5]/46,XX,Idmin[8] 46,XX[8]/46,XX,der(2)t(1;2)(q32 ;q37)[11] 46,XX,del(1)(p1Ip22)[18] 46,XY[5]/46,XY,add(2)(p25)[10] 46,XY[5]/46,XY, - 3, + mar[9] 46,XY[2]/45,XY, - 17, I drain[9] 46,XY,add(14)(q32)[9] 46,XY,del(11)(q13)[10] 46,XX[4]/46,XX,der(14)t(l 1;14) (q13;q32)[13] 46,XX[2]/47,XX, + mar[6] 46,XX,del(1)(p1Ip22)[11] 4 6 , X X [ 7 ] / 4 5 , X X , - 12181 46,XY[9]/45,XY, - 18111] 46,XY,del(1)(pllp22)[12] 46,XX,add(2)(p25) [10] 46,XY[7]/46,XY, + 3, - 17110] 46,XY,der(14)t(11;14)(q13;q32)[21] 46,XX[3]/45,XX, - 3,1dmin[7] 46,XY,del(11)(q13) [1 S] 46,XX[6]/46,XX,add(14)(q32)[12] 4 6 , X Y [ 4 ] / 4 7 , X Y , + marl8] 46,XY[10]/45,XY, - 12112] 46,XY,del(17)(p11)[8] 46,XY,add(2)(p25)[19] 46,XY[7]/46,XY,ldmin[9] 46,XY[8]/46,XY,del(1)(pllp22)[13] 4 6 , X X [ 6 ] / 4 6 , X X , + 3, - 12111] 46,XY[3]/46,XY,add(14) (q32)[11] 46,XY[8]/46,XY, - 12, + mar[12] 46,XY[4]/46,XY,der(2)t(1;2)(q32;q37) [15] 46,XX,del(11)(q13)[12] 46,XX[8]/46,XX,add(14)(q32)[14] 46,XX,del(17)(p11)[16]

Abbreviations: AN, patients with at least one normal mitosis and an abnormal clone; AA, patients with only abnormal mitoses;

mar, small metacentric marker; dmin, double-minute chromosome.

Nourandom Abnormalities in Multiple Myeloma tients. The large metacentric marker chromosomes detected in three patients were the result of the addition of chromatin material to the short arm of chromosome 2 (2 p + ]. But the source of the extra chromatin on 2 p + could not be determined. 2 p + marker chromosomes have been reported in myeloma patients by others [5, 6]. The location of genes that codify for the immunoglobulin K light chain in the short arm of chromosome 2 [7, 8] stresses the importance of rearrangement affecting the short arm of this chromosome. The origin of the small metacentric marker chromosomes observed in four patients could not be identified by G-banding. Doubleminutes (dmin} were observed in five patients. The preponderant incidence of double-minutes has been in neurogenic tumors [9], rhabdomyosarcomas [10], and acute leukemias [11]. In multiple myeloma, there has not been any previous reports of dmin. Drain have been reported to contain reiterated sequences of DNA [12]. Drug-resistance genes and oncogenes have been proven to be the structural components of dmin and homogeneously staining regions [13]. Thus the molecular anatomy of dmin may have profound importance for the etiology or biology of the tumor. The presence of pseudodiploid metaphases was another striking abnormality, observed in 27 patients. Bauke et al. [14] had reported sporadic hypodiploid, pseudodiploid, and hyperdiploid cells, particularly in groups C and G in eight multiple myeloma patients. In a study on 259 marrows from patients with multiple myeloma, Lewis et al. [15] reported that in cases with aneuploid clones, one-third had a pseudodiploid pattern. Thus, pseudodiploidy appears to be a salient feature in myeloma patients. Loss or addition of chromosomes or chromosome segments encountered in this study apparently followed a nonrandom pattern. It is of interest that many of the chromosomes and chromosomal regions involved in structural abnormalities in these myeloma patients correspond to the location of identifiable oncogenes or tumor suppressor genes. From the limited literature on cytogenetic studies of patients with multiple myeloma, it seems that a 14q + anomaly is the most common abnormality, occurring in almost 30% of such cases [16, 17]. In our study, 14q+ was observed in six (17%) patients. In two patients, the 14q + anomaly was derived from a translocation between chromosomes 11 and 14, i.e., t(11;14) (q13;q32). This type of translocation has been reported in patients with multiple myeloma [18, 19], plasma cell leukemia [20], and B-cell lymphoproliferative disorders [21]. Nishida et al. [22] reported a t(14;18}(q32;q21) in pleura] effusion cells from a patient with nonsecretory myeloma. Nishida et al. [23] also reported a chromosome rearrangement, t(6;14} (p21;q32), in multiple myeloma. The extra chromatin on 14q + in the other four patients in the present study could not be established because of the poor quality of the banded chromosomes. The location of the immunoglobulin heavy chain locus on chromosome 14 implicates the importance of this chromosomal involvement in myeloma patients. Other chromosomes involved nonrandomly were numbers 1,2,3,11,12,17, and 18. Chromosome 1 was involved in six patients, out of whom four patients showed deletion of the short arm, i.e., del(1}(pllp22). Dewald et al. [17] also reported abnormalities of chromosome 1 in patients with multiple myeloma, amyloidosis, and plasma cell leukemia.

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Recently, the N-ras oncogene has been located on chromosome 1 and the lp21 region has been reported [24] to be a known breakpoint region in direct myeloma specimens. A translocation between chromosomes 1 and 2, i.e., t(1;2) (q32;q37), was detected in two patients. Chromosome 3 was involved in five patients, either as a trisomy or monosomy, in the production of pseudodiploid metaphasas. Philip [25], reviewing the literature on chromosome abnormalities in monoclonal gammopathies, stated that, in addition to abnormalities of chromosome 14, abnormalities of chromosomes I and 3 were preferentially involved, which is in conformity with our findings. Three of our patients showed deletion of the long arm of chromosome 11, i.e., del(ll)(q13). Abnormalities of chromosome 11 have not been strongly linked with plasma cell disorders. Dewald et al. [17] reported involvement of chromosome 11 in nine of 82 patients with multiple myeloma and the abnormalities consisted of translocations and deletions. Oncogene Hu-ets-1, located on band 11(t23 has been shown to translocate, rearrange, or amplify in human hematologic malignancies involving band 11q23 [26-26]. However, the role of this oncogene in myeloma has not been reported. Even though chromosome 12 is rather infrequently involved in human neoplasia, we observed monosomy 12 in four myeloma patients. An oncogene c-ki-ros has been identified [24] on 12p12-~pter, but its role in myeloma remains unknown. Involvement of chromosome 17 was observed in four patients, of which two patients showed monosomy 17 and two showed deletion of the short arm of chromosome 17, i.e., del(17](p11). Ranni et al. [29] reported a 17p - in four cases of myeloma, whereas Dewald et al. [17]reported 17p + in four cases of multiple myeloma. The tumor suppressor gene p53 is located on the short arm of chromosome 17, which many investigators have found to be abnormal and which is also a chromosome that is often deleted in myeloma metaphases [24]. Thus, alterations of the short arm of chromosome 17 might be especially associated with multiple myeloma. Two patients in the current study showed monosomy 18. An oncogene BC1-2 has been located on chromosome 18 [24] and several investigators have noted increased expression of BC1-2 protein in myeloma cells [30, 31]. The reason for this increased expression is unknown. The location of a tumor suppressor gene anti-ros on 18q [24] stresses the importance of monosomy 18, a condition which may have profound influence on inactivating the 'antiras" No specific numerical or structural change occurs exclusively in multiple myeloma. But a pattern, which includes the frequent involvement of chromosomes 1, 3, and 14, especially 14q + abnormality, may be associated with multiple myeloma. The presence of marker chromosomes was also a typical feature. It is hoped that with the advent of new knowledge about important stimulators of proliferation for the plasma cell, larger numbers of patients can be studied carefully and some common cytogenetic features may emerge. However, these abnormalities may have a significant role in altering the structure or function of oncogenes and tumor suppressor genes. It therefore seems that various chromosomal changes observed in myeloma may contribute to the evolution and persistence of the malignant myeloma cell clone(s).

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REFERENCES 1. Durie BGM, Salmon SE (1977): Multiple myeloma, macroglobulinemia, and monoclonal gammopathies. In: Recent Advances in Hematology, Hofibrand AV, Brown MC, Hirsch J, eds. Churchill Livingston, Edinburgh, pp. 243-261. 2. Durie BGM, Salmon SE (1975): A clinical staging system for multiple myeloma. Cancer 36:842-854. 3. Seahright M (1971): A rapid banding technique for human chromosomes. Lancet ii:971-972. 4. ISCN (1991): Guidelines for Cancer Cytogenetics, Supplement to An International System for Human Cytogenetic Nomenclature, Mitelman F, ed. S. Karger, Basel, 1991. 5. Philip P, Drivsholm A (1976): G-banding analysis of complex aneuploidy in multiple myeloma bone marrow cells. Blood 47:69-73. 6. Van Den Berghe H, Vermaelen K, Louwagie A, Criel A, Mecucci C, Vaerman JP (1984): High incidence of chromosome abnormalities in IgG3 myeloma. Cancer Genet Cytogenet 11:381-384. 7. Malcolm S, Barton P, Bentley DL (1981}: Assignment of a kappa locus for immunoglobulin light chain to the short arm of chromosome 2 (2 cen to 2 p13). Human gene mapping conference VI. National Foundation, White Plains, NY. 8. Lenoir GM, Preud'homme JL, Berheim A, Berger R {1982): Correlation between immunoglobulin light chain expression and variant translocation in Burkitt's lymphomas. Nature 298:474-476. 9. Biedler JL, Ross RA, Shanske S, et al. (1980): Human neuroblastoma cytogenetics: search for significance of homogeneously staining regions and double minute chromosomes. Adv Neuroblastoma Res 4:81-96. 10. Barker PE {1982): Double minutes in human tumour cells. Cancer Genet Cytogenet 5:81-94. 11. Sandberg AA (1990): Chromosomes in Human Cancer and Leukemia. Elsevier-North Holland, NY. 12. George DL, Powers VE (1981): Cloning of DNA from double minutes of Y1 adrenocortical tumor cells. Evidence for gene amplification. Cell 24:117-123. 13. Stark GR, Wahl GM (1984): Gene amplification. Ann Rev Biochem 53:447-492. 14. Bauke J, Kaiser G, Schoffling K (1972): Chromosome studies in plasmacytoma and plasma cell leukemia. Verb Dtsch Ges Inn Med 78:122-125. 15. Lewis FJW, MacTaggart M, Crow RS, Will RS (1963): Chromosomal abnormalities in multiple myeloma. Lancet ii: 1183-1184. 16. Mitelman F {1981): Marker chromosome 14q+ in human cancer and leukemia. Adv Cancer Res 34:141-170.

R. A n k a t h i l et al.

17. Dewald GW, Kyle RA, Hicks GA, Greipp PR (1985):Clinical significance of cytogenetic studies in 100 patients with multiple myeloma, plasma cell leukemia or amyloidosis. Blood 66: 380-390. 18. Gahrton G, Robert KH, Friberg K, Zech L, Bird A G {1980): Nonrandom chromosomal aberrations in chronic lymphocytic leukemia revealed by polyclonal B-cell-mitogen stimulation. Blood 56:640-645. 19. Venti G, Mecucci C, Donti E, Tahilio A (1984): Translocation t (11;14) and trisomy 11q13-qter in multiple myeloma. Ann Genet 27:53-58. 20. Ueshima Y, Fukuhara S, Nagai K, Takatsuki K, Uchino H (1983): Cytogenetic studies and clinical aspects of patients with plasma cell leukemia and leukemic macroglobulinemia. Cancer Res 43:905-908. 21. Schroder J, Vuopio P, Autio K (1981): Chromosome changes in human chronic lymphocytic leukemia. Cancer Genet Cytogenet 4:11-13. 22. Nishida K, Taniwaki M, Misawa S, Abe T, Takino T, Fujii H (1987): T~nslocetion (14;18) found in a patient with multiple myeloma. Cancer Genet Cytogenet 25:375-37Z 23. Nishida K, Yashige H, Maekawa T, Fujii H, Taniwaki M, Horiike S, Misawa S, Inazawa J, Abe T (1989): Chromosome rearrangement t (6;14) {p21.1;q32.3) in multiple myeloma. Br J Haematol 71:295-296. 24. Durie BGM (1992): Cellular and molecular genetic features of myeloma and related disorders. Haemetol/Oncol Clin of N A 6:463-477. 25. Philip P (1980): Chromosomes of monoclonal gammopathies. Cancer Genet Cytogenet 2:79-81. 26. Sacchi N, Watson DK, Guerts van kessel AMH, et al. {1986): Huets-1 and Hu-ets-2 genes are transposed in acute leukemias with (4;11) and 8;21) translocations. Science 231:379-382. 27. Rovigatti U, Watson DK, Yunis JJ {1986): Amplification and rearrangement of Hu-ets-1 in leukemia and lymphoma with involvement of 11q23. Science 232:398-400. 28. Yunis ~, Brnnning RD (1986): Prognostic significance of chromosomal abnormalities in acute leukemias and myelo dysplastic syndromes. Clin Hematol 15:597-620. 29. Ranni NS, Slavutsky I, Wechster A, Brieux de Salum S (1987): Chromosome findings in multiple myeloma. Cancer Genet Cytogenet 25:309-316. 30. Durie BGM, Mason DY, Giles F, et al (1990): Expression of the Bcl-2 oncogene protein in multiple myeloma. Blood 76:347. 31. Durie BGM, Vela EE, Baum V (1991): Cytogenetic abnormalities in multiple myeloma. Epidemiology and Biology of Multiple Myeloma. Springer-Verlag, New York, pp. 137-141.