Chromosome aberrations in a series of 120 multiple myeloma cases with abnormal karyotypes

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1 Chromosome aberrations in a series of 120 multiple myeloma cases with abnormal karyotypes Anwar N. Mohamed, 1,2 * Gail Bentley, 1 Michelle L. Bonnett, 1 Jeff Zonder, 3 and Ayad Al-Katib 3 1 Department of Pathology, Detroit Medical Center, Wayne State University School of Medicine, Detroit, Michigan 2 Cytogenetics Laboratory, Hutzel Professional Building, Detroit, Michigan 3 Department of Internal Medicine, Wayne State University, Detroit, Michigan We identified 120 multiple myeloma (MM) cases with satisfactory cytogenetic evaluation and abnormal karyotypes. Hyperdiploid karyotype was found in 77 cases (64%), hypodiploid in 30 cases (25%), and the remaining 13 cases (11%) had a pseudodiploid karyotype. The most common numerical abnormalities were gains of chromosomes 15, 9, 3 followed by chromosomes 19, 11, 7, 21, and 5. Whole chromosome losses were also frequent involving primarily chromosomes X/Y, 8, 13, 14, and 22. Most cases showed also structural rearrangements leading to del(1p), dup(1q), del(5q), del(6q), del(8p), del(9p), del(13q), and del(17p). Chromosome 13/13q deletion was found in 52% of cases; complete loss of 13 was observed in 73% of cases, whereas 27% had interstitial deletions. In addition, 13/13q deletions occurred in 75% of nonhyperdiploid myeloma but only 39% of the hyperdiploid had 13/13q deletions. Translocations affecting 14q32/IGH region was seen 40 cases; t(11;14)(q13;q32) in 17 cases, t(14;16)(q32;q23) and t(8;14)(q24;q32) in three cases each, and t(6;14)(p21;q32) and t(1;14)(q21;q32) in two cases each. The remaining 14q32 translocations had various t(v;14) partners or of an undetermined origin. Remarkably, the 14q32/IGH translocations were less frequent in the hyperdiploid karyotypes than the nonhyperdiploid karyotypes (17 vs. 63%). Fourteen cases showed break at 8q24/CMYC site; seven of those had Burkitt s-type translocations. Our results revealed that conventional cytogenetics remains an important tool in elucidating the complex and divers genetic anomalies of MM. Cytogenetics identifies two distinct groups of MM, hyperdiploid and nonhyperdiploid, and establishes the presence of prognostic chromosomal markers such as 13/13q, 17p, 8q24, and 16q aberrations. Am. J. Hematol. 82: , VC 2007 Wiley-Liss, Inc. Introduction Multiple myeloma (MM) is a differentiated B-cell neoplasm characterized by dissemination of malignant plasma cells that produce monoclonal immunoglobin (Ig) or Ig fragments (M protein). In the United States, the annual incidence of MM is 30 per 1,000,000 with a ratio in the African descent doubles of the Caucasian [1 3]. The higher level of physiological immunoglobulin in the Blacks relative to Whites may reflect a larger B-cell population at risk of neoplastic changes. MM is usually incurable, with a median survival of 3 years, and 10% of patients survive more than 10 years. The disease has a variable clinicobiological features, genetic abnormalities, and response to treatment [4]. However, the clinical staging and other parameters such as serum albumin, B2 microglobulin, and plasma cell labeling index remain the most useful parameters in predicting the prognosis in MM [5,6]. Conventional cytogenetics in MM is difficult as terminally differentiated plasma cells have a low proliferation activity, and the disease can be patchy in the bone marrow [7,8]. On the basis of banded chromosomes, abnormal karyotypes are found in 30 50% of cases; more often in advanced stages than in newly diagnosed patients [7 9]. Successful cytogenetic analyses have been achieved in a number of patient series. These and other related studies have demonstrated that MM has a highly complex karyotype in the majority of patients. These include a hyperdiploid clone characterized by a distinct pattern of chromosome gains, and a hypodiploid clone often accompanied by 13/13q deletion [9 11]. Recent advances in molecular cytogenetic techniques particularly interphase fluorescence in situ hybridization (FISH) and comparative genomic hybrid- VC 2007 Wiley-Liss, Inc. ization (CGH), have demonstrated that chromosome aberrations can be found in the majority of MM cases [12 14]. The most significant FISH marker is monosomy 13 or 13q14 deletion which has been reported in a frequency of 30 50% in MM [15]. In addition, several studies have revealed that 13/13q14 deletions confer a negative impact on prognosis of MM [11,15,16]. As a result of these findings, FISH assay has become the method of choice in detection of 13/13q deletions in newly diagnosed cases and monitoring the clinical course of MM. In this study, we evaluated a series of 120 MM cases with abnormal karyotypes that displayed clonal aberrations. We identified the most common chromosomal abnormalities in our MM population, as well as, report the incidence of several abnormalities suggested in the literature to have prognostic implications in this malignancy. Results A series of 120 cases with the diagnosis of MM have been identified with abnormal karyotypes. Sixty one patients were female and fifty nine were male; all were *Correspondence to: Anwar N. Mohamed, MD, Professor of Pathology, Wayne State University School of Medicine, Detroit Medical Center, 4727 St. Antoine, Detroit, MI amohamed@dmc.org Received for publication 13 March 2007; Revised 22 April 2007; Accepted 23 April 2007 Am. J. Hematol. 82: , Published online 25 July 2007 in Wiley InterScience ( com). DOI: /ajh American Journal of Hematology

2 adults ranging in age from 39 to 86 years. Most patients had clinical stage II or III at diagnosis. Fifty six patients were newly diagnosed and the remainder were previously diagnosed and treated for MM. Clonal chromosomal abnormalities were detected by G- or Q-banding in all 120 cases. The karyotypes of these cases were classified into three categories according to the modal chromosome number of the malignant cell population (Table I). Hyperdiploid with chromosome number > 47 were seen in 77 cases (64%), hypodiploid 45 in 30 cases (25%), and pseudodiploid with 46 chromosomes but, with structural and numerical aberrations in 13 cases (11%). Most cases presented with multiple and complex chromosomal abnormalities with the exception of 4 cases; 2 cases had two abnormalities and the other 2 had one. Recurring numerical gains and losses detected in the entire 120 cases are summarized in Fig. 1. The most common whole chromosome gains were 115 (n 5 63, 53%), 19 (n 5 58, 48%), 13 (n 5 55, 46%), 119 (n 5 54, 45%), 111 (n 5 49, 41%), 17 (n 5 46, 38%), 121 (n 5 47, 39%), and 15 (n 5 44, 37%). As expected, the frequencies of these chromosome gains were higher in the hyperdiploid population than the entire cases; chromosomes 15 (76%), 9 (69%), 3 (68%), and 19 (65%). Excess of chromosomes 15, 9, and 3 accounted for 97% of all gains in the hyperdiploid clone. No gain of chromosome 8 was observed in the entire cases. Losses of whole chromosomes in this series were also nonrandom; 213 (n 5 45, 38%), 28 (n 5 30, 25%), and 214 (n 5 23, 19%), and, 222 (n 5 25, 21%). Losses of X/Y chromosomes were more frequent than gains seen (n 5 48, 40%) cases. In our series, nonhyperdiploid karyotype (pseudodiploid and hypodiploid) was found in 43 of 120 (36%) MM cases with abnormal karyotype. The majority had chromosomes with the exception of one case (Case 81) showed a severe hypodiploid karyotype containing chromosomes. Among the hypodiploid population, loss of chromosome 13 was more evident, seen in 23/30 (77%) cases while in the hyperdiploid cases under-representation of chromosome 13 was seen in 20/77 cases (26%). Structural chromosomal abnormalities were noted in most cases (n 5 116, 97%). Rearrangements of chromosome 1 was the most frequent (n 5 77, 64%), and some cases showed more than one abnormality of chromosome 1. The most common aberrations of chromosome 1 were partial deletions of 1p (n 5 49, 41%), resulted from simple deletions or unbalanced rearrangements, followed by duplication of 1q (n 5 46, 38%). The later abnormality caused either from simple partial duplications of 1q or from unbalanced translocations leading to gain of 1q (Table II). The unbalanced der(16)t(1;16) abnormality was observed in 6 cases. The translocation had variable breakpoints; 1q12-25 and 16q12-24, in all resulted in partial gains of 1q and partial losses of 16q. Of the six patients with t(1;16); three were relapsed after chemo and radiation therapies, two relapsed after autologous bone marrow transplant, and one patient progressed from monoclonal gammopathy (MGUS). Other frequently occurring structural changes led to deletions of 6q (n 5 39, 33%), 5q (n 5 18, 15%), 8p (n 5 15, 13%), 13q (n 5 17, 14%), 9p (n 5 13, 11%), 17p (n 5 18, 15%), and 22q (n 5 8, 7%). Of the 18 cases with del(17p); 11 cases (26%) had nonhyperdiploid karyotypes while in the hyperdiploid population del(17p) was noted in (7/77 cases) less than 1%. Homogenously staining region (hsr) was observed in one case (Case 29). Forty three translocations involving the 14q32/IGH region were observed in 40/120 (34%) cases; three of these cases contained two different 14q32 translocations in the same karyotype, one case showed t(11;14) and t(1;14), one had t(14;16), and add(14)(q32), while the third case had t(14;16) and t(2;14) (Table I). Only 13/77 (17%) hyperdiploid MM contained 14q32 translocations while the remaining 27 (63%) cases with 14q32 translocations had a nonhyperdiploid karyotypes. Of the 43 14q32 translocations; t(11;14)(q13;q32) was the most frequent seen in 17 (40%) cases. The karyotypes of the t(11;14)-bearing MM was nonhyperdiploid in 16 cases while one was classified as a hyperdiploid. In one case, the t(11;14) translocation was a variant three-way t(1;11;14) (Case 80). Targeted FISH on t(1;11;14) metaphases confirmed the presence IGH/ CCND1 gene fusion. The karyotype of another case (Case 84) showed t(11;18)(q13;q22); however, FISH revealed no IGH/CCND1 gene fusion, instead 3 signals for CCND1 were noted, one of them hybridized on the translocated 18. This suggested that t(11;18) was a variant t(11;14) translocation. The Burkitt s type translocation t(8;14)(q24;q32) and t(14;16)(q32;q23) were seen in three cases each, and t(1;14) and t(6;14)(p21;q32) were observed in two cases each. FISH revealed IGH/CMYC fusion signals in the three cases bearing t(8;14). One additional case, the 14q32 translocation was suspected by G-banding. Targeted FISH using the break-apart IGH probe revealed a green signal on 4p, and one orange signal on 14q confirming the pattern of t(4;14) translocation. Add (14q32) was found in 11 cases, where the donor chromosomes could not be identified mostly because of complex karyotypes. The remaining 14q32 translocations each had different partner chromosomes (Table I). Other significant translocation affecting band 8q24, the site of CMYC oncogene, was observed in 14 cases (Table II). Seven of them contained the Burkitt s-type translocations; t(8;22)(q24;q11.2) in four cases and t(8;14)(q24;q32) in three cases (mentioned earlier). The other seven cases had a t(1;8)(p21;q24) in two cases, t(6;8)(p21;q24) in one case, and add(8q24) in the remaining four. The karyotypes of five cases with 8q24 translocations contained also 14q32 translocations. Discussion Metaphase chromosome analyses on a large series of MM have been limited to a few reports because of inability of plasma cells to divide in culture [7 9]. Despite that, chromosome analysis provides a broad image of chromosome aberrations in proliferating plasma cells from patients with MM. FISH, on the other hand, has greatly enhanced the detection rate of genetic abnormalities in MM. However, FISH provides limited information [13,14,17,18]. In the present study, we reviewed 120 cases of MM with abnormal karyotypes. Approximately, half of the cases were newly diagnosed. According to their chromosome number, two groups were identified; a hyperdiploid group with chromosome number greater than 46 was seen in 64% of cases and the second group (36%) had a nonhyperdiploid karyotype (Table I). The majority of the hyperdiploid group had chromosome number in the 50. The karyotypes of the hyperdiploid clones demonstrated a characteristic pattern of multiple trisomies with remarkable additions of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Chromosome15 was commonly present in a tetrasomic pattern. Gain of chromosomes 15, 9, and 3 was found in 97% of the hyperdiploid cases. Therefore, a combination of three probes CEP 3, 9, and 15 can detect the majority of the hyperdiploid MM. Hyperdiploidy in MM is a dominant finding, being reported with frequency vary from 40 65% of cases [9,10,18]. In a study of 280 MM cases, hyperdiploidy was found in 44% of cases with abnormal karyotypes a frequency lower than in our series [9]. A similar pattern of chromosome gains has been observed by other previous studies [9,10,19,20]. We American Journal of Hematology DOI /ajh 1081

3 TABLE I. Abnormal Karyotypes of 120 Cases With Multiple Myeloma Hyperdiploid Age/sex Karyotypes 1 81F 53 57,XX,del(1)(p36),12,del(3)(p21)x2,15,add(5)(q35),17,19,111,add(13)(q21),121,122,13 4mar[cp13]/46,XX[7] 2 51F 48,XX,22,13,add(3)(q21),27,del(8)(p11.2),der(9)t(1;9)(q12;q33),111,111,add(16)(q13),i(17)(q10),119[3]/46,XX[17] 3 51M 53 57,Y,der(X)t(X;8)(p22;q13),11,dup(1)(q23q44),13,14,t(5;11)(q13;p15),17,28,19,add(9)(p24),110,111,add(14)(q32),118,119,220,121,1mar[cp24]/46,XY[6] 4 71F 54,X,2X,13,17,19,111,111,115,118,119,121[4]/46,XX[16] 5 42F 50 52,X,add(X)(q28),11,13,19,ins(11;1)(q13;p21p36),der(12)t(8;12)(q11.2;p12),115,216,118,1mar[cp11]/46,XX[24] 6 54F 53,XX,11,der(1;13)(q10;q10),13,16,17,19,213,118,119,121[4]/46,XX[16] 7 63F 64 67<3n>, XX,2X,del(1)(p22),1der(1;19)(q10;p10)x3,24,17,210,213,214,216,220,222[16]/46,XX[9] 8 43F 48 49,X,2X,der(1;9)(q10;p10),t(1;8)(p21;q24),13,add(5)(q35),216,118,119,121[8]/46,XX[22] 9 67F 48,X,2X,111,213,115,115,119[9]/46,XX[23] 10 56M 61 63<3n>,XX,2Y,del(1p22),add(3q)(q29),15,26,28,212,214,216,217,119,220,222[6]/46,XY[15] 11 63M 48 49,XY,add(1)(p36),t(13;15)(q34;q22),add(14)(q32),115,add(17)(q25),der(20)(1;20)(q12;p13)x2,121,11 mar [cp20] 12 56M 53, X,2Y,13,15,15,t(6;20)(q15;q13),19,213,115,der(15)t(1;15)(q12;p13),118,119,121[cp8]/46,XY[12] 13 55M 51 53,XY,13,15,i(8)(q10),19,111,115,115,119[cp7]/46,XY[19] 14 86M 48,XY,dup(1)(q21q32),115,119[cp19]/46,XY[1] 15 61F 73 75<3n>,XX,2X,11,dup(1)(q12q44)x2,13,15,26,17,28,19,210,add(10)(p13)x2,111,213,214,115,117,118,119,220,121,222[cp9]/46,XX[11] 16 62M 50,XY,13,19,111,115[cp2]/46, XY[18] 17 54F 52,X,del(X)(q24),der(1)del(1)(p22)dup(1)(q21q32),add(2)(p25),13,add(3)(q23),16,17,17,del(7)(q32),28,19,del(9)(q33)x2,del(12)(q22),del(13)(q14q31),add(17) (q25),add(18)(q23),121,121[cp14]/46,xx[4] 18 71M 70 75<3n>,X,2Y,i(1)(q10)x2,13,24,15,del(6)(q22)x2,17,19,111,213,214,119,120[cp18]/46,XY[2] 19 57M 50 52,XY,del(1)(p21)x2,13,15,16,del(6)(q13q27),der(7)t(7;11)(p22;q11),213,115,216,117,119,119,222 [cp18]/46,xy[2] 20 69F 73 76<3n>,XXX,add(1)(p22),dic(1;2)(p13;q37),add(2)(q37),13,13,15,add(5)(q31),16,28,210,111,112,213,115,117,119,121[cp20] 21 77F 52,XX,13,15,16,del(6)(q15q25),i(8)(q10),19,115,115,add(15)(p11.1),216,del(17)(p11.2),add(19)(q13),121[cp7]/46,XX[13] 22 66F 52 55,X,2X,t(2;12)(p12;q24),15,del(5)(q12q14),16,der(6;14)(p10;q10),17,19,111,115,115,t(16;20)(p13;q13),117,119,220,121,add(21)(p11)[6]/46,XX[14] 23 61F 51 52,X,del(X)(q22),del(1p13p36),13,15,del(5)(q22),del(6)(q16),del(9)(p21),111,add(11)(p15),add(14)(q32),115,115[cp12]/46,XX[8] 24 42F 57,XX,del(1)(p22p36),12,add(2)(q37),13,add(3)(q26),17,del(7)(q22),19,add(9)(p24),111,115,115,119,120,121,121[cp6]/46,XX[14] 25 55M 49 51,X,2Y,der(1)t(1;13)(p36;q14),del(6)(q21),28,del(10)(q22q26),add(11)(p15),add(12)(p13),214,add(14)(q32),der(16)t(Y;16) (q12;p13), 118,119,121,121,11 3mar[cp12]/46,XY[4] 26 71M 50 51,XY,del(1)(q21),der(1;7)(q10;p10),del(2)(p16),add(5)(q12),15,add(6)(p24),17,111,del(11)(p15),add(12)(p13),214,115,119, der(19)t(18;19)(q11.2;q13),221,del(22)(q12)[,11 2mar[cp20] 27 83F 71 74<3n>,XX,2X,11,del(1)(p22),add(1)(q21),13,del(6)(q15q27),28,19,212,115,216,217,118,119,121[cp2]/46,XX[18] 28 52M 51,XY,del(5)(q13q31),17,110,111,del(11)(p14p15),del(12)(p12),add(13)(q34),115,add(17)(p11.2),119,121,121[cp19]/46,XY[1] 29 63F 47 50,XX,t(1;5)(p22;q24),dup(2)(p22p25),del(3)(p14p25),17,add(9)(p22),add(9)(p23),1add(9)(q12),del(11)(p11.2p15),del(13)(q12q31)x2,213,216, add(17)(q25),add(19)(p13),der(21)t(9;21)(q12;p12),del(22)(q12),hsr(11)(q25),12 3mar[cp15]/46,XX[5] 30 65F 50 52,X,2X,add(1)(p36),del(1)(p21)x2,13,25,17,add(8)(p21),19,111,add(11)(p15),213,115,der(16)t(5;16)(q13;q24),119,121[cp17]/46,XX[3] 31 60M 58 60<2n>,Y,add(X)(q26),1Y,add(2)(q37),13,add(3)(q29),14,add(4)(q35),15,17,del(7)(p12),19,111,der(11)t(9;11)(q11;q25),112,add(12)(p13), 213,214,115,add(17)(p13),118,118,119,121,12 5mar[cp15]/46,XY[5] 32 54M 48 49,X,2Y,t(1;4)(p12;q33),del(1)(p21),15,19,del(13)(q13),115,121[cp11]/46,XY[9] 33 43F 52,XX,13,17,19,del(9)(p21),111,115,1mar [cp5]/46,xx[15] 34 55M 55,X,2Y,add(1)(p22),add(2)(p23),13,del(4)(q31),15,add(5)(q22),19,111,add(11)(q23),del(12)(p11;p13),t(14;16)(q32;q23),115,115,217,118,119, add(19)(q13),120,121[cp17]/46,xy[3] 35 56M 48,XY,13,26,19,del(13)(q12q22),der(16)t(1;16)(q21;p13),217,119[cp4]/46XY[16] 36 46M 51,X,2Y,dup(1)(q21q32),der(1;8)(q10;q10),del(8)(p12),13,del(8)(q22),del(9)(q22),111,der(11)t(9;11)(q13;q23),214,115,216,118,121,12 3mar[cp7]/46,XY[13] 37 83M 47,XY,del(6)(q22),add(7)(p22),28,der(8;14)(q10;q10),19,214,add(16)(q24),119,add(22)(p11.2),der(22)t(1;22)(p13;q12),1mar[cp15]/46XY[5] 38 64F 72<3n>,XX,1X,1X,t(11;14)(q13;q32)x2[5]/46,XX[17] 39 61F 49,X,2X,t(1;8)(p22;q24),15,del(14)(q22),115,115,add(22)(q11),1mar[cp20] (Continued) 1082 American Journal of Hematology DOI /ajh

4 TABLE I. Continued Hyperdiploid Age/sex Karyotypes 40 65F 48,X,2X,13,add(3)(p26),14,17,add(7)(q36),19,del(12)(p12),del(13)(q12q21),222[cp3]/46,XX[17] 41 47F 54,XX,15,t(6;14)(p21;q32),17,19,19,111,119,121,121[6]/46,XX[14] 42 54M 54,XY,13,15,19,19,115,115,del(18)(q22),119,der(20)t(1;20)(q21;q13.3),121[cp3]/46,XY[13] 43 53F 53 54,X,2X,add(1)(p13)x2,13,16,add(7)(p15),t(8;22)(q24;q11.2),111,del(13)(q12q21)x2,115,115,216,add(17)(q25),add(18)(q23), 121,121,1r,12mar[cp13]46,XX[6] 44 54M 55 56,XY,add(2)(p23),13,14,15,add(5)(q35),17,28,19,19,111,115,119,der(20)t(8;20)(q11.2;q13.3)x2,121[cp10]/46,XY[10] 45 66F 77<3n>,XXX,1X,12,add(2)(q31),13,add(4)(p16)x2,15,16,17,28,19,110,111,del(11)(p11.2p13)x2,213,214,115,216,der(16)t (1;16)(q12;q24)x2,118,119,121,222[cp6]/46,XX[14] 46 59M 54,XY,13,del(3)(p13),15,add(5)(q35),add(6)(p25),17,19,del(9)(p22)x2,115,add(18)(q23),add(19)(p13),12mar[cp9]/46,XY[11] 47 66F 55 57,XX,13,13,t(4;14)(q23;q32),15,15,17,19,111,115,117,119,119[cp3]/46XX[17] 48 64F 53 55,2X,add(X)(p22),del(1)(q22),der(5)t(5;13)(q35;q14),16,add(6)(q25),17,28,19,der(9)t(9;13)(q34;q14),add(12)(p12), 213,115,115,118,119,add(19)(q13),121[cp13]46,XX[7] 49 61M 58,XY,12,add(2)(p12),der(2;17)(q10;q10),1der(3;8)(q10;q10),add(3)(p12),13,15,16,17,28,19,19,111,115,115,119,120,121[cp2]/46,XY[18] 50 63M 56,XY,12,13,15,16,t(8;14)(q24;q32),19,111,115,der(15)t(1;15)(q12;q26),117,119,der(19)t(1;19)(q12;q13),120[cp12]46,XY[8] 51 39F 50,X,2X,13,add(3)(q27),24,15,der(5)t(1;5)(q21;q35),16,add(6)(q27),17,del(7)(q32),t(8;22)(q24;q11.2),del(13)(q12q21),add(14)(q32),115,119[cp13]/46,XX[7] 52 57F 56 58,XX,del(2)(p16),13,15,28,19,19,111,111,del(13)(q14),115,add(15)(p11.2),add(18)(p11)x2,119,der(19)t(7;19)(p11;q13)x2,121[cp7]/46,XX[13] 53 53M 55,XY,11,13,del(3)(p21),add(3)(q13),15,del(6)(p22),17,28,19,19,del(9)(p13),111,del(13)(q12q34),114,115,119,121,222[9]/46,XY[11] 54 64F 51 54,XX,1X,del(1)(p13),13,15,16,17,28,19,111,del(11)(p11.2),214,115,119[cp7]/46,XX[13] 55 75M 57 59,XY,12,13,15,16,17,28,19,der(9)t(1;9)(p24;q21),111,111,113,114,115,115,119,121[cp6]/46,XY[15] 56 58M 50,XY,15,del(6)(q15),add(6)(q25),der(6;8)(p10;q10),28,19,add(12)(p13),115,115,121,add(22)(q13)[cp4]/46,XY[16] 57 63F 51,XX,13,17,19,213,115,115,der(16)t(1;16)(q25;q24),121[cp4]/46,XX[16] 58 54F 54 56,XX,21,add(1)(q22),15,26,17,add(7)(p22),19,add(9)(p21),111,115,115,121,121,1r,12 3mar[cp4]/46,XX[16] 59 54M 48 49,X,2Y,11,del(1)(p13),inv(1)(p13q34),del(5)(q31q34),16,del(6)(q15q27),111,add(19)(q13),121[cp4]/46,XY[16] 60 80F 59,X,2X,1der(2)t(1;2)(q12;q21),13,15,1der(6)t(4;6)(q12;q13),17,t(8;22)(p12;q12),19,111,115,115,117,118,119,121[cp15]/46,XX[5] 61 70F 51 54,X,del(X)(q13),11,del(1)(p13),13,24,15,del(6)(q15),17,19,111,115,115,121[cp3]/46,XX[9] 62 43M 54,X,2Y,12,15,17,19,19,111,115,115,119,119,222[2]/46,XY[cp18] 63 58M 52,X,2Y,13,15,17,del(8)(p11.2),19,111,add(12)(q24),115,add(15)(11.2),add(16)(q24),117,119,222[cp7]/46,XY[13] 64 42M 56,XY,13,15,16,t(8;14)(q24;q32),19,111,115,115,119,120,121[7]/46,XY[14] 65 59M 63 65<3>,X,2Y,der(1)t(1;8)(q10;q10),15,inv(8)(p22q11.2),dup(11)(q13;q25),213,214,115,216,119,220,222[cp5]/46,XY[15] 66 65M 48,X,2Y,t(6;13)(p23;q21),28,19,19,add(9)(p22),111,add(12)(p13),213,115,der(15)t(15;15)(q26;q12),del(17)(p12),119,add(19)(p13),222[cp18]/46,XY[2] 67 61M 51,XY,del(1)(q12),del(1)(p13)x2,1der(1;21)(q10;q10),der(6)t(1;6)(p13;q27),16,17,28,111,112,213,115,1mar[cp16]/46,XY[4] 68 80F 51 53,X,2X,1i(1)(q10),1del(1)(p12p36),15,del(5)(q11.2q33),16,17,19,115,der(16)t(1;16)(q31;q23),del(17)(p12),add(20)(q13)[cp3]/46,XX[17] 69 55M 56 58,X,2Y,12,13,15,del(6)(q13q23),x2,17,dup(8)(q13q22),19,111,115,115,119,121,121[cp3]/46,XY[17] 70 80M 56,XY,13,15,16,17,19,115,115,117,119,121[cp7]/46,XY[13] 71 65M 55,XY,1der(1;15)(q10;q10),12,13,17,28,19,111,115,115,117,119[cp3)/46,XY[17] 72 58F 50,XX,13,15,16,add(6)(q27),19,dup(11)(p11.2p15),i(15)(q10),add(17)(p13)[2]/46,XX[18] 73 69M 49,XY,der(1;15)(q10;q10)x2,13,der(7)t(1;7)(p13;p15),t(8;14)(q24;q32),19,213,214,119,add(19)(p13)[cp12]/46,XY[8] 74 57M 56,XY,1Y,del(1)(p13p22),15,16,der(6)t(1;6)(q21;q13),17,19,der(9)t(1;9)(q12;q34),111,213,add(14)(q32),115,115,119,121,121[6] /46,XY[14] 75 88M 52,X,add(Y)(q12),13,15,16,17,add(8)(q24),add(8)(p21),19,213,115,119[2]/46,XY[18] 76 64F 51,XX,13,15,del(6)(9q13),28,19,111,213,115,115,der(16)t(1;16)(q25;q23),119[cp4]/46,XX[16] 77 69M 50,XY,del(1)(p34),add(2)(q37),13,i(3)(p10),add(6)(q21),19,111,119,der(19)t(1;19)(q12;p13),121[cp8]/46,XY[12] Hypodiploid 78 59F 42 44,XX,del(1)(p22p32),der(12)t(1;12)(q21;q13),213,add(14)(q32),222[cp12]/46,XX[8] 79 49M 42, X,2Y,der(1)dup(q21q42)del(p22p32),add(7)(p22),der(9)add(p13)add(q34),t(10;14)(q24;q32),212,213,214,add(17)(p12),del(20)(q12)[18]/46,XY[2] 80 75F 44,X,2X,add(1)(p13),t(1;11;14)(p36;q13;q32),213[16]/46,XX[3] (Continued) American Journal of Hematology DOI /ajh 1083

5 TABLE I. Continued Hyperdiploid Age/sex Karyotypes 81 52F 28 31<1n>,X,13,17,del(7)(p15),19,111,112,115,118,119,i(21)(q10)[cp18]/46,XX[2] 82 54M 42,X,1X,2Y,22,213,add(14)(q32),222,add(22)(q11)[7]/46,XY[11] 83 47M 44, X,2Y,del(1)(p12p31),del(6)(q15q25),t(11;14)(q13;q32),213[15]/46,XY[5] 84 79F 44 45,XX,der(1;21)(q10;q10),del(3)(q21),t(3;14)(p21;q32),add(8)(q24),t(11;18)(q13;q22),213,del(14)(q24),der(19)t(1;19)(q12;p13),222[12]/46,XX[8] 85 56M 42,X,2Y,del(6)(q21),27,der(7)t(7;8)(q36;q13),28,add(8)(q24),del(12)(q21),add(12)(q24),213,add(15)(p11),del(17)(p12),add(21)(p13)[cp15]/46,XY[7] 86 61M 43,XY,i(1)(q10),del(2)(p32),del(6)(q15),213,214,216, 121,222[7]/46,XY[7] 87 60F 42,X,2X, t(1;15)(q12;q26),210,t(11;14)(q13;q32),213,add(17)(p12),218,220,121[9]/46,xx[7] 88 56F 42 43,X,2X,del(1)(p21),der(1;2)(q10;q10),28,add(14)(q32),add(17)(p12),218,add(21)(p11),222[cp11]/46,XX[3] 89 49F 44,XX,del(1)(p22p35),22,der(2)t(1;2)(q12;p25),t(7;14)(q22;q32),t(8;12)(q24;p12),t(8;22)(q24;q11.2),del(12)(p11.2p12),213,der(18;21)(q10;q10),del(22)(q11)[cp10]/46,XX[10] 90 52F 43,X,2X,1add(1)(p36),dic(1;17)(p36;p13),add(1)(p32),add(6)(q23),213,214,217[cp20] 91 74F 44,X,2X,21,inv(2)(p13;q37),der(5)t(1;5)(q21;q35),der(6)t(1;6)(q21;q21),111,del(11)(q21),213,214,der(17)(1;17)(p31;p13),118,220[cp6]/46,XX[14] 92 56F 42 44,X,2X,der(1;5)(q10;q10),del(6)(q13),212,213,217,r(17)(p13;q25)[cp5]/46,XX[16] 93 50M 42,X,2Y,i(6)(p10),t(11;14)(q13;q32),213,214,add(17)(p12),222[cp3]/46,XY[17] 94 64F 43 44,X,2X,t(1;11)(q12;q23),inv(2)(p13p24),add(4)(q35),add(6)(q34),add(9)(q34),t(11;14)(q13;q32),213,222[cp3]46,XX[18] 95 78F 45,X,2X,del(6)(q21q25),19,der(9;15)(q10;q10),del(10)(q22),t(11;14)(q13;q32),213,der(14)t(1;14)(q21;q32),del(17)(p12)[cp15]/46,XX[5] 96 62M 42 44,XY,add(1)(p32),22,add(4)(q33),add(6)(q21),del(8)(p21),add(11)(q23),del(11)(q21q23),del(12)(p12),add(12)(p12),213,214,der(15)t(1;15)(p10;q10), der(16)t(1;16)(q25;q24),217,add(20)(q13),11 2mar[cp14] 97 52F 45,X,2X,i(6)(p10),der(6)t(1;6)(p25;q12),t(8;22)(q24;q11.2),19,del(13)(q12q21),der(14)t(1;14)(q21;q32)[cp2]/46,XX[18] 98 67M 43,XY,dup(1)(q21q32),t(1;11)(q12;p15)add(1)(p36),t(2;4)(p21;q23),add(3)(q26),der(6)t(6;8)(p25;q12),28,add(12)(q24),del(13)(q14q34),der(14)t(6;14)(p21; q32),inv(16)(p11q24),add(18)(q23),add(19)(q13),220,222[cp11]/46,xy[9] 99 63M 45,XY,der(1;19)(q10;p10),del(3)(p12p21),del(6)(q13),17,der(8)t(8;22)(p11.2;q12),t(11;14)(q13;q32),del(13)(q14q22),216,222[cp4]/46,XY[11] M 42,X,2Y,del(1)(p13p22),der(1;7)t(1;?:7)(p13;?p22),del(2)(q31q37),der(7;9)(p15;q34),28,29,der(10)(8;10)(q11.2;p12),213,add(15)(q26),der(20)t(1;20)(q12;q13.3)[cp13]/46,XY[7] F 41,X,2X,del(1)(p13p36),28,213,add(13)(q31),214,222[4]/46,XX[16] F 45,X,2X,t(11;14)(q32;q13)[cp3]/46,XX[27] M 44,X,2Y,28,213,214,121,1mar[cp6]/46,XY[14] M 44,XY,der(3)t(1;3)(q21;q29),t(4;14)(p16;q32),der(9)t(1;9)(q12;q34),210,213[19]/46,XY[1] M 44,XY,der(1;6)(q10;p10),del(5)(q31),del(6)(q13q23),del(8)(p21),add(8)(q24),del(10)(q24),t(11;14)(q13;q32),217,add(17)(q25)[cp6]/46,XY[17] F 44,XX,21,26,der(8)t(1;8)(q21;p23),der(8)t(6;8)(p12;q24),der(11)t(11;15)(p11.2;q11.2),del(11)(q22),del(12)(q23),213,add(15)(q21),i(16)(p10),add(17)(p13), 218,der(18)t(1;18)(q12;p11.2),121,121,121,222[cp5]/46,XX[16] F 45,XX,der(1;15)(q10;q10),t(2;14)(p12;q32),del(3)(q23),213,t(14;16)(q32;q23)[cp12] Near-diploid M 46,XY,del(6)(q13q23),t(7;15)(q22;q26),del(17)(p11.2)[12]/46,XY[4] M 46,XY,13,t(3;15)(q29;q14),del(3)(q24),16,del(6)(q15),der(6)del(p24)del(q21),add(10)(q22),t(11;14)(q13;q32),214,add(16)(q24),add(21)(p11.2),222[cp13]/46,XY[16] M 46, XY,del(1)(p31),del(3)(p12),add(14)(q32)[3]/46,XY[17] F 46,XX,add(1)(p22),der(5)t(1;5)(q13;q31),del(3)(p24),25, 28,214,add(17)(p13),add(19)(p13),11 4mar [cp14]/46,xx[6] F 46,XX,del(6)(q15q25),19,del(13)(q12q14),t(14;16)(q32;q23),der(18)t(1;18)(q12;q22),der(22)t(11;22)(p11;q13)[13]/46,XX[7] F 46,XX,28,t(11;14)(q13;q32),del(13)(q12;q21),1der(14)t(11;14),add(15)(q22),del(16)(p11)[3]/46 XX[18] F 46,XX,del(6)(p24),der(14)t(11;14)(q13;q32)[cp20] F 46,XX,t(7;14)(p22;q32)[2]/46XX[19] F 46,XX,dup(1)(q21q24)[4]/46,XX[16] F 46,X,add(X)(q22),11,add(1)(p22),t(11;14)(q13;q32),213,der(20)t(13;20)(q21;q13)[7]/46,XX[13] F 46,X,t(X;9)((q26;q12),add(1)(p36),t(2;12)(q21;p13),dup(3)(p23p26),del(6)(q21),17,del(8)(p22),add(9)(q33),del(10)(p13),213,115,del(16)(p11.2),217,218,121[cp8]/46XX[17] M 46,X,2Y,del(5)(q23q35),der(7)t(1;7)(q12;p12),add(10)(q26),111,der(11)t(1;11)(q12;p15),del(13)(q12q21),der(14;21)(q10;q10),115,der(15;21)(q10;q10),216,217,119[cp8]/46,XY[12] M 46,XY,t(11;14;?)(q13;q32;?)[2]/46,XY[18] 1084 American Journal of Hematology DOI /ajh

6 TABLE II. The Frequency of Structural Abnormalities in 120 Multiple Myeloma Cases Abnormality Total number % Del 1p Dup 1q Del 5q Del 6q Del 8p T 8q Del 9p Del 11p 10 8 Del 11q 7 4 Del 13q T 14q Del 16q Del 17p Del 22q 8 7 Figure 1. Numerical abnormalities in 120 MM cases. observed that the IGH/14q32 translocation is infrequent in the hyperdiploid group MM seen only in 17%, and only one of those had t(11;14)(q13;q32). At Mayo Clinic, abnormal karyotypes and FISH-detected IGH translocations revealed that MM patients with IGH translocations were more likely to be nonhyperdiploid [21]. In a study of a series of 55 MM cases with abnormal karyotype, Smadja et al. found hyperdiploidy in 29 cases (53%), and only two of these cases displayed a 14q32/IGH translocation by conventional banded chromosomes [22]. Among the 27 hyperdiploid MM without obvious 14q32 translocation, FISH detected two cases carrying t(4;14). Accordingly, Samdja et al. concluded that 14q32 translocation is infrequent in hyperdiploid MM [22]. Therefore, our results and those by others suggest that 14q32 translocation is unevenly distributed among the MM cases being infrequent in the hyperdiploid clones [21 24]. In hematological malignancies, hyperdiploidy is a common cytogenetic finding in pediatric acute lymphoblastic leukemia (ALL) seen in approximately one third of cases. A clear picture of nonrandom pattern of chromosome gains 4, 6, 10, 14, 17, 18, 21, and X are established for ALL [25,26]. Obviously, the pattern of trisomies in MM and ALL are different with the exception of chromosome 21 being common in both. This suggests that the cumulative dosage and expression effects of groups of genes on multiple different chromosomes can define the pathobiological routes of these two lymphoid leukemias, ALL and MM. Similar to the hyperdiploid ALL, hyperdiploid MM have been associated with a favorable response to therapy and prolonged survival [11,27]. In our series, nonhyperdiploidy was found in 36% of MM cases with abnormal karyotype. In this group, structural aberrations were predominant, and greater in number than in the hyperdiploid group. The 14q32 translocation was found in 27/43 (63%) of cases; 16 (59%) of those had t(11;14) translocation. By use of conventional cytogenetics, Samdja et al. found hypodiploidy in 47% of MM cases, a higher frequency than seen in this study. However, comparable to our observation, 60% of their hypodiploid cases contained 14q32 translocation [22]. A recent study by Wuilleme et al., flowcytometry (FCM) was utilized to measure the DNA content in a large series of MM cases, and further they extended their analysis using FISH probes to validate the sensitivity of this approach [24]. Hyperdiploidy was detected in 50% of their cases, and in addition they found most recurrent 14q32 translocations occurred in the nonhyperdiploid clones. Wuilleme et al. concluded that the combination of FCM and FISH would permit the detection of ploidy in MM independent of plasma cell proliferation and without the need of karyotyping. However, the prognostic value of this approach still needs to be validated to address whether it is associated with the proliferation activity or the DNA index of plasma cells. Loss of chromosome 13/13q has been associated with an adverse clinical outcome in MM [15,16]. FISH is able to detect 13/13q deletions 2 3 times more than metaphase cytogenetic. Furthermore, studies have shown that survival of patients with 13/13q deletions was not significantly different when detected by karyotype or interphase FISH [28]. In contrast, MM patients with disomy 13 by either technique experienced prolonged survival. In our series, 52% of cases contained 13/13q deletion; 73% of those had complete loss of chromosome 13, while the remaining 27% cases had interstitial deletions of 13q. In addition, the distribution of the 13/13q deletions was unequal; in the nonhyperdiploid MM, 13/13q deletions occurred in 75% of cases, but only 39% of the hyperdiploid had 13/13q deletions. These results appeared to support other previously published studies which showed a strong association between 13/13q deletion and hypodiploidy. Both hypodiploidy and 13/13q deletion bearing MM cases appear to have a poor prognosis [11,22,27 29,38]. However, it remains uncertain if hypodiploidy and loss of chromosome 13 are independent risk factors or have additive effects. Presently many clinical trials are using FISH to determine the status of chromosome 13 for the consideration of risk stratification. We found translocations affecting the IGH/14q32 in 34% of cases. These results are relatively in accordance with those reported by Calasanz et al. and Smadja et al., but not with recent studies reporting over 50% detection rate by use of interphase FISH [9,13,23,30]. Five recurrent chromosomal partners are involved in 14q32 translocations in MM including 11q13, 4p16, 6p21, 16q23, and 20q11 [31,39]. These translocations are considered as early primary events that are mediated by errors in IGH switch recombination during the maturation of B-cells in germinal centers [31,32]. In our study, the most frequent partner chromosome in 14q32 translocations was t(11;14)(q13;q32), seen in 14% of cases. Recently Dewald et al. reported t(11;14)/ IGH-CCND1 fusion in 7.8% of their cases while, other studies have reported a higher frequency of 20% [7,17]. At chromosomal level, the t(11;14) in MM is similar to that of mantle cell lymphoma but the IGH breakpoint is different American Journal of Hematology DOI /ajh 1085

7 IGHS versus IGHJ. The t(4;14) and t(4;16) were under-represented in our series, because these translocations are cryptic, and FISH was applied only on selected cases. Secondary IGH translocations that dysregulate the CMYC oncogene on 8q24 are seen in bout 3% of MM cases [33]. Those types of translocations are considered late progression events in the pathogenesis of MM, and likely will have a negative impact on the overall prognosis. Of interest 6% of our cases showed Burkitt s translocations t(8;14) or t(8;22), and all had advanced disease. Another less frequent but nonrandom aberration observed in our series was der(16)t(1;16). In all six cases, the translocation was unbalanced with variable breakpoints, always resulted in partial loss of 16q and partial gain of 1q. The t(1;16) was observed in cases previously treated and in MM progressed from MGUS suggesting that this abnormality is merely a marker of clonal evolution associated with relapse or progression. Losses of 17p was observed in 15% of cases; however, it is higher in the nonhyperdiploid cases (26%) than the hyperdiploid (1%). The tumor suppressor gene p53/17p13 is the most common genetic defect in human malignancies. In MM, deletion of 17p/p53 is detected in a frequency ranging from 9 30% [29,34]. As in chronic lymphocytic leukemia, 17p/p53 deletion provides a powerful independent prognostic factor associated with a shorter survival and disease progression [11,34,35]. Most of our cases that showed 17p deletion were relapsed after treatment or bone marrow transplant. In summary, this report focuses on a large series of MM cases with abnormal karyotype primarily using conventional banding technique. The types of chromosomal abnormalities are similar to those reported in the literature, although the frequency of some differs. In the present study, hyperdiploidy was dominant characterized by multiple chromosomal gains, but less structural aberrations. Unlike the hyperdiploid, the nonhyperdiploid MM had less numerical but more structural abnormalities such as 14q32 translocation, 13/13q deletion, and 17p. Clearly, this and previous studies have revealed two main pathways involved in the early events of myelomagenesis; multiple trisomies in hyperdiploid and IGH translocation in nonhyperdiploid. These alterations exert their actions through one of two mechanisms; changes in quantitative gene ratio or dysregulation of gene expression. Both mechanisms cause subsequent genomic instability leading to acquisition of additional abnormalities such as dup1q, deletions 1p, 8p, 9p, and 13q that provide a proliferative advantage. Secondary CMYC translocation, t(1;16), and/or 17p deletion are late genetic events seen in progression of myeloma. In conclusion, MM is best characterized by conventional cytogenetics that identifies subgroups with distinct pattern of chromosomal aberrations. FISH is highly recommended as a complement for specimens that yield normal karyotypes and ambiguous findings. FISH testing for gain of chromosomes 3, 9, and 15, 13/13q deletion, 17p deletion, and t(4;14) translocation are strongly encouraged for risk assessment and stratification. Materials and Methods A retrospective search of our clinical cytogenetics database was conducted to identify patients with a referral diagnosis of MM or other monoclonal gammopathy. A total 297 cases were submitted for cytogenetic evaluation over a period extended from 1995 to Six cases were excluded from the study because their cytogenetics abnormalities were consistent with myelodysplasia. Of the 291 remaining MM cases; 120 (41%) cases had clonal chromosomal abnormalities and 179 (59%) yielded a normal karyotype. Specimens were collected from Wayne State University/Detroit Medical Center and Karmanos Cancer Institute, and from specimens sent to us for consultation. The bone marrow specimens were processed for histologic, cell surface marker, and cytogenetic studies. The morphological diagnosis of MM was based on established criteria [2]. For conventional cytogenetics, bone marrow aspirates were processed using two different culture methods; 24 hr unstimulated culture medium consisted of RPMI and 20% fetal bovine serum, and 48 hr culture supplemented with 10% giant cell conditioned medium. At the end of incubation, cultures were exposed to colcemid for 45 min and then harvested according to the current protocols established in the lab [36]. Twenty or more G- or Q-banded metaphase cells were analyzed when possible. When a single abnormal metaphase cell was identified, an additional 20 cells were examined for evidence of clonality. Interpretation of chromosome abnormalities followed the criteria established by International System for Human Cytogenetics Nomenclature (ISCN) 2005 [37]. FISH was performed on bone marrow aspirate left-over from conventional cytogenetic harvests, while targeted FISH was performed on destained, banded and karyotyped metaphases. Interphase and metaphase FISH were done using DNA specific probes; D13S319 13q14/ 13q34 for the detection of 13/13q14 deletions, IGH/CCND1 for t(11;14)(q13;q32), or IGH/CMYC for t(8;14)(q24;q32). All probes were purchased from Vysis. (Downers, Grove, IL). Details concerning pretreatment of slides and hybridization were carried out according to instructions accompanying the probes. The chromosomes were counterstained with 4 0, 6-diamidino-2-phenylondole and viewed with Zeiss Axioskop fluorescence microscope. At least 200 interphase cells and 10 metaphases were analyzed for each probe. Results were abnormal when the percent of cells with any given chromosome abnormality exceed the normal cut-off value. All FISH probes were validated using negative and positive controls. References 1. Bataille R, Harousseau JL. 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