Clinicopathological Characteristics of Hyperdiploidy with High-Risk Cytogenetics in Multiple Myeloma

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Brief Communication Diagnostic Genetics CROSSMARK_logo_3_Test 1 / 1 Ann Lab Med 2018;38:160-164 https://doi.org/10.3343/alm.2018.38.2.160 ISSN 2234-3806 • eISSN 2234-3814

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Naery Yang, M.D.1, Yeung Chul Mun, M.D.2, Chu-Myong Seong, M.D.2, Hee Jin Huh, M.D.3, and Jungwon Huh, M.D.1 Department of Laboratory Medicine1, College of Medicine, Ewha Womans University, Seoul; Department of Internal Medicine2, College of Medicine, Ewha Womans University, Seoul; Department of Laboratory Medicine3, Dongguk University, Ilsan Medical Center, Goyang, Korea

In multiple myeloma (MM), hyperdiploidy (HD) is known to impart longer overall survival. Received: February 27, 2017 However, it is unclear whether coexistent HD ameliorates the adverse effects of known Revision received: June 25, 2017 Accepted: October 25, 2017 high-risk cytogenetics in MM patients. To address this issue, we investigated the clinicopathological characteristics of HD with high-risk cytogenetics in MM. Ninety-seven patients Corresponding author: Jungwon Huh Department of Laboratory Medicine, with MM were included in the study. For metaphase cytogenetics (MC), unstimulated cells College of Medicine, Ewha Womans from bone marrow aspirates were cultured for either 24 or 48 hours. To detect HD by in- University, 1071 Anyangcheon-ro, terphase fluorescence in situ hybridization (iFISH), we assessed trisomies of chromosomes Yangcheon-gu, Seoul 07985, Korea Tel: +82-2-2650-5320 5, 7, 9, 11, 15, and 17. Of the 97 MM patients, 40 showed HD. The frequency of co-oc- Fax: +82-2-2650-5091 currence of HD and high-risk cytogenetics was 14% (14/97). When the clinicopathologi- E-mail: [email protected] cal characteristics were compared between the two groups of HD with high-risk cytoge- Co-corresponding author: Hee Jin Huh netics vs. non-HD (NHD) with high-risk cytogenetics, the level of beta 2 microglobulin and Department of Laboratory Medicine, stage distribution significantly differed (P =0.020, P =0.032, respectively). This study shows Dongguk University School of Medicine, that some of the clinicopathological characteristics of MM patients with high-risk cytoge- Ilsan Hospital, 27 Dongguk-ro, Ilsandong- gu, Goyang 10326, Korea netics differ according to HD or NHD status. Tel: +82-31-961-7893 Fax: +82-31-961-7902 E-mail: [email protected]

© Korean Society for Laboratory Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom- mons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the Key Words: Hyperdiploidy, Multiple myeloma, Cytogenetics, High risk original work is properly cited.

Risk stratification of patients with multiple myeloma (MM) is im- one or more odd-numbered chromosomes (chromosome 3, 5, portant to predict survival and define a treatment strategy. Cyto- 7, 9, 11, 15, or 17). Previous studies have shown that abnor- genetic abnormalities are clinically relevant prognostic factors in malities such as t(4;14), t(14;16), and deletion of the short arm MM [1, 2]. Patients with MM can be categorized into hyperdip- of chromosome 17 (17p) are predictive of significantly short- loidy (HD) and non-hyperdiploidy (NHD) groups according to ened survival (defined as high-risk cytogenetics), whereas HD is the primary cytogenetic abnormalities. The definition of HD in associated with cytogenetics with a favorable outcome [1, 2]. MM has varied across studies; one study defined HD as numer- There is a small subset of patients with MM that shows evidence ous chromosomal trisomies and low prevalence of IgH translo- of both HD and high-risk cytogenetics, but its clinical significance cations [3], and another as a chromosome count of 48–65, with and prognostic impact is controversial [5-8]. a gain of at least two odd chromosomes [4]. HD in this study This study aimed to investigate clinicopathological character- was defined by a chromosome count of 47 or more, with gain of istics associated with HD with high-risk cytogenetics in MM, com-

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pared with those of NHD with high-risk cytogenetics in MM. In- (14/97); t(4;14), (14;16), and del(17p) were observed in 7% terestingly, del(5q) was detected in two patients, which corre- (7/97), 2% (2/97), and 8% (8/97) of the patients, respectively sponded to an EGR1 deletion. We examined the clinical signifi- (Table 1). Three patients presented concurrent, multiple high- cance of either del(5q) or an EGR1 mutation in MM and reviewed risk cytogenetics (See Supplemental Data Table S1; cases 12, the literature. 18, and 35). We also reviewed the frequencies of high-risk cyto- A total of 97 patients were newly diagnosed as having MM, genetics in the NHD group (See Supplemental Data Table S2) according to the International Myeloma Working Group diagnos- and compared them with those in the HD group. The difference tic criteria [9], at two institutions in Korea (93 from Ewha Wom- in the frequencies of high-risk cytogenetics was not statistically ans University Mokdong Hospital, Seoul, Korea; four from Dong- guk University Ilsan Hospital, Goyang, Korea) between 2010 Table 1. Comparison of clinical and laboratory characteristics of and 2015. The demographics, laboratory, and clinical data of patients with hyperdiploidy with high-risk cytogenetics and non-hy- the patients were retrospectively reviewed. This study was ex- perdiploidy with high-risk cytogenetics empted from approval from the institutional review board, as it HD with high- NHD with high- Variable risk cytogenetics risk cytogenetics P * was a retrospective review of existing medical records, and all (n=14) (n=16) information was de-identified. Demographic and laboratory Male:female, number (% male) 12:2 (85.7) 8:8 (50.0) 0.042 characteristics of the patients are summarized in Supplemental Age (yr) Data Tables S1 and S2. HD was confirmed by metaphase cytogenetics (MC) and/or median (range) 66 (57–85) 67 (48–82) 0.546 interphase fluorescence in situ hybridization (iFISH). For MC, Albumin (g/dL) unstimulated cells from bone marrow aspirates were cultured median (range) 3.1 (2.2–4.5) 3.1 (1.6–3.9) 0.755 for either 24 or 48 hours. All data were retrospectively reviewed β2 microglobulin (mg/L) and reanalyzed according to the International System for Hu- median (range) 4.5 (2.4–39.6) 11.8 (2.7–71.2) 0.020 man Cytogenetic Nomenclature (ISCN 2013) guidelines [10]. Serum LDH (IU/L) iFISH was performed using the following probes (Abbott/Vysis, median (range) 189 (114–611) 254 (170–1,250) 0.056 Downers Grove, IL, USA; Metasystems, Heidelberg, Germany; t(4;14) or Kreatech, Amsterdam, the Netherlands): IgH dual color break number (%) 7 (50.0) 3 (18.8) 0.075 apart rearrangement probe, TP53 dual color probe, CEP7/D7S486 t(14;16) dual color probe, 13q14/13q34 dual color probe, IgH/CCND1 number (%) 2 (14.3) 1 (6.3) 0.472 dual color probe, dual fusion probe, IgH/FGFR3 dual color probe, del(17p) dual fusion probe, IgH/MAF dual color probe, dual fusion probe, number (%) 8 (57.1) 12 (75.0) 0.309 1q21/1p32 dual color probe, EGR1/D5S721,D5S23 dual color 1q21 amplification probe, p16/CEP9 dual color probe, and/or CEP15 single color number (%) 11 (78.6) 8 (50.0) 0.111 probe. To detect HD by iFISH, we assessed trisomies of chro- ISS stage number (%) 0.051 mosomes 5, 7, 9, 11, 15, and 17, using targeted probes. At least I 3 (21.4) 0 (0.0) 200 interphase cells from each case were counted. II 5 (35.7) 3 (18.8) Of the 97 patients with MM, 57 (59%) were classified into the III 6 (42.9) 13 (81.3) NHD group, and HD was detected in 40 patients (41%) by MC ISS-R stage number (%) 0.032 and/or iFISH. At diagnosis, iFISH in two patients (See Supple- I 0 (0.0) 0 (0.0) mental Data Table S1; cases 6 and 9) revealed HD, whereas a normal karyotype was revealed by MC. However, after the dis- II 8 (57.1) 3 (18.8) ease progressed, HD was also detected by MC. III 6 (42.9) 13 (81.3) We defined high-risk cytogenetics as the presence of t(4;14), *P values were calculated by the χ2 test for categorical variables and the Mann-Whitney U test for continuous variables in a comparison between pa- t(14;16), or del(17p), based on the revised International Staging tients with HD with high-risk cytogenetics and those with NHD with high-risk System proposed by the International Myeloma Working Group cytogenetics. Significant values are shown in bold. Abbreviations: HD, hyperdiploidy; ISS, International Staging System; ISS-R, [1]. Among patients newly diagnosed with MM, the frequency Revised International Staging System; LDH, lactate dehydrogenase; NHD, of co-occurrence of HD and high-risk cytogenetics was 14% non-hyperdiploidy. https://doi.org/10.3343/alm.2018.38.2.160 www.annlabmed.org 161 Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM

significant (P =0.467) between the two groups. cording to the revised International Staging System (ISS-R) [11] Table 1 summarizes the comparative analysis between the was significantly different between the two groups (P =0.032) two groups of HD with high-risk cytogenetics vs. NHD with high- (Table 1). It is noteworthy that 57% of patients with HD and high- risk cytogenetics. risk cytogenetics belonged to ISS-R stage II, whereas 81% of pa- The difference in stage distribution, determined according to tients with NHD and high-risk cytogenetics belonged to stage III. the International Staging System (ISS), did not reach statistical Gain of chromosome 9 (22 patients) was the most commonly significance (P =0.051), but stage distribution determined ac- observed aneuploidy in the HD group, identified by iFISH, fol-

A B

C Left Right D

Fig. 1. Karyotype and interphase fluorescence in situ hybridization* using a D5S721/ D5S23/EGR1 probe in multiple myeloma patients with chromosome 5 aberrations. (A) Case 9 showing partial trisomy 5; two normal chromosome 5s and one del(5q). (B) Case 38 showing del(5q). (C) Case 38 showing one red signal loss (2G1R), suggestive of del(5q). (D) Case 6 showing three green and red signals (3G3R), suggestive of trisomy 5 (left panel); and four green and three red signals (G4R3), suggestive of partial tetrasomy 5 (three normal chromosome 5s and one del(5q)) (right panel). (E) Case 16 showing G4R3, suggestive of partial tetrasomy 5. *FISH probe design is as follows: Chromosome 5p15 E shows a green signal (G), and 5q31 shows a red signal (R). The normal FISH pattern of chromosome 5 is two green signals and two red signals (2G2R).

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lowed by gain of chromosome 5 (n=21), 15 (n=19), similar to rence of HD [7, 8]. In contrast, one study showed that patients the frequencies reported in previous studies [12, 13]. iFISH us- with high-risk cytogenetics have higher survival rates when triso- ing probes targeting chromosomes 5, 9, and 15 for the detec- mies are present than when they are absent [6]. Another study tion of HD showed various patterns. In one patient (See Supple- showed that trisomy 15 increases the rate of progression-free mental Data Table S1; case 20), iFISH showed trisomy of chro- survival in patients with del(17p), whereas HD does not improve mosome 5 (43.5%), whereas both trisomy and tetrasomy were clinical outcomes in patients harboring t(4;14) [5]. These dis- observed for chromosomes 9 (trisomy, 13.0%; tetrasomy, 48.5%) crepant findings may be attributed to different treatment regi- and 15 (trisomy, 18.5%; tetrasomy, 22.5%). In another patient mens. Certain therapies (i.e., bortezomib-based therapies) can (See Supplemental Data Table S1; case 32), trisomies of chro- mitigate the risk associated with specific abnormalities, as in the mosomes 9 (72.0%) and 11 (61.0%) were revealed by iFISH, case of t(4;14) or del(17p) [1, 2]. whereas both trisomy and tetrasomy were observed for chromo- Our study showed that a considerable subset—14% (14/97)— some 15 (trisomy, 30.0%; tetrasomy, 29.5%). In one patient of all MM patients had HD and coexistent high-risk cytogenet- (See Supplemental Data Table S1; case 6), both trisomies and ics. Of the 40 HD patients, 35% (14/40) presented high-risk cy- tetrasomies of chromosome 5 (trisomy 26.5%, tetrasomy 78.5%) togenetics, whereas 28% (16/57) of NHD patients presented and 9 (trisomy 14.5%, tetrasomy 73.5%) were observed at re- high-risk cytogenetics. Moreover, the distribution of stages ac- lapse by iFISH, whereas only trisomies of chromosome 5 (28.0%), cording to ISS-R differed between the groups with HD with high- 9 (77.5%), and 15 (61.5%) were observed at diagnosis. risk cytogenetics and NHD with high-risk cytogenetics. This study As for chromosome 5 abnormalities, the del(5q) was observed indicates that the clinicopathological characteristics of MM pa- in two patients by MC (See Supplemental Data Table S1; cases tients with high-risk cytogenetics may differ according to HD or 9 and 38). One patient (case 9) showed a normal karyotype at NHD status. diagnosis, but partial trisomy 5 (two normal chromosome 5s Further comprehensive studies in larger cohorts, using the and one with a 5q deletion) was detected by MC at nine months same treatment strategies, are needed to delineate the prognos- (Fig. 1A). In another patient (case 38), del(5q) was identified by tic impact of concurrent HD and high-risk cytogenetics com- MC and 5q signal loss, corresponding to an EGR1 deletion of pared with that of NHD and high-risk cytogenetics. Assessment 5q31 (21.6%), was confirmed by iFISH (Fig. 1B and C). Fur- of HD by MC and iFISH may be mandatory for patients newly thermore, in one patient (case 40), iFISH indicated two clones: diagnosed as having MM. one with trisomy 5 (15.0%), and the other with partial trisomy 5 [two normal and one with del(5q)] (11.5%), whereas only trisomy Authors’ Disclosure of Potential Conflicts of 5 was revealed by MC. Two patients (cases 6 and 16) showed Interest partial tetrasomy 5 by iFISH: three normal and one with del(5q) (78.5% in case 6; 22.0% in case 16) (Fig. 1D and E). No potential conflicts of interest relevant to this article were re- Similar to our findings, one study showed partial trisomy 5 ported. (two normal and one with a 5q deletion) in clonal plasma cells of patients newly diagnosed as having MM [14]. EGR1 as a can- Acknowledgements didate gene for del(5q) in MDS has been associated with an in- dividual’s response to lenalidomide [15]. However, another study This study was supported by the Basic Science Research Pro- showed that an EGR1 mutation is highly associated with HD in gram through the National Research Foundation of Korea, funded MM [16]. In MM, EGR1 was recently shown to be involved in by the Ministry of Education, Science, and Technology (NRF- recruiting MYC to the promoters of NOXA and BIM and induc- 2012R1A1A2044138). ing p53-independent apoptosis [17, 18]. The clinical significance of del(5q) or an EGR1 mutation on 5q in MM remains to be as- REFERENCES certained. There is no consensus on the prognostic impact or risk asso- 1. Rajkumar SV. Myeloma today: disease definitions and treatment advanc- ciated with concurrent HD and high-risk cytogenetics. A few es. 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164 www.annlabmed.org https://doi.org/10.3343/alm.2018.38.2.160 Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM ) I I II II II II II II II II II II III III III III III III ISS-R I I I I II II II II II II II II III III III III III III ISS Continued to the next page ( -13 -13 -13 -13 Other , t(11;14), IgH del(8p) del(13q) del(13q) IgH rearr. MAF gain MAF deletion del(8p), del(13q) deletion, -13, -15 -13, MAF deletion gain, MAF deletion del(5q) IgH rearr., MAF deletion IgH rearr., del(1p), t(11;14), 5´ IgH (%) 1) 20 95 76 63 18 17 61 NT neg neg neg neg neg neg neg neg neg neg 1q21 ampl

1) 7 39 37 NT neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) del(17p)

1) 10 NT neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(14;16)

Aberrations identified by interphase FISH 1) 17 47 NT neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(4;14) , +9 , +9, , +9, , +9, , +9, , +9, , +9, , +9, +7 +5 +9 HD , +11, +11 +15 +15 +15 +15 +15 +5 +5 +5 +5 +5 +5 +5 +5 +7, +9, +7, +13 +7, +11 +9, +15 +5 +11, +15 +11, +15 +11, +15 +15, +17 ,+6,

+5 ,+7,-8,+9,+9, ,-6,add(6)(q22),-8, +5 +5,del(5)(q22q33) 46,XX[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XX[20] 46,XX[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] G-banded chromosome study +mar2,+mar3[cp10]//46,XY[6] (q25),+19,+21,+mar[3]/46,XX[17] add(22)(p12),+mar1,+mar2[2]/46,XX[7] add(6)(q22),+7,add(7)(p15)x2,+9,+9,+11,-12,+13,add(13) Characteristics of patients with multiple myeloma and hyperdiploidy, identified by a G-banded chromosome study and/or interphase FISH Characteristics of patients with multiple myeloma and hyperdiploidy, 52,X,-X,del(1)(q41),?inv(1)(p22q11),+2,-4, +9,+11,+12,+12,add(12)(p10),add(13)(p10),+14,+15,-16,+19, der(9)t(9;11)(p13;q13),add(10)(q22),del(13)(q12q22),+15,add(17) 53~60,X,-X,+1,add(1)(p13), del(1)(q41),+2,+3,+4,del(4)(q21q25), 53,X,del(X)(q22), der(1)t(1;?8)(p13;?q13), (p10),+14,+14,+15,-17,+18,+19,+20,+21,+21,+22,add(22)(p12),+mar1, 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 20 34 Date (mo.) 49 79 41 81 82 57 52 55 62 63 57 66 57 69 56 Age F F F F F F M M M M M M M M M Sex

Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Supplemental Data Table S1. Supplemental Data Table https://doi.org/10.3343/alm.2018.38.2.160 www.annlabmed.org Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM ) I II II II II II II II II II III III III III III III III ISS-R I I II II II II II II II III III III III III III III III ISS

Continued to the next page ( -13 -13 -13 Other del(1p) del(13q) IgH rearr. MAF gain MAF deletion -13, MAF gain 5´ IgH deletion 5' IgH deletion del(1p), IgH rearr., del(1p), IgH rearr., MAF gain, +18, -X Whole IgH deletion del(13q), IgH rearr. -13, -15, MAF deletion -13, whole IgH deletion (%) 1) 14 43 53 67 13 14 14 28 23 71 neg neg neg neg neg neg neg 1q21 ampl

1) 7 6 36 neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) del(17p)

1) neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(14;16)

Aberrations identified by interphase FISH 1) 46 34 63 25 neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(4;14) , +9 , +9, , +9, , +9, , +9, , +9, , +11 NT NT +5 +9 HD +17 +15 +15 +17 +17 +11 +15 +5 +5 +5 +5 +5 +5 +5 +9, +11, +9, +11, +11, +15 +11, +15 +11, +15,

,-8,-10,-12,-13,-14,+15,-16,-20, -5 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] -22[6]/46,XX[14] +mar[5]/46,XY[15] -16[cp13]/46,XY[7] 45,X,-Y[11]/46,XY[9] G-banded chromosome study ,+6,+9, add(9)(p24),+10,+10,+11,+12,-13,dic(14;15) +5 add(17)(p11.2)t(15;17)x2,+18,-19,-21,-22[2]/46,XY[18] (q32;q26),+16, add(17)(q25),-17,+18, +19,+19[1]/46,XX[19] -13,add(13)(p10),+15,+15,-17,+19,?del(20)(q13.1),+21,+21, Continued 63~66,X,-X,-X,del(1)(q21), -2,add(2)(31),+3,add(3)(q27), del(3) +11,+15,add(16)(q13~q21),+17,+19, +20,+21[cp6]/46,XX[14] 62~65,XXY,+Y,-1, t(1;12)(q32;q24),-2,-6,-8, add(11)(q25),-12,-15, 62~65,XXY,+Y,-1, 57,XX,+3,+4,+7,+9,+11,+11,+15,+19,+20,+21,+22[5]/46,XX[15] 58,X,-X,-X, der(1)t(1;5)(p12;p13),-2, 56,XX,+3,+3, +11,-12,-13,-14,-14, +15,der(15)t(15;17)(q22;q21)x2,-16,+17, der(17) 57~59,X,-X, ?psu dic(1;5)(p13;q35),+2,+3, +del(6)(q14q25),+7,+9,+9, 54,XY,+1,del(1)(p13), t(1;11)(p22;q14),+3,+7,-8,+9, +11,del(12)(p11.2), 54,XY,+1,del(1)(p13), -8,add(9)(p24),+10,-13,-14,-17, +18,+18,-19, +1~9mar[cp4]/46,XX[10] 68,XXY,+Y,+1,+2,+3,add(4)(q12),add(4)(q21),add(6)(q13)x3,+9,-10,-10, 68,XXY,+Y,+1,+2,+3,add(4)(q12),add(4)(q21),add(6)(q13)x3,+9,-10,-10, (q21q23),i(3)(p10), add(4)(p16),-6,add(6)(q21),-7,add(7)(p22), del(7)(q31), 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Date (mo.) 70 70 73 75 66 82 60 98 63 64 75 52 80 52 66 85 71 Age F F F F F M M M M M M M M M M M M Sex

Case no. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Supplemental Data Table S1. Supplemental Data Table www.annlabmed.org https://doi.org/10.3343/alm.2018.38.2.160 Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM II II II II II III III III III ISS-R II II II II III III III III III ISS -13 Other deletion , t(11;14), 5´ IgH IgH rearr. deletion, -15 IgH deletion, MAF -13, MAF deletion FGFR3 deletion, -13 IgH gain, MAF deletion del(5q) del(1p), del(13q), whole (%) 1) 33 13 56 80 98 16 neg neg neg 1q21 ampl

1) 5 3 33 neg neg neg neg neg neg (%) del(17p)

1) 11 neg neg neg neg neg neg neg neg (%) t(14;16)

Aberrations identified by interphase FISH 1) 86 89 neg neg neg neg neg neg neg (%) t(4;14) , +9, , +9, , +9, , +9, , +9, HD +15 +17 +5 +5 +5 +5 +5 +11, +15 +11, +15 +11, +15 +11, +15,

, +5 ,+7,+9, +5 ,+6, der(6)t(6;9)(q13;q12), +5 ,+11,der(14)t(11;14)(q13;q32),+19, der(19) +22[11]/46,XX[9] -20,+21[5]/46,XX[15] +mar2[cp3]/46,XY[17] G-banded chromosome study t(1;19)(q21;q13.3)[cp12]/46,XY[8] (q21;q13.4),-21-22,+mar[2]/46,XY[18] +11,+15,+18,+19,+21,+22[cp6]/46,XX[14] 50~55,X,-X,+3, der(3;15)(q10;q10), del(5)(q31~q32) +7,add(7)(p21),+9, +mar1,+mar2[cp15]/46,XY[5] ,del(6)(q13q21),+7,+9,+11,-13,+15, der(16) t(1;16)(q12;q24), 45~48,XY,add(1)(q23),+8,add(8)(p11.2),-13,+15,add(16) 45~48,XY,add(1)(q23),+8,add(8)(p11.2),-13,+15,add(16) +5 49,X,-X,der(1)t(1;?)(p22;?),+9,+11,del(12)(p13),+14,+15,-16, Continued -17,add(19) (p13.3),-20,-22,+mar1,+mar2,+mar3[2]/46,XY[18] 46,XY,add(1)(q23),+8,-13,+15, add(16)(q12~q13),der(19)t(1;19) 46,XY,add(1)(q23),+8,-13,+15, 50~52,XY,+1,der(1;22)(q10;q10),+3, -22,+mar1,+mar2[cp3]/76,XXY,+Y,add(1)(q23),+2,+3,-7,+8,+add(8) 48~49,XY,del(1)(q32q42), der(1)dup(1)(q21q32), add(3)(q27)x2,-4, 48~49,XY,del(1)(q32q42), 47~48,XY, t(1;19)(q21;q13.4),+20,add(21)(p11.1),-22,+mar1,+mar2[2]/46,XY[15] 50,XY, (p11.2),+10,-13,-14,+15,add(15)(p10~p11.2),add(16)(q13),+19,der(19) (q12~q13),add(17)(q24)der(17) del(17)(p11.2),der(19)t(1;19) (q21;q13.4), 49,XX,del(1)(p13.2),del(1)(p21),+6,-8,-10,+11,+12, +15,+16,del(17)(p13), add(6)(q15),-8,+9,+11,add(12) (p11.2),+15,add(16)(p12),-17,+19,+mar1, 0 0 0 0 0 0 0 0 28 Date (mo.) 72 77 71 66 66 58 62 68 Age F F F M M M M M Sex

Positive cut-off values for t(4;14), t(14;16), del(17p), and 1q amplification were 0.6%, 0.6%, 1.8%, and 1.8%, respectively. Positive cut-off values for t(4;14), t(14;16), del(17p), and 1q amplification were 0.6%, 1.8%, respectively. Case no. 33 34 35 36 37 38 39 40 Supplemental Data Table S1. Supplemental Data Table 1) female; FISH, fluorescence in situ hybridization; HD, hyperdiploidy; ISS, International Staging System; ISS-R, Revised M, male; Abbreviations: ampl, amplification; F, rearrangement. not tested; rearr., mo., months; neg, negative; NT, https://doi.org/10.3343/alm.2018.38.2.160 www.annlabmed.org Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM ) I I I I II II II II II II II II II II II III III III III III III III III III ISS-R I I I I I II II II II II II II II III III III III III III III III III III III ISS

-15 Continued to the next page gain ( rearr. Other deletion t(11;14) del(13q) del(13q) del(13q) IgH rearr. MAF gain IgH rearr., deletion,-13 del(1p), p16 del(13q),IgH del(13q),-15 del(13q), IgH del(1p), 5´ IgH -13, IgH rearr., -13, IgH rearr., rearr.,-15, MAF rearr.,-15, FGFR3 deletion del(8p), del(13q) del(13q), t(11;14) (%) 1) 5 4 47 49 51 19 34 19 15 97 47 neg neg neg neg neg neg neg neg neg neg neg neg neg 1q21 ampl

1) 9 8 9 6 5 59 62 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) del(17p)

1) 25 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(14;16) Aberrations identified by interphase FISH

1) 39 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(4;14) 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XX[20] 46,XX[20] 46,XY[20] 45,X,-Y[9]/46,XY[11] 46,XX,inv(9)(p11q13)c[20] G-banded chromosome study 46,XX,del(1)(p21)[2]/46,XX[18] 46,XY,dup(1)(q44q11)[14]/46,XY[6] 46,XY,del(13)(q12q14),del(17)(p13)[1]/46,XY[19] (p11.2),del(17)(q25), +add(19)(p13),+mar1,+mar2[cp5]/46,XY[35] Characteristics of patients with multiple myeloma and non-hyperdiploidy, identified by a G-banded chromosome study and/or interphase FISH Characteristics of patients with multiple myeloma and non-hyperdiploidy, 44~46,XY,der(1;14)(q10;q10),add(2)(p13),add(3)(q29),add(4)(p14~p16),add(8) 45,X,-Y,-11,der(14)t(11;14)(q13;q32),der(16)t(1;16)(q21;q12.1),+mar[9]/46,XY[11] (p12~p21),add(9)(p13~p22),del(12)(q24.1),-13,del(14)(q32),-16,+17,der(17)del(17) 53 70 70 81 53 51 82 79 48 78 52 46 66 89 78 75 67 53 55 69 72 65 55 68 Age F F F F F F F F F F M M M M M M M M M M M M M M Sex

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Case no. Supplemental Data Table S2. Supplemental Data Table www.annlabmed.org https://doi.org/10.3343/alm.2018.38.2.160 Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM ) I I II II II II II II II II II II II II II II II II II III III III III III ISS-R I I I I II II II II II II II II II II II II II II III III III III III III ISS

-13 -13 Continued to the next page gain ( rearr. rearr. rearr. Other t(11;14) t(11;14) t(11;14) del(13q) IgH rearr. IgH rearr. deletion,-17 -13, t(11;14) del(13q), IgH del(13q), IgH -13, -14, MAF del(1p), FGFR3 del(1p), -13, IgH (%) 1) 7 3 10 65 37 11 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg 1q21 ampl

1) 9 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) del(17p)

1) neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(14;16) Aberrations identified by interphase FISH

1) 62 29 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg (%) t(4;14)

46,XX[20 46,XX[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XX[20] 46,XX[20] 46,XX[20] 46,XX[20] 46,XX[20] 46,XY[20] 46,XX[20] 46,XX[20] -14[3]/46,XX[7] +mar1,+mar2[cp6]/46,XX[14] G-banded chromosome study 46,XX,t(11;14)(q13;q32),-16,+mar[12]/46,XX[8] Continued 43~45,X,-X,-1,+8,add(8)(q22),i(8)(q10), add(12)(p11.2~13),-13,-18,-20,add(21)(p11.2), 43,X,-X,del(1)(p13p22),der(1)t(1;1)(p36.3;q12),del(8)(q11.1q22),der(9)t(1;9)(q12;p22),-13, 57 78 69 59 85 57 68 78 57 72 53 35 52 56 88 79 74 66 60 59 77 60 72 78 Age F F F F F F F F F F F F F F M M M M M M M M M M Sex

Case no. 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 Supplemental Data Table S2. Supplemental Data Table https://doi.org/10.3343/alm.2018.38.2.160 www.annlabmed.org Yang N, et al. Hyperdiploidy with high-risk cytogenetics in MM II II II II II III III III III ISS-R I II II II III III III III III ISS -17 Other deletion t(11;14) t(11;14) IgH deletion del(1p), p16 del(1p), -13, t(11;14), whole t(11;14), 5´ IgH FGFR3 deletion, -13, IgH rearr., 5´ -13, IgH rearr., IgH deletion, -15, deletion, t(11;14) (%) 1) neg neg neg neg neg neg neg neg neg 1q21 ampl

1) 3 9 7 6 neg neg neg neg neg (%) del(17p)

1) neg neg neg neg neg neg neg neg neg (%) t(14;16) Aberrations identified by interphase FISH

1) neg neg neg neg neg neg neg neg neg (%) t(4;14)

46,XY[20] 46,XX[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 46,XY[20] 45,X,-Y[18]/46,XY[2] (p13.3),+mar[cp6]/46,XX[5] G-banded chromosome study Continued -13,der(14)t(11;14)(q13;q32),add(16)(p13.3),?der(17)add(17)(p12)add(17)(q21),add(19) 44~45,X,-X,-1,del(2)(q33),+7,add(7)(p22), del(7)(p21),add(8)(q24),?ins(11;?)(q13;?),-12, 61 70 59 79 60 75 87 62 80 Age F F M M M M M M M Sex