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Identification of recurrent chromosomal breakpoints in multiple myeloma with complex karyotypes by combined G-banding, spectral karyotyping, and fluorescence in situ hybridization analyses
Cancer Genetics and Cytogenetics 169 (2006) 143e149
Identification of recurrent chromosomal breakpoints in multiple myeloma with complex karyotypes by combined G-banding, spectral karyotyping, and fluorescence in situ hybridization analyses Borja Sa´eza,b,*, Jose´ I. Martı´n-Suberob, Cristina Largoc, Marı´a C. Martı´nc, Marı´a D. Oderoa,d, Felipe Prosperd, Reiner Siebertb, Marı´a J. Calasanza, Juan C. Cigudosac a Department of Genetics, University of Navarra, Irunlarrea s/n, Pamplona, Navarra 31008, Spain Institute of Human Genetics, University Hospital Schleswig-Holstein, Schwanenweg 24, Campus Kiel, Kiel, Germany, D-24105 c Cytogenetics Unit, Spanish National Cancer Research Center, Melchor Fernandez Almagrd 3, Madrid, Spain, 28029 d Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Irunlarrea s/n, Pamplona, Navarra 31008, Spain b
Received 30 January 2006; received in revised form 7 April 2006; accepted 11 April 2006
Abstract
The description of novel chromosomal aberrations in multiple myeloma (MM) remains necessary to fully understand the pathogenesis of this heterogeneous disease. Therefore, we have used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) with locus-specific probes to characterize the chromosomal abnormalities in 11 MM cases in which G-banding revealed a complex karyotype. SKY refined G-banding karyotypes in all cases. Recurrent breakpoints involved bands Xp11, 8q24, 11q13, 12q13, 13q21, and 14q32. In addition, combined SKY and FISH analyses permitted us to identify a subset of patients harboring 22q11.2 rearrangements not involving the IGL locus. This finding suggests the presence of other gene(s) in band 22q11 that might be implicated in MM pathogenesis. Moreover, band 1p13 was identified as a novel partner of immunoglobulin (IG) translocations in MM. Finally, using interphase FISH, we have detected interstitial deletions in 13q14 and 17p13, as well as cryptic translocations affecting IGH, which were neither detected by G-banding nor by SKY. The results of the present study suggest the existence of hitherto unknown nonrandom chromosomal changes that may play a role in the pathogenesis of MM. Our findings underline the importance of the combination of banding, SKY, and FISH analyses to increase the accuracy of karyotype interpretation in plasma cell neoplasias. Ó2006 Elsevier Inc. All rights reserved. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction The clinical importance of specific chromosome abnormalities in multiple myeloma (MM) has been highlighted by several studies. Translocations with breakpoints in immunoglobulin (IG) genes (i.e., IGH in 14q32, IGK in 2p12, and IGL in 22q11.2), such as the t(4;14)(p16;q32) and the t(14;16)(q32;q23), are associated with an adverse impact on prognosis [1,2]. Likewise, deletions of the tumor suppressor gene TP53 (17p13), as well as chromosome 13 abnormalities, mainly monosomy 13, are also associated with poorer outcome in MM [1e3]. However, the description of novel chromosomal aberrations that might be implicated in MM pathogenesis remains necessary for the full characterization of the genetic basis of this heterogeneous * Corresponding author. Tel.: þ34-948-425600; fax: þ34-948-425649. E-mail address: [email protected] (B. Sa´ez). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.04.008
disease. Nowadays, two groups of cases are of major interest for the identification of genetic markers in MM: those with locus breakpoints involving novel partner genes and those with recurrent breakpoints not involving IG genes, which might be associated with the nonhyperdiploid and the hyperdiploid subsets of MM, respectively [4,5]. Multicolor spectral karyotyping (SKY) [6] has repeatedly shown its ability to detect novel chromosomal aberrations in MM misidentified by or cryptic to G-banding analysis [7,8]. Therefore, through the use of SKY, we have evaluated 11 MM with complex karyotypes. Subsequent interphase fluorescence in situ hybridization (I-FISH) analyses were applied for the detection of IG gene breakpoints as well as 13q and TP53 deletions in all those cases with available material. Using this approach, we have identified hitherto unknown chromosomal sites at which translocation breaks cluster in MM.
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2. Materials and methods 2.1. Patients In this study, we analyzed MM samples from the bone marrow of 11 patients in which conventional G-banding analysis, performed as described elsewhere [9], revealed a highly complex karyotype. All cases were retrieved from the cytogenetic database of the Department of Genetics of the University of Navarra (Pamplona, Spain). This study was performed in the context of the G03/136 Red tematica FIS, Spanish Ministry of Health, and of the Health Department of the Gobierno de Navarra (946/2005), from which Review Board approval was received. Informed consent according to institutional guidelines was obtained for using the specimens for research purposes. 2.2. SKY Material left from cytogenetic analyses was used for SKY analyses. Slides were hybridized using the SKY method according to the manufacturer’s protocol (Applied Spectral Imaging, Migdal Ha’Emek, Israel). Images were acquired with an SD300 Spectra Cube (Applied Spectral Imaging) mounted on a Zeiss Axioplan microscope using a custom-designed optical filter (SKY-1; Chroma Technology, Brattleboro, VT). Up to 20 metaphase cells were captured and analyzed for each case whenever possible. SKY is somewhat limited in the determination of breakpoints and in the identification of intrachromosomal changes such as duplications, deletions, and inversions [6,7]. As a result, breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding and by comparison with G-banded karyotype of the same tumor. The chromosomal aberrations were described according to ISCN (1995) guidelines [10]. 2.3. I-FISH I-FISH was performed as described elsewhere [11] on bone marrow cells left from routine cytogenetic analyses by using break-apart probes for the IGH (Vysis, Downers Grove, IL), IGK, and IGL loci [11]. In addition, the LSI IGH-CCND1; the LSI IGH-FGFR3 double-color, doublefusion translocation probes (Vysis), and a FISH probe for the detection of IGH-MAFB translocations (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) were used. For the detection of chromosome 13 abnormalities, a multicolor FISH assay consisting of probes for RB1, D13S319, D13S25 (all of them in 13q14), and D13S327 (13q34) (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) was applied. The BAC clone RP11199F11, which spanned the TP53 gene [12], in combination with a probe for chromosome 17 centromere (CEP17; Vysis), was applied for the detection of TP53 deletions. A total
of 100 nuclei were evaluated per case by two independent observers. The cut-off levels for each probe are given in Table 2 (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) [11,13]. The LSI MYC Dual-Color break-apart rearrangement probe (Vysis) and the BAC clones flanking the breakpoints of the BCL10, FGFR1, and CCND1 locus (Nagel I, Sa´ez B, Martı´n Subero JI, Siebert R, unpublished data) [14,15] were applied for supplementary FISH analysis. In addition, a combination of the LSI MYC Dual-Color break-apart probe with the IGL break-apart probe was used for the detection of IGLMYC fusions. 3. Results 3.1. Recurrent findings detected by G-banding G-banding chromosome analysis of the 11 MM studied revealed complex changes that were not always entirely defined. The karyotypes are given in Table 1. Recurrent chromosomal breakpoints affected bands 14q32 (four cases), 1p21 (three cases), and 11q13 and 16q22~q23 (two cases each). Unresolved marker chromosomes were detected in five cases. 3.2. Recurrent findings detected by SKY As shown in Table 1, SKY analysis confirmed some aberrations detected by G-banding, unmasked the origin of marker chromosomes, and redefined various structural changes. The most frequent breakpoint cluster detected by combined SKY and G-banding mapped to the chromosomal band 1p13, which was affected in 8/11 cases. In addition, breakpoints at 22q11.2 (six cases); 8q24 and 14q32 (four cases each); Xp11, 11q13, and 13q21 (three cases each); and 12q13 (two cases) were recurrent (Table 2). 3.3. FISH for the detection of chromosome 13 abnormalities and TP53 deletions Interphase FISH analyses for the detection of 13q and TP53 deletions were performed in seven cases with available material (Table 2). In two of these MM cases, I-FISH suggested a complete loss of chromosome 13. In another three of the seven cases, I-FISH detected interstitial deletions of band 13q14 missed by G-banding and SKY, one of which was present in a hexasomic clone for chromosome 13. In the remaining two cases, I-FISH revealed the normal signal constellation, indicating the presence of two intact chromosomes 13. In four MM cases, I-FISH revealed aberrations of chromosome 17. I-FISH suggested the occurrence of a TP53 deletion missed by combined G-banding and SKY in two cases, one of which was present in a trisomic clone for chromosome 17. In the other two cases, I-FISH revealed the existence of extra copies of the TP53 locus. The remaining three cases retained both TP53 alleles, as shown by I-FISH.
Table 1 Summary of G-banding and SKY results in 11 MM cases Recurrent breakpoints detected by combined G-banding and SKY G-banding karyotype
SKY karyotype
1
48,XY,del(3)(p21),10,14, 15,del(22)(q11),D5mar[4]
48,Y,der(X)t(X;3)(q22;q21), der(1)t(1;6)(p13;q?), del(14)(q21q32), der(15)t(15;16)(q22;q?), 16,3dmin/58,idem,þ3,þ5,þ9, þ10,þ15,þ17,þ17,þ18,þ19,þ21, der(22)t(1;13;22)(?;?;q22)[4]
X
2
56,XY,þder(1)(q)x2, þder(6)(q),þ7,þ8,þ9, þ11,þ15,þ18, þ19[19] 53,Y,X,del(1)(p32), add(2)(q33),4,þ5,þ9,þ11, add(17)(p13),þ19, add(20)(q13),þ22, D4mar[4]
56,XY,þdic(1;6)(p13;q25)2, þ7,þ7,t(8;22)(q24;q11.2), þ9,þ11,þ15,þ18,þ19, þ21[10] 50~54,Y,der(X)t(X;15)(q21;?), t(1;2)(p13 ;p21), þ5,þ7,del(8)(p12),þ9,þ9, þ11,der(11)t(X;11) (q21;q23),þ15, der(16)t(X;16)(p11.2;q11.2), þ19[cp7] 43,X,Y,der(4)t(4;6)(q28;?), der(11)t(11;20)(q13;q13.1), t(12;13)(q13;q21), 13,der(14)t(11;14)(q13;q32), der(16)t(13;16)(q21;q ?), 20,der(21)t(4;21)(q?;q22)[12] 61!3nO,XY,X, der(1)t(1;19)(p13;p13.1) x2,4,del(6)(q21),t(6;17)(p21;q21), del(8)(q21),t(8;22)(q24;q11.2),10, 12,13,þ15,16,17,19,20, 22[cp11] 52,XX,t(1;2)(p13;q36), þi(3)(p10),þ5,þ7, t(8;22)(q24;q11.2), þ9,þ14,þ15[10] 45,XX,del(1)(p13), der(3)t(3;6)(p25;q?), der(6)t(X;1;6)(?;p25;q?), der(9)t(7;9)(q?;p11.2),13, t(14;20)(q32;q11.2)[10]
X
3
4
43,X,Y,der(1)(p), der(4)(q),t(11;14)(q13;q32), 13,del(16)(q22),20, add(21)(q22)[24]
5
61,!3nO,XY,X, þdel(1)(p21)x2,4,5,der(7q), 10,12,13,der(14)(q32), 16,17,19,20,22, D2mar[19]
6
52,XX,þdel(1)(p21)x2, add(2)(q37),þder(3),þ4,þ7, þ9,13,16,19,þ21,22, D3mar[10] 45,XX,del(1)(p?),add(3)(p26), der(6),add(9)(p24),13, add(14)(q32),del(16)(q23)[41]
7
Xp11
X
1p13
8q24
11q13
12q13
13q21
14q32
22q11.1
X
X
X
X
X,X
X
X,X
X
X
X
X
X
X
X
X
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Case no.
X
(Continued)
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Table 1 Continued Recurrent breakpoints detected by combined G-banding and SKY G-banding karyotype
SKY karyotype
Xp11
1p13
8
56,XY,þdel(1)(p21), þ3,þ5,þ9,þadd(11)(p15), þ15,þ18,þ19,þ21, Dmar[9]
X
X
9
3940,XY,cx[28]
10
45,X,Y;8892,!4O,XXYY, t(11;14)(q13;q32), cx[50]
11
50,XY,þ5,del(6)(q22)2, þ9,der(12),t(12;15?) (q24;q11),add(13)(q34), þ15,del(18)(q12), þ19[16]
56,XY,þ1,þ der(1)t(1;22)(p13;q11.2), þ3,þ5,þ9,i(11)(q10), þder(11)t(X;11)(p11.2;q13), þ15,þ18,þ19,þ21 [cp5] 39,X,Y,2,der(5)t(X;5;9) (?;?;q35),der(8)t(3;8)(?;p22), der(8)t(8;22)(p11.2;q11.2), der(10)t(9;10)(?;p11.2), der(11)t(11;22)(p11.2;q11.2), del(12)(q13),13,13,14,15, der(16)t(X;13;16)(?;?;q21), der(17)t(4;17)(q11.2;p11.2), 22[15] 90,!4nO,XXYY, del(1)(p13),2,4, der(7)t(7;15)(p11.2;q?), t(8;13)(q24;q14L21),del(9)(q12), der(10)t(1;10)(p?;p?), t(11;14)(q13;q32), der(19)t(13;19)(?;q13), der(22)t(10;22)(q?;q11.2) [cp12] 50,XY,þ5,der(6)t(X;6) (p11.2;q11.2)2, þ9,der(12)t(1;12)(q21;q24), dup(13)(q21q34), þ15,del(18)(q21),þ19[12]
8q24
11q13
12q13
13q21
14q32
X
X
X
X
X
G-banding and spectral karyotyping are described according to the ISCN (1995) guidelines [10]. Bold indicates recurrent cytogenetic aberrations detected by means of G-banding and SKY analysis.
X
X
22q11.1
X
X
dup(13)(q21q34)
X
X
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Case no.
Table 2 Chromosome 13q14 and 17p13 (TP53) deletions and breakpoints affecting the IG genes detected by means of I-FISH Interphase FISH resultsa Case
3
4
5
6
7
8 9
10
11
nd nd
nuc ish(RB1x2, D13S319x2,D13S25x2, D13S327x2) nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x2)[65%] nuc ish(RB1x2, D13S319x2,D13S25x2, D13S327x2) nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x2)[15%] nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x1)[61%] nd nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x1)[85%] nuc ish(RB1x4, D13S319x4,D13S25x4, D13S327x6)[62%] nd
TP53-CEP17 [13] (10%) nd nd
LSI IGH break apart (Vysis) [13] (1%)
Oncogene fused to IGH
nd nd
nd nd
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2) [61%]
CCND1 and MAFB
nuc ish(TP53x3-4, CEP17x2)[49%]
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x3)[44%]
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2) (IGH-cen sep IGH-telx1)[51%] nd nuc ish(IGHx1) (IGH-cen sep IGH-telx1)[80%] nuc ish(IGHx2~3, IGH-telx2)(IGH-cen sep IGH-telx2)[57%] nd
nd nuc ish(TP53x1, CEP17x2)[74%] nuc ish(TP53x4, CEP17x4)[55%] nd
IGL break apart [11] (1.4%)
Oncogene fused to IGL
IGK break apart [11] (0.3%)
n.a. nd
nd nd
nuc ish(IGLx2) nuc ish(IGLx2) (IGL-cen sep IGL-telx1)[40%] nuc ish(IGLx2)
n.a.
nuc ish(IGK-cen x1, IGK-telx2)
nuc ish(IGLx2)
n.a.
nuc ish(IGKx2)
MYC
nuc ish(IGKx3) [50%]
MYC
nuc ish(IGKx2)
MAFB
nuc ish(IGLx2) (IGL-cen sep IGL-telx1) [52%] nuc ish(IGLx2) (IGL-cen sep IGL-telx1) [67%] nuc ish(IGLx2)
n.a.
nuc ish(IGKx2)
nd FGFR3
nuc ish(IGLx2) nuc ish(IGL-cenx1)
n.a. n.a.
nd nuc ish(IGKx2)
CCND1 and FGFR3
nuc ish(IGL-cenx4)
n.a.
nuc ish(IGKx3-4) [50%]
nd
nd
nd
B. Sa´ez et al. / Cancer Genetics and Cytogenetics 169 (2006) 143e149
1 2
chr. 13 probe* (2%)
nd
Recurrent abnormalities appear in bold type. Recurrent breakpoints were detected by combined G-banding and SKY. a Described according to the ISCN-95 guidelines [10]. cen: probe located centromeric to the gene; tel: probe located telomeric to the gene; nd: not done; n.a.: not applicable. * Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation.
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3.4. Immunoglobulin gene rearrangements
3.5. FISH analyses for the MYC and CCND1 oncogenes
I-FISH analyses for the detection of IG breakpoints were performed in seven cases with available material (Table 2). I-FISH confirmed the t(11;14)(q13;q32) and the t(14;20)(q32;q13) detected by combined G-banding and SKY analysis, leading to IGH-CCND1 and IGH-MAFB fusions, respectively. Two cases showed IGH breakpoints missed by G-banding and SKY. In both cases, an IGHFGFR3 fusion was identified by I-FISH. Interestingly, in one of these cases, an IGH-FGFR3 and an IGH-CCND1 fusion were shown to coexist in the same tumor clone. Six MM showed a rearrangement at 22q11 by combined G-banding and SKY; three of these resulted from a t(8;22)(q24;q11.2). Subsequent FISH analyses detected involvement of the IGL locus in all three cases. An IGL-MYC fusion was further confirmed in the two cases where material was available. Remarkably, the other three MM with 22q11 aberrations lacked evidence for IGL involvement by I-FISH. In these three cases, SKY revealed four nonrecurrent unbalanced translocations, namely der(1)t(1;22) (p13;q11), der(22)t(10;22)(q?;q11), der(8)t(8;22)(p11;q11), and der(11)t(11;22)(p11.2;q11). Involvement of FGFR1 (8p12) was excluded in the der(8)t(8;22)(p11.2;q11.2) by means of I-FISH. An IGK (2p12) breakpoint was detected in a single case with a t(1;2)(p13;p21) (Table 2). Additional I-FISH analyses excluded the involvement of BCL10 (1p22) in that particular case.
FISH was performed in those cases with available material and in which combined G-banding and SKY revealed breakpoints close to the MYC and CCND1 oncogenes, which are known to be involved in lymphomagenesis. MYC (8q24) was found rearranged and gained by means of FISH in a case carrying a t(8;13)(q24;q14~q21), but it was not possible to prove whether MYC was implicated in the translocation with band 13q14~q21. Combined G-banding and SKY showed two different rearrangements affecting band 11q13 as a result of a der(11)t(11;20)(q13;q13.1) and der(14)t(11;14) (q13;q32) in one case (case no. 4). FISH confirmed the presence of an IGH-CCND1 fusion, as discussed above, and also revealed a second rearrangement affecting the derivative chromosome 11 from the t(11;14)(q13;q32). Subsequent FISH analyses showed the involvement of the IGH and MAFB genes in the der(11)t(11;20)(q13;q13.1) described by SKY (Fig. 1). 4. Discussion Through the application of SKY, a range of chromosomal rearrangements that were either misclassified or unrecognizable by G-banding analyses was detected in the present study. SKY analyses identified several chromosomal sites at which translocation breaks clustered. The most frequent breakpoint location was in band 1p13, which has been recurrently affected in MM [16]. Other
Fig. 1. (A) Combination of G-banding, SKY, and FISH analysis in case 4 increases the accuracy of karyotype interpretation. (A1) G-banding analysis revealed a balanced t(11;14)(q13;q32). (A2) Posterior SKY analysis redefined the abnormality as a der(11)t(11;20)(q13;q13.1) and a der(14)t(11;14)(q13;q32). (A3) Finally, FISH accurately demonstrated an IGH-CCND1 fusion (arrow) and an IGH-MAFB fusion (arrowhead) as a result of the der(14)t(11;14)(q13;q32) and of the der(11)t(11;20)(q13;q13.1), respectively. (B) SKY analysis showing three of the 22q11 rearrangements identified in the present study, in which FISH analysis revealed lack of IGL involvement. (From left to right) der(1)t(1;22)(p13;q11), der(11)t(11;22)(p11.2;q11), and der(22)t(10;22)(q?;q11).
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chromosomal sites, including Xp11, 8q24, 11q13, 12q13, 13q21, and 14q32, have been recurrently affected in our series. In addition, three chromosomal translocations that affect band 22q11 but lack IGL involvement have been identified, suggesting the implication of alternative gene(s) located in this chromosomal band in MM. It is noteworthy that anomalies in 22q11 detected by G-banding have been associated with adverse prognosis in MM [17], therefore a profound study of the gene(s) involved in these aberrations and their implication in myeloma pathogenesis is mandatory. Furthermore, a novel t(1;2)(p13;p21) was identified by SKY in one case, in which subsequent I-FISH analysis revealed an IGK rearrangement. Despite these findings, SKY, as well as G-banding first, failed to detect interstitial deletions affecting 13q14 or 17p13, as well as cryptic rearrangements leading to the IGH-FGFR3 and IGH-MAFB fusions, which were only detected by applying I-FISH. In summary, our results provide evidence of the ability of SKY to detect novel recurrent chromosomal rearrangements that might have pathogenic potential in MM and, therefore, warrant future investigations in the biologic effects of such aberrations. In addition, combination of SKY with I-FISH increases the accuracy of karyotype interpretation, permitting a more precise breakpoint mapping and the detection of small interstitial deletions and cryptic translocations in MM, which is important for proper diagnosis and patient management.
Acknowledgments The authors thank the scientific and technical staff of the Department of Genetics of the University of Navarra (Pamplona, Spain); of the Cytogenetic Unit of the CNIO (Madrid, Spain); and of the Institute of Human Genetics (Kiel, Germany). This work has been partially supported by G03/136 Red Tema´tica FIS, Spanish Ministry of Health and by the Health Department of the Gobierno de Navarra (946/2005). Borja Sa´ez and Cristina Largo are scholars of the Gobierno de Navarra (Pamplona, Spain). References [1] Fonseca R, Blood E, Rue M, Harrington D, Oken MM, Kyle RA, Dewald GW, Van Ness B, Van Wier SA, Henderson KJ, Bailey RJ, Greipp PR. Clinical and biologic implications of recurrent genomic aberrations in myeloma. Blood 2003;101:4569e75. [2] Gertz MA, Lacy MQ, Dispenzieri A, Greipp PR, Litzow MR, Henderson KJ, Van Wier SA, Ahmann GJ, Fonseca R. Clinical implications of t(11;14)(q13;q32), t(4;14)(p16.3;q32), and e17p13 in myeloma patients treated with high-dose therapy. Blood 2005;106: 2837e40. [3] Chang H, Qi C, Yi QL, Reece D, Stewart AK. p53 gene deletion detected by fluorescence in situ hybridization is an adverse prognostic factor for patients with multiple myeloma following autologous stem cell transplantation. Blood 2005;105:358e60.
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[4] Saez B, Martin-Subero JI, Guillen-Grima F, Odero MD, Prosper F, Cigudosa JC, Harder L, Calasanz MJ, Siebert R. Chromosomal abnormalities clustering in multiple myeloma reveals cytogenetic subgroups with nonrandom acquisition of chromosomal changes. Leukemia 2004;18:654e7. [5] Debes-Marun CS, Dewald GW, Bryant S, Picken E, SantanaDavila R, Gonzalez-Paz N, Winkler JM, Kyle RA, Gertz MA, Witzig TE, Dispenzieri A, Lacy MQ, Rajkumar SV, Lust JA, Greipp PR, Fonseca R. Chromosome abnormalities clustering and its implications for pathogenesis and prognosis in myeloma. Leukemia 2003;17:427e36. [6] Schro¨ck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494e7. [7] Rao PH, Cigudosa JC, Ning Y, Calasanz MJ, Iida S, Tagawa S, Michaeli J, Klein B, Dalla-Favera R, Jhanwar SC, Ried T, Chaganti RS. Multicolor spectral karyotyping identifies new recurring breakpoints and translocations in multiple myeloma. Blood 1998;92:1743e8. [8] Sawyer JR, Lukacs JL, Munshi N, Desikan KR, Singhal S, Mehta J, Siegel D, Shaughnessy J, Barlogie B. Identification of new nonrandom translocations in multiple myeloma with multicolor spectral karyotyping. Blood 1998;92:4269e78. [9] Calasanz MJ, Cigudosa JC, Odero MD, Ferreira C, Ardanaz MT, Fraile A, Carrasco JL, Sole F, Cuesta B, Gullo´n A. Cytogenetic analysis of 280 patients with multiple myeloma and related disorders: primary breakpoints and clinical correlations. Genes Chromosomes Cancer 1997;18:84e93. [10] ISCN. An international system for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [11] Martin-Subero JI, Harder L, Gesk S, Schlegelberger B, Grote W, Martinez-Climent JA, Dyer MJ, Novo FJ, Calasanz MJ, Siebert R. Interphase FISH assays for the detection of translocations with breakpoints in immunoglobulin light chain loci. Int J Cancer 2002;98: 470e4. [12] Agirre X, Novo FJ, Calasanz MJ, Larrayoz MJ, Lahortiga I, Valganon M, Garcia-Delgado M, Vizmanos JL. TP53 is frequently altered by methylation, mutation, and/or deletion in acute lymphoblastic leukaemia. Mol Carcinog 2003;38:201e8. [13] Martin-Subero JI, Gesk S, Harder L, Grote W, Siebert R. Interphase cytogenetics of hematological neoplasms under the perspective of the novel WHO classification. Anticancer Res 2003;23:1139e48. [14] Ye H, Liu H, Attygalle A, Wotherspoon AC, Nicholson AG, Charlotte F, Leblond V, Speight P, Goodlad J, Lavergne-Slove A, Martin-Subero JI, Siebert R, Dogan A, Isaacson PG, Du MQ. Variable frequencies of t(11;18)(q21;q21) in MALT lymphomas of different sites: significant association with CagA strains of H pylori in gastric MALT lymphoma. Blood 2003;102:1012e8. [15] Rubio-Moscardo F, Climent J, Siebert R, Piris MA, Martin-Subero JI, Nielander I, Garcia-Conde J, Dyer MJ, Terol MJ, Pinkel D, MartinezCliment JA. Mantle-cell lymphoma genotypes identified with CGH to BAC microarrays define a leukemic subgroup of disease and predict patient outcome. Blood 2005;105:4445e54. [16] Nilsson T, Hoglund M, Lenhoff S, Rylander L, Turesson I, Westin J, Mitelman F, Johansson B. A pooled analysis of karyotypic patterns, breakpoints and imbalances in 783 cytogenetically abnormal multiple myelomas reveals frequently involved chromosome segments as well as significant age- and sex-related differences. Br J Haematol 2003;120:960e9. [17] Calasanz MJ, Cigudosa JC, Odero MD, Garcia-Foncillas J, Marin J, Ardanaz MT, Rocha E, Gullon A. Hypodiploidy and 22q11 rearrangements at diagnosis are associated with poor prognosis in patients with multiple myeloma. Br J Haematol 1997;98:418e25.
Identification of recurrent chromosomal breakpoints in multiple myeloma with complex karyotypes by combined G-banding, spectral karyotyping, and fluorescence in situ hybridization analyses Borja Sa´eza,b,*, Jose´ I. Martı´n-Suberob, Cristina Largoc, Marı´a C. Martı´nc, Marı´a D. Oderoa,d, Felipe Prosperd, Reiner Siebertb, Marı´a J. Calasanza, Juan C. Cigudosac a Department of Genetics, University of Navarra, Irunlarrea s/n, Pamplona, Navarra 31008, Spain Institute of Human Genetics, University Hospital Schleswig-Holstein, Schwanenweg 24, Campus Kiel, Kiel, Germany, D-24105 c Cytogenetics Unit, Spanish National Cancer Research Center, Melchor Fernandez Almagrd 3, Madrid, Spain, 28029 d Division of Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Irunlarrea s/n, Pamplona, Navarra 31008, Spain b
Received 30 January 2006; received in revised form 7 April 2006; accepted 11 April 2006
Abstract
The description of novel chromosomal aberrations in multiple myeloma (MM) remains necessary to fully understand the pathogenesis of this heterogeneous disease. Therefore, we have used spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) with locus-specific probes to characterize the chromosomal abnormalities in 11 MM cases in which G-banding revealed a complex karyotype. SKY refined G-banding karyotypes in all cases. Recurrent breakpoints involved bands Xp11, 8q24, 11q13, 12q13, 13q21, and 14q32. In addition, combined SKY and FISH analyses permitted us to identify a subset of patients harboring 22q11.2 rearrangements not involving the IGL locus. This finding suggests the presence of other gene(s) in band 22q11 that might be implicated in MM pathogenesis. Moreover, band 1p13 was identified as a novel partner of immunoglobulin (IG) translocations in MM. Finally, using interphase FISH, we have detected interstitial deletions in 13q14 and 17p13, as well as cryptic translocations affecting IGH, which were neither detected by G-banding nor by SKY. The results of the present study suggest the existence of hitherto unknown nonrandom chromosomal changes that may play a role in the pathogenesis of MM. Our findings underline the importance of the combination of banding, SKY, and FISH analyses to increase the accuracy of karyotype interpretation in plasma cell neoplasias. Ó2006 Elsevier Inc. All rights reserved. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction The clinical importance of specific chromosome abnormalities in multiple myeloma (MM) has been highlighted by several studies. Translocations with breakpoints in immunoglobulin (IG) genes (i.e., IGH in 14q32, IGK in 2p12, and IGL in 22q11.2), such as the t(4;14)(p16;q32) and the t(14;16)(q32;q23), are associated with an adverse impact on prognosis [1,2]. Likewise, deletions of the tumor suppressor gene TP53 (17p13), as well as chromosome 13 abnormalities, mainly monosomy 13, are also associated with poorer outcome in MM [1e3]. However, the description of novel chromosomal aberrations that might be implicated in MM pathogenesis remains necessary for the full characterization of the genetic basis of this heterogeneous * Corresponding author. Tel.: þ34-948-425600; fax: þ34-948-425649. E-mail address: [email protected] (B. Sa´ez). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.04.008
disease. Nowadays, two groups of cases are of major interest for the identification of genetic markers in MM: those with locus breakpoints involving novel partner genes and those with recurrent breakpoints not involving IG genes, which might be associated with the nonhyperdiploid and the hyperdiploid subsets of MM, respectively [4,5]. Multicolor spectral karyotyping (SKY) [6] has repeatedly shown its ability to detect novel chromosomal aberrations in MM misidentified by or cryptic to G-banding analysis [7,8]. Therefore, through the use of SKY, we have evaluated 11 MM with complex karyotypes. Subsequent interphase fluorescence in situ hybridization (I-FISH) analyses were applied for the detection of IG gene breakpoints as well as 13q and TP53 deletions in all those cases with available material. Using this approach, we have identified hitherto unknown chromosomal sites at which translocation breaks cluster in MM.
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2. Materials and methods 2.1. Patients In this study, we analyzed MM samples from the bone marrow of 11 patients in which conventional G-banding analysis, performed as described elsewhere [9], revealed a highly complex karyotype. All cases were retrieved from the cytogenetic database of the Department of Genetics of the University of Navarra (Pamplona, Spain). This study was performed in the context of the G03/136 Red tematica FIS, Spanish Ministry of Health, and of the Health Department of the Gobierno de Navarra (946/2005), from which Review Board approval was received. Informed consent according to institutional guidelines was obtained for using the specimens for research purposes. 2.2. SKY Material left from cytogenetic analyses was used for SKY analyses. Slides were hybridized using the SKY method according to the manufacturer’s protocol (Applied Spectral Imaging, Migdal Ha’Emek, Israel). Images were acquired with an SD300 Spectra Cube (Applied Spectral Imaging) mounted on a Zeiss Axioplan microscope using a custom-designed optical filter (SKY-1; Chroma Technology, Brattleboro, VT). Up to 20 metaphase cells were captured and analyzed for each case whenever possible. SKY is somewhat limited in the determination of breakpoints and in the identification of intrachromosomal changes such as duplications, deletions, and inversions [6,7]. As a result, breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding and by comparison with G-banded karyotype of the same tumor. The chromosomal aberrations were described according to ISCN (1995) guidelines [10]. 2.3. I-FISH I-FISH was performed as described elsewhere [11] on bone marrow cells left from routine cytogenetic analyses by using break-apart probes for the IGH (Vysis, Downers Grove, IL), IGK, and IGL loci [11]. In addition, the LSI IGH-CCND1; the LSI IGH-FGFR3 double-color, doublefusion translocation probes (Vysis), and a FISH probe for the detection of IGH-MAFB translocations (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) were used. For the detection of chromosome 13 abnormalities, a multicolor FISH assay consisting of probes for RB1, D13S319, D13S25 (all of them in 13q14), and D13S327 (13q34) (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) was applied. The BAC clone RP11199F11, which spanned the TP53 gene [12], in combination with a probe for chromosome 17 centromere (CEP17; Vysis), was applied for the detection of TP53 deletions. A total
of 100 nuclei were evaluated per case by two independent observers. The cut-off levels for each probe are given in Table 2 (Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation) [11,13]. The LSI MYC Dual-Color break-apart rearrangement probe (Vysis) and the BAC clones flanking the breakpoints of the BCL10, FGFR1, and CCND1 locus (Nagel I, Sa´ez B, Martı´n Subero JI, Siebert R, unpublished data) [14,15] were applied for supplementary FISH analysis. In addition, a combination of the LSI MYC Dual-Color break-apart probe with the IGL break-apart probe was used for the detection of IGLMYC fusions. 3. Results 3.1. Recurrent findings detected by G-banding G-banding chromosome analysis of the 11 MM studied revealed complex changes that were not always entirely defined. The karyotypes are given in Table 1. Recurrent chromosomal breakpoints affected bands 14q32 (four cases), 1p21 (three cases), and 11q13 and 16q22~q23 (two cases each). Unresolved marker chromosomes were detected in five cases. 3.2. Recurrent findings detected by SKY As shown in Table 1, SKY analysis confirmed some aberrations detected by G-banding, unmasked the origin of marker chromosomes, and redefined various structural changes. The most frequent breakpoint cluster detected by combined SKY and G-banding mapped to the chromosomal band 1p13, which was affected in 8/11 cases. In addition, breakpoints at 22q11.2 (six cases); 8q24 and 14q32 (four cases each); Xp11, 11q13, and 13q21 (three cases each); and 12q13 (two cases) were recurrent (Table 2). 3.3. FISH for the detection of chromosome 13 abnormalities and TP53 deletions Interphase FISH analyses for the detection of 13q and TP53 deletions were performed in seven cases with available material (Table 2). In two of these MM cases, I-FISH suggested a complete loss of chromosome 13. In another three of the seven cases, I-FISH detected interstitial deletions of band 13q14 missed by G-banding and SKY, one of which was present in a hexasomic clone for chromosome 13. In the remaining two cases, I-FISH revealed the normal signal constellation, indicating the presence of two intact chromosomes 13. In four MM cases, I-FISH revealed aberrations of chromosome 17. I-FISH suggested the occurrence of a TP53 deletion missed by combined G-banding and SKY in two cases, one of which was present in a trisomic clone for chromosome 17. In the other two cases, I-FISH revealed the existence of extra copies of the TP53 locus. The remaining three cases retained both TP53 alleles, as shown by I-FISH.
Table 1 Summary of G-banding and SKY results in 11 MM cases Recurrent breakpoints detected by combined G-banding and SKY G-banding karyotype
SKY karyotype
1
48,XY,del(3)(p21),10,14, 15,del(22)(q11),D5mar[4]
48,Y,der(X)t(X;3)(q22;q21), der(1)t(1;6)(p13;q?), del(14)(q21q32), der(15)t(15;16)(q22;q?), 16,3dmin/58,idem,þ3,þ5,þ9, þ10,þ15,þ17,þ17,þ18,þ19,þ21, der(22)t(1;13;22)(?;?;q22)[4]
X
2
56,XY,þder(1)(q)x2, þder(6)(q),þ7,þ8,þ9, þ11,þ15,þ18, þ19[19] 53,Y,X,del(1)(p32), add(2)(q33),4,þ5,þ9,þ11, add(17)(p13),þ19, add(20)(q13),þ22, D4mar[4]
56,XY,þdic(1;6)(p13;q25)2, þ7,þ7,t(8;22)(q24;q11.2), þ9,þ11,þ15,þ18,þ19, þ21[10] 50~54,Y,der(X)t(X;15)(q21;?), t(1;2)(p13 ;p21), þ5,þ7,del(8)(p12),þ9,þ9, þ11,der(11)t(X;11) (q21;q23),þ15, der(16)t(X;16)(p11.2;q11.2), þ19[cp7] 43,X,Y,der(4)t(4;6)(q28;?), der(11)t(11;20)(q13;q13.1), t(12;13)(q13;q21), 13,der(14)t(11;14)(q13;q32), der(16)t(13;16)(q21;q ?), 20,der(21)t(4;21)(q?;q22)[12] 61!3nO,XY,X, der(1)t(1;19)(p13;p13.1) x2,4,del(6)(q21),t(6;17)(p21;q21), del(8)(q21),t(8;22)(q24;q11.2),10, 12,13,þ15,16,17,19,20, 22[cp11] 52,XX,t(1;2)(p13;q36), þi(3)(p10),þ5,þ7, t(8;22)(q24;q11.2), þ9,þ14,þ15[10] 45,XX,del(1)(p13), der(3)t(3;6)(p25;q?), der(6)t(X;1;6)(?;p25;q?), der(9)t(7;9)(q?;p11.2),13, t(14;20)(q32;q11.2)[10]
X
3
4
43,X,Y,der(1)(p), der(4)(q),t(11;14)(q13;q32), 13,del(16)(q22),20, add(21)(q22)[24]
5
61,!3nO,XY,X, þdel(1)(p21)x2,4,5,der(7q), 10,12,13,der(14)(q32), 16,17,19,20,22, D2mar[19]
6
52,XX,þdel(1)(p21)x2, add(2)(q37),þder(3),þ4,þ7, þ9,13,16,19,þ21,22, D3mar[10] 45,XX,del(1)(p?),add(3)(p26), der(6),add(9)(p24),13, add(14)(q32),del(16)(q23)[41]
7
Xp11
X
1p13
8q24
11q13
12q13
13q21
14q32
22q11.1
X
X
X
X
X,X
X
X,X
X
X
X
X
X
X
X
X
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Case no.
X
(Continued)
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Table 1 Continued Recurrent breakpoints detected by combined G-banding and SKY G-banding karyotype
SKY karyotype
Xp11
1p13
8
56,XY,þdel(1)(p21), þ3,þ5,þ9,þadd(11)(p15), þ15,þ18,þ19,þ21, Dmar[9]
X
X
9
3940,XY,cx[28]
10
45,X,Y;8892,!4O,XXYY, t(11;14)(q13;q32), cx[50]
11
50,XY,þ5,del(6)(q22)2, þ9,der(12),t(12;15?) (q24;q11),add(13)(q34), þ15,del(18)(q12), þ19[16]
56,XY,þ1,þ der(1)t(1;22)(p13;q11.2), þ3,þ5,þ9,i(11)(q10), þder(11)t(X;11)(p11.2;q13), þ15,þ18,þ19,þ21 [cp5] 39,X,Y,2,der(5)t(X;5;9) (?;?;q35),der(8)t(3;8)(?;p22), der(8)t(8;22)(p11.2;q11.2), der(10)t(9;10)(?;p11.2), der(11)t(11;22)(p11.2;q11.2), del(12)(q13),13,13,14,15, der(16)t(X;13;16)(?;?;q21), der(17)t(4;17)(q11.2;p11.2), 22[15] 90,!4nO,XXYY, del(1)(p13),2,4, der(7)t(7;15)(p11.2;q?), t(8;13)(q24;q14L21),del(9)(q12), der(10)t(1;10)(p?;p?), t(11;14)(q13;q32), der(19)t(13;19)(?;q13), der(22)t(10;22)(q?;q11.2) [cp12] 50,XY,þ5,der(6)t(X;6) (p11.2;q11.2)2, þ9,der(12)t(1;12)(q21;q24), dup(13)(q21q34), þ15,del(18)(q21),þ19[12]
8q24
11q13
12q13
13q21
14q32
X
X
X
X
X
G-banding and spectral karyotyping are described according to the ISCN (1995) guidelines [10]. Bold indicates recurrent cytogenetic aberrations detected by means of G-banding and SKY analysis.
X
X
22q11.1
X
X
dup(13)(q21q34)
X
X
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Case no.
Table 2 Chromosome 13q14 and 17p13 (TP53) deletions and breakpoints affecting the IG genes detected by means of I-FISH Interphase FISH resultsa Case
3
4
5
6
7
8 9
10
11
nd nd
nuc ish(RB1x2, D13S319x2,D13S25x2, D13S327x2) nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x2)[65%] nuc ish(RB1x2, D13S319x2,D13S25x2, D13S327x2) nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x2)[15%] nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x1)[61%] nd nuc ish(RB1x1, D13S319x1,D13S25x1, D13S327x1)[85%] nuc ish(RB1x4, D13S319x4,D13S25x4, D13S327x6)[62%] nd
TP53-CEP17 [13] (10%) nd nd
LSI IGH break apart (Vysis) [13] (1%)
Oncogene fused to IGH
nd nd
nd nd
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2) [61%]
CCND1 and MAFB
nuc ish(TP53x3-4, CEP17x2)[49%]
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x3)[44%]
nuc ish(IGHx2)
n.a.
nuc ish(TP53x2, CEP17x2)
nuc ish(IGHx2) (IGH-cen sep IGH-telx1)[51%] nd nuc ish(IGHx1) (IGH-cen sep IGH-telx1)[80%] nuc ish(IGHx2~3, IGH-telx2)(IGH-cen sep IGH-telx2)[57%] nd
nd nuc ish(TP53x1, CEP17x2)[74%] nuc ish(TP53x4, CEP17x4)[55%] nd
IGL break apart [11] (1.4%)
Oncogene fused to IGL
IGK break apart [11] (0.3%)
n.a. nd
nd nd
nuc ish(IGLx2) nuc ish(IGLx2) (IGL-cen sep IGL-telx1)[40%] nuc ish(IGLx2)
n.a.
nuc ish(IGK-cen x1, IGK-telx2)
nuc ish(IGLx2)
n.a.
nuc ish(IGKx2)
MYC
nuc ish(IGKx3) [50%]
MYC
nuc ish(IGKx2)
MAFB
nuc ish(IGLx2) (IGL-cen sep IGL-telx1) [52%] nuc ish(IGLx2) (IGL-cen sep IGL-telx1) [67%] nuc ish(IGLx2)
n.a.
nuc ish(IGKx2)
nd FGFR3
nuc ish(IGLx2) nuc ish(IGL-cenx1)
n.a. n.a.
nd nuc ish(IGKx2)
CCND1 and FGFR3
nuc ish(IGL-cenx4)
n.a.
nuc ish(IGKx3-4) [50%]
nd
nd
nd
B. Sa´ez et al. / Cancer Genetics and Cytogenetics 169 (2006) 143e149
1 2
chr. 13 probe* (2%)
nd
Recurrent abnormalities appear in bold type. Recurrent breakpoints were detected by combined G-banding and SKY. a Described according to the ISCN-95 guidelines [10]. cen: probe located centromeric to the gene; tel: probe located telomeric to the gene; nd: not done; n.a.: not applicable. * Sa´ez B, Martı´n-Subero JI, Odero MD, Prosper F, Cigudosa JC, Schoch R, Calasanz MJ, Siebert R, manuscript in preparation.
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3.4. Immunoglobulin gene rearrangements
3.5. FISH analyses for the MYC and CCND1 oncogenes
I-FISH analyses for the detection of IG breakpoints were performed in seven cases with available material (Table 2). I-FISH confirmed the t(11;14)(q13;q32) and the t(14;20)(q32;q13) detected by combined G-banding and SKY analysis, leading to IGH-CCND1 and IGH-MAFB fusions, respectively. Two cases showed IGH breakpoints missed by G-banding and SKY. In both cases, an IGHFGFR3 fusion was identified by I-FISH. Interestingly, in one of these cases, an IGH-FGFR3 and an IGH-CCND1 fusion were shown to coexist in the same tumor clone. Six MM showed a rearrangement at 22q11 by combined G-banding and SKY; three of these resulted from a t(8;22)(q24;q11.2). Subsequent FISH analyses detected involvement of the IGL locus in all three cases. An IGL-MYC fusion was further confirmed in the two cases where material was available. Remarkably, the other three MM with 22q11 aberrations lacked evidence for IGL involvement by I-FISH. In these three cases, SKY revealed four nonrecurrent unbalanced translocations, namely der(1)t(1;22) (p13;q11), der(22)t(10;22)(q?;q11), der(8)t(8;22)(p11;q11), and der(11)t(11;22)(p11.2;q11). Involvement of FGFR1 (8p12) was excluded in the der(8)t(8;22)(p11.2;q11.2) by means of I-FISH. An IGK (2p12) breakpoint was detected in a single case with a t(1;2)(p13;p21) (Table 2). Additional I-FISH analyses excluded the involvement of BCL10 (1p22) in that particular case.
FISH was performed in those cases with available material and in which combined G-banding and SKY revealed breakpoints close to the MYC and CCND1 oncogenes, which are known to be involved in lymphomagenesis. MYC (8q24) was found rearranged and gained by means of FISH in a case carrying a t(8;13)(q24;q14~q21), but it was not possible to prove whether MYC was implicated in the translocation with band 13q14~q21. Combined G-banding and SKY showed two different rearrangements affecting band 11q13 as a result of a der(11)t(11;20)(q13;q13.1) and der(14)t(11;14) (q13;q32) in one case (case no. 4). FISH confirmed the presence of an IGH-CCND1 fusion, as discussed above, and also revealed a second rearrangement affecting the derivative chromosome 11 from the t(11;14)(q13;q32). Subsequent FISH analyses showed the involvement of the IGH and MAFB genes in the der(11)t(11;20)(q13;q13.1) described by SKY (Fig. 1). 4. Discussion Through the application of SKY, a range of chromosomal rearrangements that were either misclassified or unrecognizable by G-banding analyses was detected in the present study. SKY analyses identified several chromosomal sites at which translocation breaks clustered. The most frequent breakpoint location was in band 1p13, which has been recurrently affected in MM [16]. Other
Fig. 1. (A) Combination of G-banding, SKY, and FISH analysis in case 4 increases the accuracy of karyotype interpretation. (A1) G-banding analysis revealed a balanced t(11;14)(q13;q32). (A2) Posterior SKY analysis redefined the abnormality as a der(11)t(11;20)(q13;q13.1) and a der(14)t(11;14)(q13;q32). (A3) Finally, FISH accurately demonstrated an IGH-CCND1 fusion (arrow) and an IGH-MAFB fusion (arrowhead) as a result of the der(14)t(11;14)(q13;q32) and of the der(11)t(11;20)(q13;q13.1), respectively. (B) SKY analysis showing three of the 22q11 rearrangements identified in the present study, in which FISH analysis revealed lack of IGL involvement. (From left to right) der(1)t(1;22)(p13;q11), der(11)t(11;22)(p11.2;q11), and der(22)t(10;22)(q?;q11).
B. Sa´ez et al. / Cancer Genetics and Cytogenetics 169 (2006) 143e149
chromosomal sites, including Xp11, 8q24, 11q13, 12q13, 13q21, and 14q32, have been recurrently affected in our series. In addition, three chromosomal translocations that affect band 22q11 but lack IGL involvement have been identified, suggesting the implication of alternative gene(s) located in this chromosomal band in MM. It is noteworthy that anomalies in 22q11 detected by G-banding have been associated with adverse prognosis in MM [17], therefore a profound study of the gene(s) involved in these aberrations and their implication in myeloma pathogenesis is mandatory. Furthermore, a novel t(1;2)(p13;p21) was identified by SKY in one case, in which subsequent I-FISH analysis revealed an IGK rearrangement. Despite these findings, SKY, as well as G-banding first, failed to detect interstitial deletions affecting 13q14 or 17p13, as well as cryptic rearrangements leading to the IGH-FGFR3 and IGH-MAFB fusions, which were only detected by applying I-FISH. In summary, our results provide evidence of the ability of SKY to detect novel recurrent chromosomal rearrangements that might have pathogenic potential in MM and, therefore, warrant future investigations in the biologic effects of such aberrations. In addition, combination of SKY with I-FISH increases the accuracy of karyotype interpretation, permitting a more precise breakpoint mapping and the detection of small interstitial deletions and cryptic translocations in MM, which is important for proper diagnosis and patient management.
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