Deletions of chromosome 13 in multiple myeloma identified by interphase FISH usually denote large deletions of the q arm or monosomy

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1 (2001) 15, Nature Publishing Group All rights reserved /01 $ Deletions of chromosome 13 in multiple myeloma identified by interphase FISH usually denote large deletions of the q arm or monosomy R Fonseca 1, MM Oken 2, D Harrington 3, RJ Bailey 1, SA Van Wier 1, KJ Henderson 1, NE Kay 1, B Van Ness 4, PR Greipp 1 and GW Dewald 1 1 Mayo Clinic Department of Hematology and Internal Medicine, Department of Laboratory Medicine and Pathology; 2 Virginia Piper Cancer Institute, Minneapolis, MN; 3 ECOG Statistical Center, Dana Farber Cancer Institute, Boston, MA; and 4 University of Minnesota, Cancer Center, Minneapolis, MN, USA Deletions of the long arm of chromosome 13 (13q ) are observed in patients with multiple myeloma (MM), are rarely observed in the monoclonal gammopathy of undetermined significance (MGUS) and have been associated with a worsened prognosis in MM. However, no minimally deleted region in the13q arm has been defined at 13q, and consequently no tumor suppressor genes have yet been identified that are important for disease pathogenesis. We attempted to characterize these chromosome 13q deletions at the molecular cytogenetic level. We studied 351 newly diagnosed patients, entered into the E9486/E9487 clinical study of the Eastern Cooperative Oncology Group. Fluorescent in situ hybridization (FISH) combined with immune fluorescent detection (cig-fish) of clonal plasma cells (PC) and cytomorphology were used to analyze interphase, bone marrow (BM) cell, cytospin slides. We simultaneously used DNA probes for the following locus specific probes (LSI); LSI 13 (Rb) and D13S319, which hybridize to 13q14. We subsequently studied distal deletions using the D13S25 probe (13q14.3) and a subtelomeric probe (13qSTP) for the 13q-arm (D13S327) in 40 cases with documented LSI 13 (Rb)/D13S319 deletion and 40 without deletion of these loci. Of 325 evaluable patients, we found 13q deletions in 176 (54%) using LSI 13 (Rb) and D13S319 probes. Of 40 patients with LSI 13 (Rb)/D13S319 deletions, 34 (85%) had coexistent deletion of both D13S25/13qSTP. These results indicate that chromosome 13 deletions in MM involve loss of most if not all of the 13q arm perhaps even indicating monosomy. In six cases the 13qSTP signal was conserved, but D13S25 was lost indicating large interstitial deletions involving 13q14. In 39 of the 40 cases without LSI 13 (Rb)/D13S319 deletions, the normal pattern of two pairs of signals was observed for D13S25/13qSTP. Deletions involving 13q14 are very common in MM as detected by cig- FISH. These deletions appear to predominantly involve loss of large segments of the 13q arm or monosomy 13, and only occasionally represent an interstitial deletion. (2001) 15, Keywords: multiple myeloma; chromosome deletion; paraproteinemias; in situ hybridization. 13q14 are infrequent in MGUS 13 and are thus believed to be associated with the more advanced stages of disease. Nevertheless, it is unclear whether 13q is important for disease pathogenesis. Moreover, a minimally deleted region in chromosome 13 has not been defined and there is no proven pathogenetic role for the abnormality in MM. A detailed study by Shaughnessy and colleagues 14 identified a high prevalence of 13q in the clonal PC of patients with MM when multiple FISH probes were used. They reported observing complex FISH patterns that were consistent with large deletions of 13q The characterization of 13q is further complicated by the lack of appropriate centromeric DNA probes for chromosome 13 to aid in the study of aneuploidy in interphase nuclei using FISH. Current centromeric probes for chromosome 13 cross-hybridize with the centromere of chromosome 21. To better characterize these deletions we studied patients with deletions of 13q14 using DNA probes to determine if 13q-arm deletions region extend from 13q14 to the 13q-arm telomere. Patients and methods Patients Patients were entered into the Eastern Cooperative Oncology Group clinical trial E9486 and associated correlative study E9487. The clinical description of these patients has been previously published. 15 Briefly, these were newly diagnosed patients with MM treated with conventional chemotherapy (Table 1). Conventional cytogenetic analysis was not required at the time of study entry. Introduction Deletions of the long arm of chromosome 13 (13q ), mostly at the q14 site, and monosomy of chromosome 13 are commonly described in MM PC as determined by conventional metaphase analysis, 1 3 comparative genomic hybridization 4,5 and multicolor metaphase FISH (SKY). 6,7 Tricot and colleagues have associated the presence of 13q with an adverse prognosis in patients with MM 8,9 and proposed this genomic abnormality as one of the most important prognostic factors for MM patients. Interphase FISH studies have described a prevalence of the abnormality of 30 50% and also suggest an association with an adverse outcome. 11 Deletions of Correspondence: R Fonseca, Division of Hematology and Internal Medicine, Mayo Building W10B, Rochester, MN, 55905, USA; Fax: Received 7 November 2000; accepted 26 January 2001 BM samples and in situ hybridization BM samples of patients with MM were obtained at the time of routine clinical procurement and prior to study entry. The study was conducted under IRB approval and informed consent was obtained prior to enrollment. BM samples were enriched for mononuclear cells using the Ficoll-gradient centrifugation method. Cytospin slides were made and stored at 70 C for future use. Slides were thawed and subjected to immune fluorescent detection of the cytoplasmic light chain with clone-specific, 7-amino-4-methylcoumarin-3-acetic acid (AMCA) labeled antibodies (kappa or lambda). 16 The signal was amplified with a second AMCA labeled anti-igg antibody. Subsequently the cells were fixed with 2% paraformaldehyde and permeabilized with proteinase K (10 g/ml) at room temperature. Nick-translated probes were co-denatured under a cover slip using a hybridization mixture containing 50% formamide and 10% dextran sulfate. The cells were

2 982 Table 1 Descriptive features of patients Deletions of 13q14 in myeloma Variable All (n = 325) % Median age (mean) 61.2 (62.6) (range) (34 86) Gender Male 62.1 Female 37.9 Performance status Plasmacytoma No 90.0 Yes 10.0 Weight loss 10% 7.2 Lytic bone lesions 79.4 Hypercalcemia (Ca mg%) 24.5 Serum M component 1 g/dl 82.9 Urine M component 1 g/dl 74.2 Light chain type kappa 64.8 lambda 35.2 hybridized overnight and washed with 0.4 SSC for 2 min at 72 C, anti-fade was added. Probes To test for 13q deletions we used the DNA probes LSI 13 (Rb) (440 kb probe), D13S319 (130 kb), and D13S25 (160 kb) from Vysis, Inc, Wetzlar, Germany (Figure 1). These probes have been extensively tested in normal human cells and localize to the 13q14 13q14.3 region. We also used a subtelomeric BAC clone as control for telomeric deletions (Plate 45, 440-K13, Genome Systems, BAC Human Release II). To screen the BAC library we used the following PCR primers, 5 -CAG AGGTAGCTTCATAAAG-3 and 5 -CTATCTGCAACTTA TTTACC-3, for the marker D13S327 (approximately 100 kb, GDB: ). We confirmed the correct hybridization to the subtelomeric region of 13q of the DNA obtained from the clone (Figure 2). Scoring These probes were found to have an upper limit of normal for loss of signal of 5% for each pair (mean + 3 standard deviations). In addition, in patients without abnormalities detected, the probes produced a mean plus three standard deviations measurement of 2.9% (n = 146) for the LSI 13 (Rb)/D13S319 pair and 4% for the D13S25/13qSTP pair (n = 39). These probes have also been used by other investigators with similar upper limit of normal determinations (approximately 9%). 10,11,14 To further improve our specificity we decided to consider patients as positive for the deletions only if they had over 10% abnormal cells. This proved not to be critical since the vast majority of patients with abnormalities had them in 90% of PC (see Results). We attempted to score 100 PC per observer. Two experienced observers independently and blindly scored each one of the samples. The presence of signals arising from the non-involved allele in the PC, and the signals in the myeloid cells served as controls for adequate hybridization. For the 13q analysis we considered Figure 1 The chromosomal location of the 13q probes used for this study is located. The Figure shows a normal human chromosome 13 and the location of the D13 marker probes and their relative size. Because of their proximity in an interphase nucleus the normal pattern was that of two closely associated signals for the probes LSI 13 (Rb)/D13S319 and two pairs of signals for D13S25 and 13qSTP. The map of 13q14 is only at an approximate scale and has been drawn and modified from the map of Kalachikov et al 17 and the manufacturer s map of the probes ( the normal pattern to be that of two pairs of closely associated signals for both the LSI 13 (Rb)/D13S319 and D13S25/13qSTP pairs of probes. The loss of one or two of the signals was considered as indicative of heterozygous or homozygous deletion respectively (Figure 3). Results We studied a total of 351 patients of whom 325 could be analyzed for the LSI 13 (Rb)/D13S319 combination of probes (failure rate 8%). Of these 325 patients, 176 showed evidence of deletions (54%) (Figure 4). The median number of PC with deletions was 97% (range %) and 161 patients had greater than 50% abnormality. Only two patients deemed to be abnormal had less than 25% involvement and only 15 patients had less that 50% of the PC with the abnormality. In 172 cases (98%) there was simultaneous loss of LSI 13 (Rb) and D13S319 signals. Two cases had heterozygous deletions of D13S319 but no loss of LSI 13 (Rb) in 86 and 100% of their PC. One case had a heterozygous deletion of LSI 13 (Rb), but not D13S319 in 76% of PC. One case had homozygous deletion of LSI 13 (Rb) with heterozygous deletion of D13S319 in 67% of PC. No cases of nullisomy were observed. There was a high degree of correlation between the percentage of PC with abnormality detected by the two observers (Figure 5). Of 40 cases with deletions of LSI 13 (Rb)/D13S319, 34 (85%) also showed loss of both D13S25 and 13qSTP. The

3 Deletions of 13q14 in myeloma median percentage of PC with loss of D13S25/13qSTP signals was 97% (range %). This abnormal pattern is indicative of large q-arm deletions or monosomy. In six of 40 (15%) patients with evidence of 13q14 deletions by the LSI 13 (Rb)/D13S319, the probe D13S25 was deleted while 13qSTP (D13S327) was conserved indicating an interstitial deletion of 13q14 or an unbalanced translocation. Among 40 patients with a normal LSI 13 (Rb)/D13S319 pattern, 39 retained hybridization sites for the probes D13S25/13qSTP (97.5%). The one exception (2.5%) showed three signals of 13qSTP with two signals of D13S25 in 34% of PC indicating a possible trisomy of 13 with interstitial deletion involving D13S Discussion Figure 2 A normal metaphase hybridized with the probes D13S319 (red) and 13qSTP is shown. The metaphase shows the correct localization to the subtelomeric region of the chromosome 13 q arm of the 13qSTP (green signal). The correct localization of the signal is also confirmed by the co-hybridization of the probe D13S319 (red signal) in a position centromeric to 13qSTP ( 100 magnification, Leica DMRXA microscope). a c Figure 3 The normal and abnormal patterns of hybridization encountered in the study are shown. (a) The normal pattern for the association of probes LSI 13 (Rb) and D13S319 in a PC without deletion, and (b) the normal pattern of the pair of probes D13S25 and 13qSTP. (c) The most common abnormal pattern of LSI 13 (Rb)/D13S319 deletion, and (d) the most common pattern for deletion of D13S25/13qSTP in patients with co-existent LSI 13 (Rb)/D13S319 deletion ( 100 magnification, Leica DMRXA microscope). b d Our study demonstrates that large deletions of the long arm of chromosome 13 in MM are frequent, and that 13q are usually large deletions that involve loss of the 13q arm from 13q14 to 13q-ter (13q34), if not complete monosomy, in the majority of cases. It is possible that some cases have areas of noncontiguous deletion such as has been seen in chronic lymphocytic leukemia (CLL) 17 and MM, 14 but the most likely explanation from our series is that these patients had large deletions of the q-arm. In a previous study Avet-Loiseau and colleagues 13 identified telomeric extension of the deletions to 13q31. This study identifies deletions extending further to the telomeric end of the 13q arm up to 13q34 and thus is also compatible with large deletions. Our study also shows that deletions of D13S25 and D13S327 are rare in the absence of LSI 13 (Rb)/D13S319 deletions. The pattern of mono-allelic loss, and no nullisomy is compatible with what has been previously published for MM. 11,13,18 Whether 13q deletions are important for disease pathogenesis remains to be determined. If indeed 13q proves to be important in the pathogenesis of MM, it would most likely be in the context of the two-hit hypothesis for tumor suppressor genes. 19 We were unable to detect a ubiquitous area of deletion since 46% of patients had a normal pattern with the LSI 13 (Rb)/D13S319 probes. Furthermore, in 40 cases that were normal for LSI 13 (Rb)/D13S319, deletions in D13S25/13qSTP probes were rarely found. One has to consider the resolution capacity of FISH strategies and thus we cannot exclude that smaller areas of deletion are present at 13q14 in a larger proportion of patients despite a normal FISH result. The locus of deletion at D13S272 by Shaughnessy and colleagues 14 is in physical proximity to the marker D13S319 and within less than 40 kb. 20 Thus deletions of D13S319 are also reflective of D13S272 deletions. This was the most frequently deleted area in our set of patients along with D13S25. Thus, then the deleted area identified by our study would span from at least D13S319 to D13S25. In this study we cannot precisely identify the telomeric loss of DNA beyond D13S25, in cases with interstitial deletions, without loss of 13qSTP. D13S25 has been the presumed location of the putative tumor suppressor gene DBM. 17,21 DBM resides approximately 530 kb from the 3 end of the Rb gene. 21 The nature of 13q14 deletions in MM seems to be different to that of similar deletions in CLL (seen in up to 66% of cases). 17,20,22 28 In CLL, there appears to be an area of minimal deletion located at 13q14 and monosomy or large deletions appear not to be as common as in MM. 26,29 In contrast to MM, deletions at 13q14 have been associated with an improved

4 Deletions of 13q14 in myeloma 984 Figure 4 The graph depicts the percentage of cells with loss of LSI 13 (Rb)/D13S319 signals indicative of deletion. The dark line marks the upper limit of normal value used for this study. The values for percentage of abnormal cells were arranged in incremental order from left to right and all patients tested are shown. Figure 5 The plot of correlation between the percent of PC with signal abnormality according to the two observers. All 325 patients are shown in the graph but there are multiple areas of overlap for a given data point. outcome in CLL patients. 26,27 Once more, the specific roles these abnormalities play in both disorders need to be better elucidated. It is possible that the loss of material at 13q14 affects clonal cells in different manners according to the stage of B-cell differentiation, or it may signify a different biology and consequences for deletions at this site in the two diseases. The prevalence of the 13q abnormality is similar to what most other investigators have reported using interphase FISH The only significant discrepancy in prevalence is with the study from the University of Arkansas where they found deletions of chromosome 13 in 86% of patients when using a panel of FISH probes extending over the q-arm. 14 The significance of this observation is unknown and needs to be confirmed by other investigators. However the global interpretation of their data suggests that when deletions were present they were large deletions likely extending to the telomeric end of the q-arm and would thus be consistent with our data. 14 The presence of this abnormality in the majority of the clonal cells suggests that clones with 13q emerge early in the course of the disease and is selected because of clonal growth advantage or apoptosis resistance. Because 13q deletions are rare in MGUS, 13 and increase in prevalence with advancing stages of the disease it is likely that 13q clones are selected in disease progression to MM. In our study, there was little heterogeneity (that is, most cells had the abnormality when present) in the percentage of clonal cells with the abnormality, as has been reported by Avet-Loiseau et al 13 and contrasts with the report by Shaughnessy, where marked heterogeneity of 13q was observed in MM. 11,14 It is possible this results from their inclusion of patients with earlier stage PC proliferative disorders such as smoldering MM or MGUS, as several patients had a low percentage plasmacytosis with a low labeling index. In the study by Zojer and colleagues, 11 it was difficult to precisely estimate the percentage of clonal cells being scored since there was no enrichment or immuno-

5 Deletions of 13q14 in myeloma cells, but this analysis would be confounded by the initiation of chemotherapy. Deletions of 13q14 appear to have clinical significance in MM 8,9 but some issues remain unresolved. The first is whether the same significance of 13q seen in metaphase analysis 8,9 is also observed when samples are tested with interphase FISH. Given that the abnormality is so common, it has not been clear whether the abnormality per se would confer a poor prognosis or whether 13q is just a marker of a high proliferative clone, with its resulting informative abnormal metaphases. It has been previously documented that the presence of abnormal cytogenetics, regardless of the abnormality is associated with an adverse outcome. 1 We have shown a tight correlation between the presence of an abnormal karyotype in MM and a high proliferative rate of PC as assessed by the PC labeling index. 30 Recent studies have confirmed the prognostic ability of deletions of 13q when assessed by FISH. 10,11,31 We have studied the prognostic value of these abnormalities when tested in a large cohort of patients, 32 and have similarly found a strong association with an adverse outcome (RF, manuscript in preparation). The next unresolved issue is whether this adverse prognostic capacity is specific for 13q or whether it is merely indicative of a clonal process capable of surviving despite the presence of the genomic loss, in this case monosomy. To state it differently, the ongoing aneuploidy observed in neoplastic 33,34 disorders is more likely to generate a chaotic karyotype, and thus, a less likely to survive clone. Therefore the presence of growth, failure of apoptosis, or drug resistance, despite genomic loss (monosomy), whatever the chromosome, may indicate intrinsic biologic differences of a clone with a survival advantage. In fact studies assessing for loss through deletions of 17p with FISH probes specific for the p53 gene, also harbor prognostic significance in MM. 35 Is it that 13q is associated with an adverse outcome or is this common to other chromosomal/genomic DNA loss? Further work is needed to elucidate the pathogenetic mechanisms of 13q and its overall relation to the genomic instability of PC disorders. 985 Acknowledgements Figure 6 The area of minimal deletion in 13q in the 40 cases with deletions of LSI 13(Rb)/D13S319. There is loss of LSI 13 (Rb), D13S319 and D13S25 in all cases studied except one, and in only six cases there is a remaining signal from the 13qSTP probe. The percentages of abnormal cells for each one of the pairs of probes for a patient are shown in the second (LSI 13 (Rb)/D13S319) and seventh (D13S25/13qSTP) columns. In the right side of the Figure we also depict the results for three patients with variant patterns of 13q14 deletion. fluorescent detection of the clonal PC. In this last study the percentage of cells (unselected) with deletion (approximately 33%) closely matches that with the percentage of BM clonal involvement by the MM (37%), suggesting 13q14 deletions were present in the majority of PCs. It is however possible that later in the dynamic growth of a clonal population of PC those cells with 13q segregate even at later stages and with few cell divisions can predominate over cells lacking 13q. Given that all of our patients had newly diagnosed MM, this hypothesis seems less likely. We did however find that in 15 cases the proportion of cells with 13q was less than 50% of the light chain restricted clone, and it would be interesting to evaluate these patients for progressive gain of 13q clonal This work was supported in part by Public Health Service grant R01 CA from the National Cancer Institute. RF is a and Lymphoma Society Translational Research Awardee. RF and PRG are also supported by the Mayo Foundation and the CI-5 Cancer Research Fund-Lilly Clinical Investigator Award of the Damon Runyon Walter Winchell Foundation. PRG is supported in part by research grant P01 CA62242 from the National Cancer Institute. PRG and NEK are supported by the ECOG grant CA C from the National Cancer Institute. References 1 Dewald GW, Kyle RA, Hicks GA, Greipp PR. The clinical significance of cytogenetic studies in 100 patients with multiple myeloma, plasma cell leukemia, or amyloidosis. Blood 1985; 66: Sawyer JR, Waldron JA, Jagannath S, Barlogie B. Cytogenetic findings in 200 patients with multiple myeloma. Cancer Genet Cytogenet 1995; 82: Smadja NV, Louvet C, Isnard F et al. Cytogenetic study in multiple myeloma at diagnosis: comparison of two techniques. Br J Haematol 1995; 90:

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