Fluorescent in situ hybridization studies in multiple myeloma

Fluorescent in situ hybridization studies in multiple myeloma Ozge Ozalp Yuregir 1, Feride Iffet Sahin 1, Zerrin Yilmaz 1, Ebru Kizilkilic 2, Sema Karakus 2 and Hakan Ozdogu 2 1 Department of Medical Genetics and 2 Department of Adult Hematology, Baskent University Faculty of Medicine, Ankara, Turkey Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) results of bone marrow samples of 36 multiple myeloma (MM) patients at the time of diagnosis have been evaluated. Three probes for chromosome 13q (RB1, D13S319, D13S25), one for 14q32 (IgH) and one for 17p13 (p53) have been used for hybridization with fixed cells. Twenty patients (55. 5%) had normal karyotypes, whereas eight (22. 2%) had numerical or structural chromosomal abnormalities. We did not find metaphases for chromosome analysis in eight (22. 2%) patients. Fluorescence in situ hybridization analyses revealed at least one or more abnormal results in 25 (69. 5%) cases, whereas 11(30. 5%) cases had no abnormal findings. 14q32 rearrangement was the most common finding in FISH analyses and has been detected in 21 cases (58. 3%). 13q deletion and 17p deletion have been detected in 11 (30. 5%) and 5 (13. 9%) cases, respectively. Fluorescence in situ hybridization studies including 14q32 and 17p13 chromosome regions may yield quite significant results during clinical follow-up of MM. Keywords: FISH, multiple myeloma, molecular cytogenetics Introduction Multiple myeloma (MM) is characterized by the clonal proliferation of malignant, immunoglobulinproducing plasma cells in the bone marrow. The disease is generally fatal and incurable, but the clinical properties, treatment responses and survival varies considerably between individuals. MM comprises 10 15% of hematological malignancies and 1% of all adult malignancies. 1 It has been reported that, all MM patients harbor cytogenetic abnormalities sometime during the course of the disease. 2 Conventional cytogenetic analysis of the bone marrow is thus an important tool in evaluating karyotype abnormalities in MM, which may include a variety of structural and numerical aberrations. However, due to the hypoproliferative Correspondence to: Professor Dr Feride Iffet Sahin, Department of Medical Genetics, Baskent University Faculty of Medicine, Kubilay Sokak No. 36 Maltepe, 06570 Ankara, Turkey E-mail: feridesahin@hotmail.com nature of the myeloma bone marrow, it is not always possible to obtain good metaphases for analysis in a number of cases. 3 Furthermore, cryptic abnormalities not visible using light microscopy and complex unbalanced rearrangements may go undetected by conventional cytogenetic methods. Fluorescence in situ hybridization (FISH) is a high throughput molecular cytogenetic tool that is used to detect such cryptic abnormalities in hematological malignancies. 4,5 Thus, cytogenetics and FISH should be used together in the work-up of these diseases. 5 Fluorescence in situ hybridization studies in MM have shown that the incidence of abnormalities is much higher than expected. Frequent chromosome abnormalities in MM include 14q32 rearrangements, 13q deletion/monosomy 13, 1q duplication, 1p, 6p, 11q and 17p deletions. 6 In this study, we aimed to investigate the most frequent cytogenetic aberrations in MM; 13q deletion, 14q32 rearrangements and 17p deletion, by using conventional cytogenetic methods and FISH 90 ß W. S. Maney & Son Ltd 2009 Received 29 August 2008; accepted 27 October 2008 DOI 10.1179/102453309X385250 Hematology 2009 VOL 14 NO 2

and correlate the results with the clinical course of the patients. Materials and methods The study was approved by Baskent University Ethics Committee for Clinical Investigations. Patients: 36 patients, whose bone marrow samples were sent to the cytogenetics laboratory of our department with the preliminary diagnosis of MM, between July 2005 and August 2007, were enrolled. Median age was 63 (36 78) and male/female ratio was 2/1 (24/12). Conventional cytogenetics: Heparinized bone marrow samples were used; and when sufficient material was available, mononuclear cells were separated with ficoll density gradient method (Biocoll-Biochrom, Germany L6113). Stimulated and unstimulated, short and long term cultures were set up using appropriate media. After harvesting, chromosomes were Giemsatrypsin banded. For each case, 20 metaphases were scored for numerical abnormalities and five metaphases were analyzed thoroughly for structural aberrations. Karyotypes were described according to ISCN 2005. FISH: Interphase nuclei and metaphase spreads obtained from unstimulated cultures were used for FISH analysis. Slides were hybridized with fluorescent labeled commercial probes according to the manufacturers instructions. Three different probes were used for the 13q14 region; LSI 13q14 (RB1) (Vysis), LSI 13q14. 3 D13S319 (Vysis) and LSI 13q14. 3 D13S25 (Vysis), localizations from proximal to distal respectively. 14q32 break-apart probe (Cytocell) was used for 14q32 rearrangements and LSI 17p13. 1 (P53) (Vysis) probe was used for 17p deletions. Slides were analyzed using a Nikon E 600 fluorescent attachment microscope. Cut-off values were assessed by hybridizing each probe to slides obtained from healthy controls (Table 1). Two hundred nuclei were scored for each probe. Figure 1 FISH analysis of Case 20 Results Clinical evaluations of the patients were done according to the criteria proposed in Guidelines on the diagnosis and management of multiple myeloma 2005. 7 Cytogenetic analysis: 20 of the patients (55. 5%) had no structural or numerical abnormalities. Five patients had hypodiploidy but the chromosomes were structurally normal. Two patients (one of them hyperdiploid) had complex karyotypes. One case showed 45,X,-Y/ 46,XY karyotype. In eight patients suitable metaphases could not be obtained for chromosome analysis. FISH: 11 of the patients (30. 6%) had no abnormalities regarding the regions analyzed. Of these, only one patient s clinic was classified as progressive disease. One patient showed abnormal results for all of the probes analyzed (Fig. 1). This patient also had progressive disease and hypodiploidy in numerical analysis (Case 20). Twenty-five of the patients (69. 4%) had at least one type of abnormality in FISH analysis. The most frequent aberration was 14q32 rearrangement (58. 3%). In nine patients, this was the only abnormality detected. Eleven patients (30. 5%) had a deletion in the 13q14 region. Seven of these patients had a large deletion Table 1 Properties of the probes used in the study Probe Expected normal signals Expected abnormal signals Size of probe region Cut-off value LSI 13q14. 3 D13S319 Spectrum Orange/ VYSIS 2 SO 1 SO 130 kb 4% LSI 13q14. 3 D13S25 Spectrum Orange/ VYSIS 2 SO 1 SO 160 kb 5% LSI 13q14 (RB1) Spectrum Orange/ VYSIS 2 SO 1 SO 220 kb 5% LSI 17p13. 1 (p53) Spectrum Orange/ VYSIS 2 SO 1 SO 145 kb 6% 14q32 (IGH) Break Probe Spectrum Yellow/ 600 kb SO CYTOCELL 2 Y 1G 1O 1Y 400 kb SG 4% SO: spectrum orange; SY: spectrum yellow; SG: spectrum green. Hematology 2009 VOL 14 NO 2 91

spanning all three regions analyzed (D13S319, D13S25 and RB1). Of the remaining, two patients had a deletion in RB1 region (Case 2, Case 8) and one patient had a deletion in D13S319 (28); other regions being intact (Table 2). There were no solitary deletions in the D13S25 region. All patients except one with 13q deletions had an accompanying cytogenetic abnormality. The least frequent abnormality was 17p13 deletion which was detected in five patients (13. 8%). Four of these patients had an additional molecular cytogenetic aberration. One patient with 17 p deletion as the single abnormality had a progressive clinical course (Case 25) (Table 2). Discussion Karyotypic instability in MM begins in the early stages of disease development. The multi-step transformation feature of MM brings about the fact that additional genetic changes may well be seen in the progression of the disease. 8 IgH gene rearrangements, 13q deletion and 17p deletion are examples of the cytogenetic changes associated with the malignant transformation of MM. 8 The occurrence of rearrangements in the 14q32 region which involves the IgH gene is one of the early molecular events in MM pathogenesis. 8 This region takes part in translocations with specific partner genes located in regions such as 11q32, 4p16. 3, 16q23 and 6p21. These translocations are thought to activate particular oncogenes found in these regions, thereby participating in disease pathogenesis. 9 14q32 rearrangements have been documented by various authors as a frequent event in MM. Pantou et al. found this aberration in 72% of patients analyzed by interphase FISH, while Schmidt-Wolf et al. reported a frequency of 79. 1%. 3,10 Similarly in our study, 14q32 rearrangements were the most common abnormality, detected in 58. 3% of the cases. The detection of 14q32 rearrangements by Table 2 Results and clinical properties of patients Patient no. Age Karyotype 13qD13S319 13qRB1 13qD13S25 17p13 14q32 Clinical follow-up 1 43 46,XY hypodiploidy N N N N A IgG stage 3A stable 2 62 46,XX N A N N A IgG stage 3B progressive-exitus 3 58 46,XY hypodiploidy N A A N A IgA stage 3A SCT-exitus 4 66 46,XY N N N N N IgA stage 3B progressive 5 77 NA N N N N N IgG stage 3B stabil 6 55 46,XY N N N N A IgG stage 2A SCT exitus 7 67 Complex karyotype N N N A A IgG stage 2A PCL differantiation 8 74 46,XX N A N N N IgG stage 2A remision 9 71 46,XY N N N N A IgA stage 3A progressive-exitus 10 64 46,XY N N N A A IgG stage 3B progressive-exitus 11 55 45,X,-Y[5]/46,XY[5] N N N N N Light chain MM SCT-stable 12 78 Complex karyotype A A A N A IgA stage 3A progressive-exitus 13 65 46,XX N N N N A No follow-up 14 67 46,XX N N N N A IgA stage 3A MM stable 15 51 46,XX N N N N A IgG stage 2A remission 16 74 46,XX N N N N A No follow-up 17 58 NA N N N N A IgG stage 3A remission 18 64 46,XY A A A N A IgG stage 2A progressive 19 73 46,XY hypodiploidy A A A N A No follow-up 20 73 46,XY hypodiploidy A A A A A IgG stage 3A progressive 21 49 46,XY A A N N A Light chain MM-progressive 22 51 46,XY N N N A A IgG stage 2A SCT remission 23 68 NA N N N N N IgG evre 2A SCT remission 24 68 46,XY N N N N N IgG stage 3A stable 25 70 46,XX N N N A N IgA stage 3A progressive 26 72 hypodiploidy N N N N N IgA stage 3A stable 27 56 46,XX N N N N N No follow-up 28 54 46,XY A N N N N IgG stable 29 36 46,XX N N N N A No follow-up 30 76 46,XX A A A N A IgG stage 3A 31 52 NA A A A N N IgG stable 32 59 46,XY N N N N A Light chain lambda MM extramedullary recurrence 33 71 NA N N N N N IgG stage 3B stable 34 62 NA N N N N N IgA stage 3A stable 35 74 NA N N N N N IgA stage 3A stable 36 40 NA N N N N N IgG stage 3A stable N: Normal result A; Abnormal result; NA: not available; SCT: stem cell therapy; PCL: plasma cell leukaemia. 92 Hematology 2009 VOL 14 NO 2

molecular cytogenetic methods in patients with normal karyotypes, support the idea that cells with in vitro mitotic capacity may not belong to the malignant clone; and that probably normal myeloid elements have been proliferated in cultures, which may contribute to the low detection rate of certain cytogenetic abnormalities. Separation of CD138 surface marker positive cells could be a reasonable approach. In this study however, FISH analysis was carried out with cells proliferated in unstimulated cultures after Ficoll separation. The fact that FISH analysis of interphase cells yields more abnormalities than conventional cytogenetic methods may indicate that FISH is a practical approach regarding the detection of putative abnormalities especially because of the large number of cells analysed. Fluorescence in situ hybridization analysis of interphase nuclei thus is important in this group of patients, regarding the detection of such putative abnormalities; especially because of the larger number of cells analyzed. In 15% of MM patients, monoclonal Ig is not detected and there is an excessive production of light chains instead. 11 This type is called Light chain myeloma and also has a high frequency of IgH gene rearrangements; though not thought to be functional. In this study, we found IgH rearrangements in two patients with light chain myeloma (Case 11, 21) (Table 2). Case 21 also had a deletion in the 13q region. It is believed that all MM cases will develop an abnormality in this region eventually through the course of the disease. 12 Deletions in 13q are associated with poor prognosis and it is thought that there may be a tumor suppressor gene for MM in this region. 13 The presence of 13q deletions in MM in different studies have been reported to be between 20 and 86%. 13 We found a deletion in the 13q14 region in 30. 5% of the patients. Among the cases with poor disease outcomes in follow-up, 46. 1% had 13q deletion. Clinically stable cases, on the other hand had a frequency of 22. 2% (OR: 3,089, 95% CI: 0,7071-13,4657). There were no follow-up data for five patients; one of these had 13q deletion, the other four did not have any cytogenetic findings. These findings support the fact that 13q14 deletion is associated with poor prognosis. The designation of a deletion in 13q is important in diagnosis not only for its putative role in the pathogenesis of the disease but also for its prognostic significance. Fluorescence in situ hybridization method is a reliable tool in detecting this deletion, but choice of the probe region is critical. It has been reported that there are at least nine different probe regions that can be used for this purpose. 12 In a study of 29 patients with identified 13q14 deletions reported by Elnenaei et al., the frequencies of deleted D13S319, RB1 and D13S25 regions were 100, 83 and 76% respectively, so it was concluded that D13S319 is the common region deleted in all patients with 13q14 deletions. 13 On the other hand, Zojer et al. found the frequencies of deletions in RB1 and D13S319 regions to be 46. 2 and 38. 9% respectively in newly diagnosed MM patients; thus proposing that RB1 region probes could be more sensitive in detecting 13q14 deletions. 14 In our study, seven cases had deletions spanning all three regions (RB1, D13S319 and D13S25) analyzed. Besides these cases that were considered to have large deletions, some patients had deletions involving solely D13S319 or RB1 regions while other regions were intact. Therefore, these probes were also considered as highly specific for use in 13q14 deletion screening. In fact, the 220 kb long RB1 region was the most informative region for 13q14 deletion detection. The fact that no patient had a deletion involving only the D13S25 region led us to believe that this region by itself was not as informative without the use of other probes. A hypodiploid karyotype was detected in three patients with 13q14 deletions (Case 3, 19, 20) (Table 2). No other cases with the deletion had hypodiploidy or monosomy 13 and the deletion was undetectable by conventional cytogenetic methods. Therefore, it could be suggested that FISH analysis is necessary in cases where 13q14 deletion cannot be shown by cytogenetics. 17p13 deletion may be present at diagnosis in MM patients, but it has been reported to appear more frequently in the latter stages of the disease and it is then associated with a poorer prognosis. 8,9 The tumor suppressor gene p53 is found in this region and it is known to take part in the pathogenesis of many cancers because of its role in cell growth and differentiation. 8 All cases with 17p13 deletions in our study except one had adverse clinics, which supports the fact that deletions in this region are associated with poorer prognosis (Case 22). Evaluation of 14q32 rearrangements, 13q14 and 17p13 deletions by FISH method is important in terms of allowing the analysis of metaphase spreads and interphase nuclei simultaneously and revealing cryptic abnormalities undetectable by conventional karyotyping. An informative way of evaluating MM patients Hematology 2009 VOL 14 NO 2 93

genetic status at diagnosis would be to use a FISH panel including such regions. Our results also demonstrate that RB1 and D13S319 region probes, when used alone or together, are valuable molecular cytogenetic markers in the analysis of 13q14 deletions. References 1 URL: http://atlasgeneticsoncology.org/anomalies/mmulid2038. html 2 Zandcki M. Multiple myeloma: almost all patients are cytogenetically abnormal. Br J Haematol 1996; 94(2): 217 227. 3 Pantou D, Rizou H, Tsarouha H. Cytogenetic manifestations of multiple myeloma heterogeneity. Genes Chromosomes Cancer 2005; 42(1): 44 57. 4 Yilmaz Z, Sahin FI, Kizilkilic E. Conventional and molecular cytogenetic findings of myelodysplastic syndrome patients. Clin Exp Med 2005; 5(2): 55 59. 5 Sahin FI, Kizilkilic E, Bulakbasi T. Cytogenetic findings and clinical outcomes of adult acute myeloid leukaemia patients. Clin Exp Med 2007; 7(3): 102 107 6 Huang SY, Yao M, Tang JL. Clinical significance of cytogenetics and interphase fluorescence in situ hybridization analysis in newly diagnosed multiple myeloma in Taiwan. Ann Oncol 2005; 16(9): 1530 1538. 7 Smith A, Wisloff F, Samson D. Guidelines on the diagnosis and management of multiple myeloma 2005. Br J Haematol 2006; 132(4): 410 451. 8 Hallek M, Bergsagel PL, Anderson KC. Multiple myeloma: increasing evidence for a multistep transformation process. Blood 1998; 91(1): 3 21. 9 Seidl S, Kaufmann H, Drach J. New insights into the pathophysiology of multiple myeloma. Lancet Oncol 2003; 4(9): 557 564. 10 Schmidt-Wolf IG, Glasmacher A, Hahn-Ast C. Chromosomal aberrations in 130 patients with multiple myeloma studied by interphase FISH: diagnostic and prognostic relevance. Cancer Genet Cytogenet 2006; 167(1): 20 25. 11 Magrangeas F, Cormier ML, Descamps G. Light-chain only multiple myeloma is due to the absence of functional (productive) rearrangement of the IgH gene at the DNA level. Blood 2004; 103(10): 3869 3875. 12 Terpos E, Eleutherakis-Papaiakovou V, Dimopoulos MA. Clinical implications of chromosomal abnormalities in multiple myeloma. Leuk Lymphoma 2006; 47(5): 803 814. 13 Elnenaei MO, Hamoudi RA, Swansbury J. Delineation of the minimal region of loss at 13q14 in multiple myeloma. Genes Chromosomes Cancer 2003; 36(1): 99 106. 14 Zojer N, Königsberg R, Ackermann J. Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization. Blood 2000; 95(6): 1925 1930. 94 Hematology 2009 VOL 14 NO 2