Frequent Gains of the Short Arm of Chromosome 9
in Multiple Myeloma With Normal G-Banded Karyotype
Detected by Comparative Genomic Hybridization
Joëlle Tchinda, PhD,
1*Sarah Volpert, PhD,
1*Martin Kropff, MD,
2Wolfgang E. Berdel, MD,
2Joachim Kienast, MD,
2Friedhelm Meinhardt, PhD,
3and Jürgen Horst, MD
1Key Words: Multiple myeloma; Comparative genomic hybridization; CGH; Gains of 9p
DOI: 10.1309/5KWKP6UKGNXXHMYH
A b s t r a c t
A number of genetic abnormalities have been detected in multiple myeloma (MM) using cytogenetic techniques. The prominent abnormalities are deletions of 13q and translocations affecting the IgH locus on 14q32. The recurrence of chromosomal abnormalities in MM suggests a specific role for them concerning its pathogenesis. We performed comparative genomic hybridization (CGH) on samples from 53 patients with MM and 4 with monoclonal gammopathies of
undetermined significance. In 31 cases (54%), normal ratio profiles were found, whereas 26 cases (46%) had aberrant profiles. The most common aberrations were gains of 9p (n = 14), 11 (n = 9), and 21q (n = 5) and loss of 22 (n = 7). In earlier reports on cytogenetics of lymphomas, gains of 9p are described as characteristic of primary mediastinal B-cell lymphoma, but the consensus region is smaller than in the present study (9p23pter vs 9p13pter). Therefore, we suggest a stronger genetic affinity between MM and primary mediastinal B-cell lymphoma than between MM and other B-cell lymphomas. To support this suggestion, more molecular cytogenetic techniques and expression analyses have to be performed.
Multiple myeloma (MM) is a B-cell malignant neoplasm characterized by clonal expansion of terminally differentiated B cells (plasma cells) in the bone marrow, associated with the synthesis of a monoclonal immunoglobulin and a high inci-dence of osteolytic bone lesions. MM is called plasma cell leukemia (PCL) when the absolute plasma cell count is more than 2 × 109/L or when plasma cells represent more than 20%
of peripheral blood cells.1 In monoclonal gammopathies of
uncertain significance (MGUS), a monoclonal protein is pres-ent in the serum without evidence of any causal disease, and there is low plasma cell count in the bone marrow.2
MM is one of the frequent hematologic disorders and rep-resents about 10% of all hematologic malignant neoplasms.3
In lymphoproliferative disorders of the bone marrow, MM represents 42% of B-cell tumors and affects mainly older patients in their sixth or seventh decades of life.4The clinical
course of MM is very different, and survival time ranges between a few months and several years related to plasma cell mass and stage of disease.5
Cytogenetic analysis is an important part of myeloma diagnosis because monosomy 13 or deletions in the long arm of chromosome 13 have been identified to be prognostic fac-tors.6Information based on conventional karyotyping is
lim-ited because malignant plasma cells have low spontaneous proliferative activity.7By conventional G-banding,
chromoso-mal aberrations are described in about 20% to 50% of the cases.8-19The karyotypes of MM usually are greatly complex
and more similar to those of epithelial tumors than other hematopoietic disorders.
Comparative genomic hybridization (CGH) is a powerful supplement in the cytogenetic characterization of MM owing to its ability to detect genetic imbalances without tumor metaphases.
The application of molecular cytogenetic methods such as CGH or fluorescence in situ hybridization (FISH) increases the per-centage of chromosomal aberrations detected in MM.20
Chromosomal aberrations detected by CGH are known only in a limited number of cases. Five studies on series of 8 to 46 patients with MM, PCL, MGUS, and myeloma cell lines have shown gains and losses in different chromosomal regions and with varying frequency.21-25The results are controversial,
and most of the data have not been validated using FISH. The first study21describes gains of chromosomes 7 and 8 in 25% of
the cases and gains in the long arm of chromosomes 1, 2, 9, and 8 in 63%. Losses were seen in the long arm of chromosome 13 in 63% of the cases and in the long arm of chromosome 14 in 38%. CGH in the second study22showed gains of 19 and 19p
in 30%, 11q in 20%, and 17q in 13% and losses of 13 and 13q in 30%, 16 and 16q in 17%, and 6q in 13%. The most frequent chromosomal imbalances detected in the third study23 were
gains of 1q (58%), 9q (58%), 19p (58%), 7p (42%), 11q (38%), 15 (33%), 6p (25%), 8q (25%), and 5p (21%) and losses of 13q (79%), 1p (42%), 14q (33%), X (33%), 8p (25%), 6q (25%), and 4 (21%). The fourth study24described differences in
chro-mosomal abnormalities between MM and PCL. It showed gains of 15q (48%), 11q (44%), 3q (40%), 9q (40%), and 1q (36%) and losses of 13q (80% PCL vs 28% MM), chromo-some 16 (80% PCL vs 16% MM), and 2q and 6p only in patients with PCL. The latest study25with the highest number
of patients (n = 46) detected frequent chromosomal gains of 1q (26%), 9q (15%), 11q (15%), 9p (11%), 5p (11%), 5q (11%), 6p (11%), 15 (11%), and 18q (11%) and losses of 13q (37%), 6q (15%), 8p (15%), Xp (15%), Xq (15%), and 1p (13%).
We performed CGH in the largest series to date—57 patients with plasma cell neoplasms. The results were validat-ed by FISH, comparvalidat-ed with conventional cytogenetics, and correlated with clinical data. The aim of our study was to iden-tify possible chromosomal imbalance patterns and regions that could have a role in the pathogenesis of MM.
Materials and Methods
Cases
For the present study, we used bone marrow samples sent for cytogenetic analysis to the Department of Human Genetics, University Hospital, Münster, Germany. We selected 57 sam-ples on the basis of suitable material being available from diag-nostic samples for CGH and FISH analyses. The tumors were staged using the clinical system of Durie and Salmon.26Clinical
and laboratory data for the patients are given in ❚Table 1❚. Cytogenetic Analysis
Bone marrow aspirates were obtained as heparinized samples. Leukocytes were isolated by density centrifugation
as previously described.27Samples were cultured for up to 72
hours and then exposed to colcemid (Gibco, Karlsruhe, Germany) 30 minutes before harvest. After hypotonic treat-ment and fixation, GTG banding was used for karyotype analysis. Approximately 20 metaphases were analyzed. The karyotypes were reported according to the International System of Cytogenetic Nomenclature.28
Metaphase Spreads for CGH
Metaphase spreads needed for CGH were prepared from phytohemagglutinin-stimulated lymphocytes of a healthy male donor as described earlier.27Up to 200 slides were
pre-pared at once and used for this study only after CGH with dif-ferentially labeled reference DNA had been performed as quality control.
DNA Isolation and Labeling
High-molecular-weight genomic DNA was isolated by a standard phenol-chloroform extraction procedure after Proteinase K digestion.29Reference DNA was obtained from
blood samples of healthy male and female donors. Tumor DNA was isolated from methanol/acetic acid–fixed cells stored at –80°C after G-banding analysis was closed. We labeled 1 µg of each tumor and reference DNA by nick trans-lation using a kit according to the manufacturer’s instructions (Vysis, Bergisch Gladbach, Germany). Tumor DNA was
❚Table 1❚
Clinical and Laboratory Data for 57 Patients*
Variable Range (Mean) Values
Age (y) 32 -74 (55.0) Sex (M/F) 38/19 MM stage 54 I 9 II 1 IIIA 31 IIIB 10
With other neoplasms 3
MGUS 3 Therapy (no/yes) (n = 1)† 33/23 BMPC (%) (n = 5)† 0-95 (32.4) CRP (mg/dL) (n = 20)† <0.5-12 LDH (U/L) (n = 16)† 88-525 (192.9) β2-M (µg/mL) (n = 27)†‡ 0.7-13 (3.4) Immunoglobulin subtype (n = 7)† IgG/IgA/IgM (n = 5)† 39/5/1 κ/λ(n = 1)† 34/15 Creatinine (mg/dL) (n = 15)†‡ 0.7-3.5 (1.3) Calcium (mg/dL) (n = 14)†‡ 8.00-12.80 (9.60)
β2-M, β2-microglobulin; BMPC, bone marrow plasma cell index; CRP, C-reactive protein; LDH, lactate dehydrogenase; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma.
*Only data for samples biopsied at the same time as the bone marrow samples were obtained for cytogenetic analysis are included. Staging of multiple myeloma was done according to Durie and Salmon.26
†Not documented for the number of cases in parentheses.
‡Values are given in conventional units; Système International (SI) units are as follows, with mean values in parentheses: β2-M, 59-1,102 nmol/L (288 nmol/L); creatinine, 62-309 µmol/L (115 µmol/L); calcium, 2-3.2 mmol/L (2.4 mmol/L).
labeled with SpectrumGreen-conjugated deoxyuridine triphosphate and reference DNA with SpectrumRed-conjugat-ed deoxyuridine triphosphate (Vysis). The size of the labelSpectrumRed-conjugat-ed DNA was verified on a 2% agarose gel to be in the range of 300 to 1,000 base pairs.
Hybridization and Detection
For hybridization and detection, 400 ng of each, labeled tumor and reference DNA, and an excess of human cot1 DNA (20 µg) were precipitated with ethanol, air dried, and dissolved in 10.5 µL of hybridization buffer (20% dextran sulfate, 4× standard saline citrate [SSC], 50% deionized formamide). This mixture was denatured at 72°C for 5 minutes. Normal metaphase spreads were denatured in 70% formamide/2× SSC (pH 7.0) at 72°C ± 2°C for 3.5 minutes, dehydrated in a series of ice-cold ethanol (70%, 80%, and 100%) for 2 min-utes each, and dried at 60°C. The denatured CGH probe was pipetted directly onto the slides, closed with a coverslip, and sealed with rubber cement. Hybridization was performed in a humidified dark chamber at 37°C for 3 days.
Slides were washed in 0.4× SSC/0.3% Nonidet P-40 (Roche, Karlsruhe, Germany) at 72°C ± 2°C for 2 minutes fol-lowed by 2× SSC/0.1% Nonidet P-40 at room temperature for 1 minute. Slides were allowed to air dry in the dark for 20 minutes. We used 10 µL of 4,6-diamino-2-phenylindole (DAPI, 40 ng/mL) containing p-phenylenediamine dihy-drochloride (1 mg/mL) as the counterstain.
Image Acquisition and Analysis
Metaphases were acquired under ×100 magnification using an epifluorescence microscope (Zeiss Axioplan 2, Carl Zeiss Göttingen, Göttingen, Germany) equipped with a 100-W mercury lamp; filter sets for an excitation of 365, 450 to 490, and 546 nm; and a cooled charge-coupled device camera (JAM M300, JAI, Yokohama, Japan). Image analysis was performed with the Isis software (MetaSystems, Althusheim, Germany). Mean ratio profiles of SpectrumGreen to SpectrumRed were
calculated for 10 metaphases. Diagnostic thresholds used to define losses and gains were set at 0.85 and 1.17.30
Interphase FISH
FISH was used to validate the CGH results. FISH using commercially available probes was performed according to the manufacturer’s protocols. All commercially available FISH probes were obtained from Vysis. In addition, we used locus-specific probes generated from DNA of yeast artificial chromosome, P1-derived artificial chromosome (PAC), and bacterial artificial chromosome provided by Centre d’Etude du Polymorphisme Humain (Paris, France) and the University of Bari (Bari, Italy). Generation of probes was performed as previously described.31 PAC DNA was isolated using the
QIAgen Large-Construct-Kit (Qiagen, Hilden, Germany). Cells fixed on slides were denatured in 70% for-mamide/2× SSC at 70°C for 2 minutes and hybridized overnight in a humidified chamber at 37°C. Posthybridization washes were done in 0.1× SSC at 60°C 3 times. Detection was achieved with a fluorescein-labeled antibiotin antibody sys-tem (Oncor, Heidelberg, Germany). Nuclei were counter-stained with DAPI/antifade (Oncor).
Analyses were performed with an epifluorescence micro-scope as described for CGH. At least 250 nuclei were counted. Thresholds for aberrant counts were defined for each probe by evaluation of signal distribution in 10 normal samples.
Results
Conventional Cytogenetics
We studied 57 cases by conventional cytogenetics. G-band-ing analysis was successful in 53 cases (93%). Four cases could not be karyotyped owing to the absence of metaphase spreads. Of 53 cases, 48 (91%) had normal karyotypes. Of 48, 46 patients had MM and 2 had MGUS. The 5 aberrant cases were patients with MM ❚Table 2❚. Derivative or marker chromosomes
❚Table 2❚
Distribution of G-Band Karyotypes and Comparative Genomic Hybridization Profiles Among Different Disease Subtypes*
Karyotype Profile
Morphologic Diagnosis Normal Aberrant No Metaphases Normal Aberrant Total
MM (total) 46 5 3 29 25 54 Stage I 6 2 1 7 2 9 Stage II 1 0 0 1 0 1 Stage IIIA 27 2 2 17 14 (7†) 31 Stage IIIB 9 1 0 4 6 (6†) 10 Other 3 0 0 0 3 (1†) 3 MGUS 2 0 1 2 1 3 Total 48 5 4 31 26 57
MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma. *Staging of multiple myeloma was done according to Durie and Salmon.26
were found in 2 cases (1215 and 1848). Deletion of a part of the long arm of 1 chromosome 13 was found in 1 case (1422). Most of the anomalies were numeric and included loss of the Y chromosome in 3 cases (319, 1558, and 1848); trisomies for chromosomes 3, 4, 5, 7, 9, 11, 15, 19, and 21; and mono-somies for chromosomes 16 and 22. The aberrant karyotypes are detailed in ❚Table 3❚.
Fluorescence In Situ Hybridization
We performed 126 hybridizations among 52 loci and 30 cases to validate the CGH results. Three hybridizations with the same probe (PAC dJ64I15) could not be analyzed owing to lack of signals. Of 123 FISH analyses, 84 (68.3%) confirmed CGH results; 39 (31.7%) did not confirm gains or losses detected by CGH. These changes consequently were classi-fied as artifacts of the CGH.
Comparative Genomic Hybridization
CGH was successful in all 57 cases. Only losses or gains val-idated by FISH were classified as aberrations. Chromosome Y was not included in the classification owing to its generally poor hybridization caused by insufficient suppression by cot1 DNA. The others were assumed to be artifacts of CGH. Of 57 cases, 31 (54%) showed normal ratio profiles, and 26 (46%) were aberrant. Whole chromosome gains concerned chromosomes 11 (n = 9); 3, 5, 7, 8, 9, and 15 (n = 3 each); 21 (n = 2); and 16 (n = 1). Monosomies were found for chromosomes 22 (n = 7); 19 (n = 2); and 13, 16, and 17 (n = 1 each). Frequently deleted chromosomal regions were 12q (n = 3) and 1p, 9q, 16q, and 21q (n = 2 each). Gains were frequent on regions 9p (n = 11); 21q (n = 3); and 5p and 11q (n = 2 each). The distribution of gains and losses among all chromosomes is traced in ❚Figure 1❚.
Correlation of CGH Results With Conventional Cytogenetics
Nineteen cases with normal karyotype using G-banding showed aberrant ratio profiles ❚Table 4❚. All 4 cases lacking
metaphases for conventional cytogenetics were aberrant using CGH ❚Table 5❚. For cases in which both methods detected anom-alies, the failure of CGH to detect nullisomy Y was not consid-ered a discrepancy in the techniques. In 2 cases (1215 and 1848), all changes detected by CGH have already been described after G-banding, but conventional cytogenetics showed additional aberrations. In case 1215 derivative chromosomes 1, 8, 16, and 17 probably are balanced aberrations because they did not result in gains or losses. Trisomy 9 and der(13) result in a gain of 9p. In addition to other changes, trisomy 15 was found in only 1 metaphase of 20 by conventional cytogenetics. This could explain the lack of detection by CGH. In case 1848, monosomy 16, trisomy 19, and a marker chromosome were missed using CGH. In case 1422, the clone found using conventional cytoge-netics was absolutely different from the one detected by CGH. Karyotypes and CGH profiles are detailed in Table 3.
Discussion
Chromosomal aberrations such as monosomy 13, dele-tion of 13q, and IgH (14q32.3) rearrangements have a role as prognostic factors in the management of MM. In the present study, a molecular cytogenetic technique, CGH, was applied in addition to conventional cytogenetics for the analysis of cases of MM or MGUS. CGH results were validated with appropriate FISH probes. Of 57 cases, 28 (49%) showed abnormalities with at least one of these techniques. The 5 previ-ous reports of CGH on MM had a range of 63% to 100%.21-25
The low percentage in our study can be explained by the inclu-sion of more patients with normal karyotypes using G-band-ing than in the other studies (91% [48/51] vs ~40%) and by the low quantity of bone marrow plasma cells (BMPCs), ranging from 3% to 95%. We nevertheless included patients with BMPCs lower than 50% because the BMPC values have some variation depending on the anatomic location of the bone mar-row aspiration and the analyst.
❚Table 3❚
Comparison of CC and CGH Results for Cases With Aberrations Using CC*
Morphologic
Case No. Diagnosis Status BMPC (%) CC CGH
319 MM I D 15 45,X,–Y[3]/46,XY[17] Normal
1215 MM IIIA T 50 53~54,XX,+der(1),+3,+4,+der(7)t(7;?)(q32;?), +3,+4,+7,+9p13pter,+11,
der(8),+9,+11,der(13)t(9;13)(?;q10),+15, +21×2,–22
der(16),der(17),+21×2,–22[cp5]/46,XX[15]
1422 MM IIIB D 85 46,XY,del(13)(q32)[6]/46,XY[23] +2,+3,+5,+9,+11,+15,
+20p11pter,–22,–Xpter-q22.1
1558 MM I T 0 45,X,–Y[4]/46,XY[16] Normal
1848 MM IIIA D 30 49,X,–Y,+5,+9,+11,–16,+19,+mar[4]/46,XY[16] +5,+9,+11
BMPC, bone marrow plasma cell index; CC, conventional cytogenetics; CGH, comparative genomic hybridization; D, at diagnosis; MM, multiple myeloma; T, under or after therapy.
In the 5 cases with aberrations shown by conventional cytogenetics, a high level of concordance was noted between CGH profiles and G-banded karyotypes. All 4 cases without G-banded karyotypes showed chromosomal changes with CGH. CGH showed gains and/or losses in 19 (40%) of 48 cases with normal karyotypes after G-banding. Chromosomal gains were far more frequent than losses, mostly involving 9 or 9p (14/57 [25%]), 11 (8/57 [14%]), 5 or 5p (5/57 [9%]), and 21 or 21q (5/57 [9%]). Losses mostly involved chromosome 22 (7/57 [12%]) and 13 or 13q (2/57 [4%]). These data are only partially in accordance with previous data. With the exception that percentages of detected changes are higher in the other studies, this is the only study describing gains of 9p. Liebisch et al25described gains of 9p, but these were always
within gains of the whole chromosome 9. In agreement with Liebisch et al,25 who described gains of chromosome 11 in
addition to gains of 11q, CGH detected gains of the whole chromosome 11 in the present study. The other 4 studies describe gains of only 11q as a frequent event. Gains of 21q are described only by Avet-Loiseau et al21 and solely in cell
lines. Losses of 22 are mentioned only by Aalto et al.23CGH
detected losses of 13 or 13q in 2 (4%) of 57 cases, in contrast with FISH, in which deletion of 13q14.3 (locus D13S319) was
found in 10 (18%) of 55 cases. This might be due to the low number of cells carrying the deletion or to the small size of this deletion.
To our knowledge, this is the first study that describes gains of 9p in MM in addition to formerly described changes such as gains of chromosome 11 and 21 and loss of some 22. Gains of chromosome region 9p or whole chromo-some 9 were detected only in stage III (A and B) MM and in a case of MM combined with an acute leukemia. Because of its relative high frequency (14/57 [25%] in this study), gains of 9p might have an important role in the biology of this dis-ease. In a comparison between primary mediastinal B-cell lymphoma (PMBL) and other B-cell lymphomas but not MM, Bentz et al32found gains of 9p to be highly characteristic of
PMBL. In an earlier study, Joos et al33described gains of 9p
to be characteristic of PMBL, which is classified as a mature B-cell neoplasm, such as MM and most of the B-cell lym-phomas. The consensus region in both studies was assigned to be further telomeric to bands 9p23 and 9p24. In the present study, the consensus region was greater: 9p13pter. In addition to JAK2 and NFIBsuggested by Bentz et al,32 we propose
CDKN2A(INK4A, p16), CDKN2B(INK4B, p15), and PAX5
as further candidate genes on 9p. 1 6 2 3 4 5 7 13 8 10 11 X 12 9 14 15 16 17 18 Y 19 20 21 22
❚Figure 1❚Gains and losses detected in 26 of 57 cases using comparative genomic hybridization. Gains are symbolized as lines on the right side of the chromosome ideograms and losses as lines on the left side. Broad lines symbolize gains of more than 3 copies.
❚Table 4❚
Comparison of Conventional Cytogenetic and CGH Results for Cases With a Normal Karyotype*
Case No. Morphologic Diagnosis Status BMPC (%) CC CGH
274 MM ND 10 46,XX –9q32qter,–12q23qter
411 MM I T 5 46,XY Normal
430 MM IIIB T ND 46,XX Normal
444 MM IIIB T 5 46,XY +9p11pter
456 MM IIIA D ND 46,XY Normal
499 MM IIIA T 70 46,XY +1q11qter,–13q12–q14
544 MM IIIA T ND 46,XX –12q23qter,–19,–22
562 MM IIIA D 30 46,XY Normal
636 MM IIIA T 90 46,XY Normal
680 MM IIIB D 80 46,XY +5p11pter,+9p11pterx2,+11×2,+21q11qter,–22
735 MM, AL D ND 46,XX +9p11pter,–21q22qter
781 MM IIIB T 60 46,XY +3,+5p11pter,–8p11pter,+9,+9p11pter×2,
+11×2,–13,+15×2,–16q11qter,–17p11pter,+21
790 MM IIIA D 95 46,XX +14q31qter
834 MM IIIA D 25 46,XY –1p33pter,–17,–22
874 MM IIIA T 10 46,XY Normal
886 MM IIIA D 90 46,XX –12q23qter,–20q11.1qter
892 MM IIIA D 70 46,XY +9p11pter,+11
896 MM IIIA D 65 46,XY Normal
1015 MM I D 20 46,XY Normal
1019 MM IIIA T 25 46,XX +9p11pter
1028 MM IIIA T 10 46,XY Normal
1218 MM IIIA D 5 46,XX Normal
1241 MM IIIB D 3 46,XY Normal
1246 MM IIIA T 20 46,XY Normal
1248 MM IIIA D 5 46,XX +5q11.1-q23.3,+9p11pter
1278 MM I D 5 46,XX Normal
1308 MM IIIA D 80 46,XY +5,+7,+9,+11,+15,–16q11qter,+21q11qter
1341 MGUS D 3 46,XX Normal
1356 MM I D 10 46,XY +7,+8
1371 MM I/MGUS D 5 46,XY +16
1433 MM IIIA T 50 46,XY Normal
1438 MM IIIA T 65 46,XY Normal
1477 MM IIIB T 35 46,XY +12p11pter,–14q31qter,–21q22.1qter
1489 MM I D 10 46,XX Normal
1493 MM IIIA D 10 46,XX Normal
1499 MM IIIA T 50 46,XX Normal
1502 MM IIIB D 50 46,XY Normal
1518 MM IIIB T 75 46,XY +8,+9p11pter,+11,+21q11qter
1529 MM IIIA T 15 46,XY Normal
1617 MM IIIA T ND 46,XX Normal
1620 MM IIIA D 5 46,XY Normal
1643 MM IIIA D 10 46,XX Normal
1684 MGUS D 10 46,XY Normal
1718 MM I D 4 46,XY Normal
1724 MM IIIA T 5 46,XY Normal
1756 MM IIIA D 35 46,XY +9p11pter×2,–10,–16,–19,–22
1831 MM IIIB T 65 46,XY Normal
1932 MM II D 10 46,XY Normal
AL, acute leukemia; BMPC, bone marrow plasma cell index; CC, conventional cytogenetics; CGH, comparative genomic hybridization; D, at diagnosis; MM, multiple myeloma; MGUS, monoclonal gammopathy of undefined significance; ND, not documented; T, during or after therapy.
*Staging of multiple myeloma was done according to Durie and Salmon.26Differences between techniques are given in bold.
❚Table 5❚
CGH Results for Cases Without G-Banding Karyotype
Case No Morphologic Diagnosis Status BMPC (%) CC CGH
737 MGUS D 5 No results +4p11pter,+8,+11
882 MM I D 15 No results –1p32.2pter,–9q34.1qter
956 MM IIIA D 25 No results +1q11qter,–22
1020 MM IIIA T 60 No results +Xq23qter,+1q11qter
BMPC, bone marrow plasma cell index; CC, conventional cytogenetics; CGH, comparative genomic hybridization; D, at diagnosis; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; T, during or after therapy.
CDKN2Aand CDKN2B, some of the most frequently dis-rupted tumor suppressor loci in human cancer, are located on chromosome 9 in region p21. They encode 2 inhibitors of cyclin-dependent kinases (CDK4 and CDK6) and upstream regulators of Rbfunction, p16 and p15, each of which is capa-ble of inducing cell cycle arrest at the G1phase. Alterations of
p16and p15occur at high incidence in MM solely by hyper-methylation of 5' CpG islands and not by homozygous dele-tions or mutadele-tions as described for other tumors.34The
func-tions of p16and p15,therefore, are hampered by this hyper-methylation, thus allowing clonal plasma cells to enter the S phase. Trisomy 9 provides a normal extra copy of p16and p15
that increases the probability of a nonhypermethylated gene copy continuing to work normally despite the hypermethyla-tion. Pérez-Simón et al35 describe better overall survival for
patients with trisomy 9 (46 vs 26 months) and trisomy 17 (103 vs 33 months). The duration of the present study was not long enough to correlate the results of the CGH with variables such as overall survival.
The human PAX5(paired box gene 5) is located in chro-mosomal region 9p13. It encodes the transcription factor B-cell lineage–specific activator protein, which is essential for the generation and maintenance of B lineage–committed cells. PAX5 may be crucial in the control of proliferation of mature B cells, and its deregulated expression might contribute to abnormal proliferation and, thus, to lymphomagenesis.36 A
chromosomal translocation, t(9;14)(p13;q32), involving PAX5
and the potent Eµ enhancer of the IgH gene resulting in over-expression of PAX5 in B cells is reported in cases of B-cell lymphoma.37Gains of 9p increase the copy number of PAX5
and, consequently, also might result in its overexpression. With reference to prognostic factors, hypodiploidy is associated with a poor prognosis, whereas hyperdiploid variants are associated with longer survival in MM and MGUS.17-19,35,38-41In our study, 11 (19%) of 57 cases were
hyperdiploid using CGH, with 4 cases showing trisomy 9 among other changes. According to the aforementioned FISH study by Pérez-Simón and colleagues,35trisomies of
chromo-somes 6, 9, and 17 are good prognostic factors with longer overall survival rates than trisomies of other chromosomes and no trisomy at all.
Another parameter, β2-microglobulin (β2-M), is sup-posed to be an independent prognostic factor associated with poor prognosis if the level is increased.42The present study
seems to confirm this finding because 12 (75%) of 16 patients with aberrations had an increased level of β2-M (>170 nmol/L). Only 3 (20%) of 15 patients with either normal karyotypes or normal ratio profiles had an increased level of β2-M. The mean β2-M level for patients with either normal
karyotypes or normal ratio profiles was 2.3 (range, 0.7-8.1). For patients with either aberrant karyotypes or aberrant ratio profiles, the mean was 4.7 (range, 1.7-13.0). In comparison
with β2-M, the quantity of BMPCs seems to correlate less with the karyotype. Of 25 patients with aberrations, 11 (44%) had a BMPC quantity higher than 50% compared with 7 (27%) of 26 patients with either normal karyotypes or normal ratio profiles. With reference to the β2-M level and BMPCs, there was no difference between patients with and without gains of 9 or 9p, 11, and 21 or 21q or losses of 22. The gener-alization of these results is limited by the small numbers of patients for each category.
This report shows the need to supplement conventional cytogenetics with molecular cytogenetic techniques such as FISH and CGH. In addition, our findings show that all cases have to be analyzed, even though the BMPC quantity is very low. To the best of our knowledge, this is the first report on gains of 9p in MM. The chromosomal region 9p includes a large number of candidate genes. To characterize all these genes and understand the biologic and clinical significance of gains or amplifications of 9p, further molecular and molecular cytoge-netic studies and gene expression analyses have to be done.
From the Departments of 1Human Genetics, 2Medicine, Hematology and Oncology, and 3Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany.
Address reprint requests to Dr Tchinda: Institut für
Humangenetik, Universitätsklinikum Münster, Vesaliusweg 12-14, 48149 Münster, Germany.
*Drs Tchinda and Volpert contributed equally to this article. Acknowledgments: We thank Joachim Volpert for donating peripheral blood for CGH metaphase spreads. We also are grateful for the excellent technical assistance of Katja Poothuis and Marcelina Skrobek.
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