|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
NEOPLASIA
From the Myeloma and Transplantation Research Center,
Division of Biometry, University of Arkansas for Medical Sciences and
Arkansas Cancer Research Center, Little Rock, AR
Multiple myeloma (MM) is a hypoproliferative
malignancy yielding informative karyotypes in no more than 30% of
newly diagnosed cases. Although cytogenetic and molecular deletion of
chromosome 13 is associated with poor prognosis, a MM tumor suppressor
gene (TSG) has not been identified. To localize a minimal deleted
region of chromosome 13, clonotypic plasma cells from 50 consecutive patients with MM were subjected to interphase fluorescence in situ
hybridization (FISH) analysis using a panel of 11 probes spanning the
entire long arm of chromosome 13. Whereas chromosome 13 abnormalities
were absent in plasma cells from 25 normal donors, 86% of patients
with MM demonstrated such aberrations. Heterogeneity, both in deletion
frequency and extent, was confirmed by simultaneous FISH with 2 chromosome 13 probes. Deletion hot spots were noted at D13S272 (70%)
and D13S31 (64%), 2 unlinked loci at 13q14. Homozygous deletions at
these loci occurred in 12% (simultaneously in 8%) of the cases.
Molecular deletions were found in all 14 patients with morphologic
deletions, in 21 of 24 with uninformative karyotypes, and 8 of 12 patients with karyotype abnormalities lacking chromosome 13 deletion.
Homozygous deletion of any marker was noted in 4% with low and in 36%
with higher plasma cell labeling index greater than 0.4%
(P = .01). The absence of increasing deletion
incidence and extent with therapy duration suggests that the observed
lesions are not induced by treatment. The high incidence and extent of chromosome 13 deletions require the correlation of specific deletion(s) with poor prognosis. These analyses will provide valuable guidance toward cloning of an MM-TSG.
(Blood. 2000;96:1505-1511) Multiple myeloma (MM) is a clonal B-cell malignancy, putatively
originating in germinal centers of lymph nodes, that homes to and
expands in the bone marrow.1 As a tumor of terminally differentiated plasma cells, its proliferative activity is usually low
so that metaphase karyotyping is difficult and informative in only one
third of newly diagnosed patients.2,3 However, DNA content
abnormalities, as determined by fluorescence in situ hybridization
(FISH) and flow cytometry, are almost universal.4-6 In
contrast to other hematologic malignancies, karyotypes in MM are
typically complex, almost representing a hallmark of this disease.7-9 Despite the genomic chaos, several recurring
chromosomal abnormalities have been identified including structural
rearrangements (eg, translocations of 14q32), trisomies (eg, chromosome
15), and monosomies (eg, chromosome 13).9 Although
recurrent translocations of MYC, BCL1, and BCL2
into the immunoglobulin heavy chain locus on chromosome 14q32 are
fundamental to the development of many B-cell leukemias and
lymphomas,10,11 14q32 translocations in MM involve a great
diversity of partner chromosomes,12-16 none of which,
however, define a distinct clinical entity.
Recent cytogenetic studies, performed in the context of both standard
and high-dose therapy, have shown that partial or complete deletion of
chromosome 13 present in 15% to 20% of newly diagnosed MM imparts a
poor prognosis.17-20 Similar observations have also been
reported for molecular deletions of RB1 and
D13S319 in conventional therapy settings.21,22
Collectively, these data strongly suggest the presence of MM tumor
suppressor gene(s) on chromosome 13. In an effort to identify and
eventually clone these gene(s), interphase FISH with 11 probes spanning
the long arm of chromosome 13 was used to establish a first-generation
molecular deletion map of this chromosome in myeloma.
Cytogenetic deletion map
Probe isolation and labeling
Triple color interphase FISH (TRI-FISH)
Myeloma bone marrow Bone marrow aspirates were collected in heparinized containers from patients with active myeloma during scheduled clinic visits. Signed informed consents for use of samples in research studies were obtained and kept on record. Bone marrow cells were separated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). The cells in the light density fraction (specific gravity 1.077) were resuspended in cell culture media plus 10% fetal bovine serum. Approximately 40 000 mononuclear cells were cytocentrifuged at 1000g for 5 minutes at room temperature.
Twenty-four slides from each sample were prepared. The slides were air
dried overnight, then fixed in 100% ethanol for 5 minutes at room
temperature and baked in a dry 37°C incubator for 6 hours. Slides
were either used immediately in TRI-FISH experiments or stored at
 20°C then used later without loss of signal.
TRI-FISH analysis of normal bone marrow Twenty-five bone marrow aspirates from normal donors were analyzed to determine the random loss or gain of signals in plasma cells and nonplasma cells for each of the 11 chromosome 13 probes used in this study. The age of the normal allogeneic bone marrow donors ranged from 1 to 40 years with a mean of 30 years, representing 10 female and 15 male donors. An average of 85 plasma cells (range, 30-129) and 104 nonplasma cells (range, 55-128) were examined with each of the 11 probes for chromosome 13. This analysis revealed a deletion or duplication incidence of less than or equal to 6% (SD, 4%) for all probes. The incidence of homozygous deletion for any probe was exceedingly rare (1%). A threshold of 20% for loss or gain in chromosome 13-probe signal in plasma cells was considered evidence that trisomy or monosomy had occurred. The 20% cut-off was also chosen for homozygous deletions to facilitate the localization of potentially important regions of chromosome loss.Spectral karyotyping (SKY) and metaphase FISH The SKY procedure has been previously described.34 Metaphase chromosomes were pretreated for FISH by incubation in prewarmed 2 × SSC (pH 7.0, 37°C) for 15 minutes, followed by 0.1 N HCl/0.05% Triton X-100 for 15 minutes, then in 2 × SSC, and finally washed in 1 × PBS. Slides were then immersed in 1% formaldehyde for 10 minutes, washed in 1 × PBS twice and 2 × SSC once, then an ethanol series (70%, 85%, 100%, 2 minutes each), and air dried at room temperature. The BAC probes for D13S221 and D13S285 were directly labeled with Spectrum-Green-dUTP and Spectrum-Red-dUTP, respectively. The chromosome 8 probe, directly labeled with Spectrum Orange-dUTP was obtained from Vysis. The chromosome probes were mixed with Hybrisol VII (Oncor) at a concentration of 5 ng/µL and 10 µL was added to areas containing abundant metapahases. Slides were brought to 75°C for 10 minutes, then to 37°C for more than 16 hours in a humidified chamber. Slides were immersed in prewarmed 65% Formamide/2 × SSC (pH 7.0) for 15 minutes, at 43°C followed by a wash in 2 × SSC for 8 minutes at 37°C, then washed in 1 × PBD (Oncor) twice at room temperature, and finally washed in 1 × PBS. Ten microliters of DAPI/antifade (1:20) was added to each area and the slides viewed.
Conventional cytogenetics of 13q arm deletions in 1000 myeloma patients The cytogenetic deletion map of chromosome 13 is depicted in Figure 2. This map shows 13q arm deletions in 106 patients ordered with respect to the centromeric and telomeric deletion boundaries. Ninety-eight (90%) involved 13q14 and 73 (68%) involved the 13q21 region. Except for 8 cases with deletions distal to q14, the 13q14 band was deleted in each case and represented the minimal region of deletion overlap.
Chromosome 13 deletion analysis of monoclonal plasma cells from MM A panel of 11 probes spanning the long arm of chromosome 13 (Figure 1) was used to analyze 50 patients with MM. TRI-FISH analysis (Figure 3A) showed molecular abnormalities in 86% of patients (Table 2). Deletion frequency for any given probe varied and ranged from 20% (case 30) to more than 90% (case 9). Variation in the frequency of heterozygous and especially homozygous deletions of probes along the chromosome was observed within a given patient. Loss of all 11 probes, suggesting monosomy 13, was observed in 15 patients and of at least 8 probes in an additional 5 so that 40% had major molecular deletions. The most commonly deleted markers were D13S272 at 13q14 (76%), representing the sole anomaly in 2 cases (Figure 3B), and D13S31 at the 13q14-q21 band (68%), which were concurrently deleted in 59%. Homozygous deletions (complete loss of signal), present in 9 patients altogether, involved these 2 probes in 6 cases each, of which 4 revealed concurrent deletions of both probes. Homozygous deletions were also observed for the BRCA2 locus at 13q12 and the marker D13S1619 at 13q22, but not for RB1. Homozygous deletions were never detected in the absence of heterozygous deletions of other probes. A normal diploid region flanked by trisomic markers was evidence of interstitial deletions in cases 13, 14, 15, 40, and 41. Trisomy restricted to telomeric markers, suggesting translocations of duplicated chromosomal material, was observed in cases 3, 27, 28, 32, and 48.
To demonstrate sensitivity of the TRI-FISH method, the results of
TRI-FISH, SKY, and metaphase FISH from the same patient were compared.
TRI-FISH analysis of case 46 showed that all probes from BRCA2 to the
telomeric marker, D13S285, showed only one signal in more than 20% of
the cells, whereas the probes 13-07-5'/3' and D13S221 showed 2 signals
in more than 80% (Table 2). The data suggested that although a large
portion of one chromosome 13 had been deleted, the centromeric region
remained disomic. SKY and metaphase FISH on chromosomes from the same
patient showed the presence of an apparently normal chromosome 13 and a
t(8;13) translocation that was not recognized by conventional G-banding (Figure 4). Eleven separate metaphase
FISH experiments were performed using a commercial probe for the
centromere of chromosome 8 labeled in orange, each of the chromosome 13 probes labeled in green, and the telomeric chromosome 13 probe labeled
in red. The data showed that both 13-07-5'/3' and D13S221 hybridized to
a normal chromosome 13 (colocalization of a green and red signal on the same chromosome) and to chromosome 8 (colocalization of a green and
orange signal) (Figure 4). Although all remaining probes along the
chromosome showed colocalization of a green and red signal on the same
chromosome (normal chromosome 13) they failed to cohybridize with the
chromosome 8 probe. Thus, SKY and metaphase FISH confirm and extend the
TRI-FISH data in this case, in that a q arm deletion had occurred, but
that the centromeric region was disomic and involved in a subtle
t(8;13) translocation.
Chromosome 13 deletion heterogeneity in MM Because all TRI-FISH experiments used a single chromosome 13 probe, these analyses could not address the question of whether deletions of multiple probes occurred in the same or different tumor cells. Simultaneous hybridization of 2 probes carrying different fluorophores was therefore carried out in 3 patients. A representative case (case 50) revealed 5% of the clonotypic plasma cells to be diploid for both probes (colocalization of 2 probes in 2 distinct regions of the nucleus), 48% to be monosomic for both probes (a single red and green colocalizing signal), 12% monomeric for only D13S272 and nullisomic for D13S31 (single red signal, no green signal), and 23% to be nullisomic for both probes (no signals). Such heterogeneity in this and the other 2 cases (not shown) is consistent with the presence of distinct subpopulations of tumor cells with varying degrees of deletion in this chromosome.Association of molecular chromosome 13 deletion and clinical parameters There was no statistically significant association of molecular deletion to age, sex, Durie-Salmon tumor stage, immunoglobulin isotype, or serum 2-microglobulin (data not shown). Molecular deletions were seen in all 14 patients with cytogenetic chromosome deletions, in 21 of 24 of those with uninformative diploid karyotypes, and even in 8 of 12 with myeloma karyotypes without obvious
abnormalities of chromosome 13 (Table 2). Molecular deletions were more
frequently observed in case of higher tumor proliferative activity,
because 9 of 25 patients with plasma call labeling indices more than
0.4% (above the median value) had homozygous deletions compared to 1 of 24 with values of 0.4% or less (P = .01). Deletion of
chromosome 13 markers occurred with similar frequency regardless of
prior therapy in 22 of 24 untreated patients, 13 of 15 treated for up to 12 months, and 9 of 11 treated for more than 12 months
(P = 1.0) (Table 2).
This first comprehensive analysis of molecular chromosome 13 deletions in MM, using an 11-probe panel spanning the long arm of this chromosome, has revealed a surprisingly high incidence of abnormalities. Although 86% of patients demonstrated at least one abnormality, 75% of the deletions were accounted for by the probe D13S272 and 66% by D13S31, both of which were concurrently deleted in 59%. Together with the observation of homozygous deletions of these 2 probes in 12%, our findings suggest a pathogenetic role of the loss of these 2 loci in MM, especially because deletion extent and frequency were not influenced by prior therapy and hence treatment induced. However, a more complex scenario with participation of other deletions in disease progression is likely because both RB1 and BRCA2 were also deleted in 60% and 48%, respectively. The high incidence of deletion detected here (86%) poses a dilemma with regard to the adverse prognostic implications recently reported using single probe FISH for the RB1 and D13S319 loci (40% of patients).21,22 In this study, these 2 probes were deleted in 52% and 70%, respectively, of newly diagnosed patients (data confirmed in an additional 35 newly diagnosed patients, J.S., unpublished data). Prospective investigations are in progress to determine which of these and additional deleted regions, alone or in combination, confer the observed poor prognosis. The analysis of concurrent deletions of 2 or more loci suspected in MM progression requires mutlicolor analysis. Preliminary data with the D13S272 and D13S31 probes indeed indicated marked deletion heterogeneity in individual patients comprising cells with homozygous deletions for one and both probes, monosomy, as well as complete lack of deletion. Given the higher proliferative activity in patients exhibiting homozygous chromosome 13 probe deletions, a proliferation hierarchy may also exist within an individual patient's tumor population, with a cell division advantage conferred by the extent of chromosome 13 deletion. To resolve this issue, concurrent cytokinetic analysis will be required, probably best performed by in vivo BrdU labeling to obtain authentic information. Contrary to other chromosomal aberrations, especially numeric anomalies (B.B., unpublished data), chromosome 13 deletion not only implies rapid disease recurrence but also initial drug resistance.26 Studies of apoptotic pathways in patients lacking chromosome 13 deletion and in those with varying deletion extent should be informative. Studies are in progress to determine whether the observed chromosome 13 deletion frequency and intrapatient deletion heterogeneity is chromosome or disease specific, especially vis-a-vis chronic lymphocytic leukemia (CLL) in which chromosome 13 deletion has been shown not to confer a dismal prognosis. The divergent clinical implications of chromosome 13 loss in MM and CLL27,28 may thus result from the deletion(s) of additional gene(s) in MM. If the inactivated gene happens to be the same in these 2 diseases, several alternative explanations can be envisioned to explain the difference in clinical phenotype. The loss of one functional copy of a gene (haploinsufficiency) may be sufficient to impart the poor clinical phenotype seen in MM. It is also possible that redundant biochemical pathways functional in CLL, but absent in myeloma, compensate for the loss of function incurred by gene inactivation. Additionally, postgerminal center hypermutations, recently shown to also affect the BCL6 proto-oncogene,29 are more frequent in MM than CLL,30 and may affect chromosome 13 genes. Thus, the stage of B-cell differentiation may play a critical role in modulating the effects of a commonly deleted gene, and chromosome 13 tumor suppressor gene(s) may be more prone to inactivating base substitution mutations in MM than in CLL. The observation of a lack of molecular chromosome 13 deletion in 19% of patients with either uninformative diploid karyotypes (3 of 24 patients) or especially MM-typical chromosome abnormalities without chromosome 13 involvement (4 of 12 patients) (Table 2) is consistent with the existence of a distinct nonchromosome 13 deletion entity. However, interstitial deletions (involving additional chromosome 13 regions not covered by our 11-probe panel), microdeletions (not detectable but rather masked by the large probes used) or even more subtle mutations (eg, base substitutions) may exist. Additionally, patients with a seemingly normal diploid chromosome 13 may in fact harbor duplication of an abnormal chromosome that may be identifiable by loss of heterozygosity studies. Based on our data, progression of chromosome 13 deletion-positive monoclonal gammopathies of undetermined significance (MGUS) to MM, as recently demonstrated by Avet-Loiseau,31 may require a deletion evolution. The observed heterogeneity in deletion extent in individual patients with frank MM may thus reflect different stages of disease progression, including the persistence of an original MGUS clone with minimal deletion. The lower proliferative activity of a MGUS subclone may account for its survival even in the face of high-dose therapy, thus explaining the persistence of low-level monoclonal gammopathy in remission. The molecular mechanism(s) of such deletion evolution may be traced to a genomic instability associated with DNA replication in terminally differentiated plasma cells or loss of genome fidelity maintenance genes such as P53, BRCA1, BRCA2, and ATM. To address these critical issues, multiprobe chromosome 13 FISH will be
applied in the following scenarios: (1) long-term MGUS without
progression (Q: no chromosome 13 deletion?) versus MGUS to MM
progression (Q: chromosome 13 deletion evolution?); (2) long-term MM
survivors after high-dose therapy with minimal residual disease (Q: no
or minimal chromosome 13 deletion?); and (3) early relapse after
high-dose therapy (Q: marked deletion heterogeneity and evolution?).
Systematic studies of molecular deletions of chromosome 13 in CLL
(especially in the recently identified subsets of unmutated
CD38+ and hypermutated CD38
We would like to thank Todd Bell, Peter Kim, Janet Lukacs, Alian Li, Yan Xiao, and Elizabeth Williamson for their excellent technical assistance. We thank the members of the Myeloma and Transplantation Research Center clinical staff and data management group. Finally, we would like to thank the patients and their families for their support of our myeloma research program.
Submitted September 22, 1999; accepted April 7, 2000.
Supported in part by CA55819 from the National Cancer Institute, Bethesda, MD.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: John D. Shaughnessy Jr, Myeloma and Transplantation Research Center, University of Arkansas for Medical Sciences and Arkansas Cancer Research Center, 4301 West Markham Street, Little Rock, AR 72205.
1.
Hallek M, Bergsagel PL, Anderson KC.
Multiple myeloma: increasing evidence for a multistep transformation process.
Blood.
1998;91:3-21
2.
Latreille J, Barlogie B, Johnston D, Drewinko B, Alexanian R.
Ploidy and proliferative characteristics in monoclonal gammopathies.
Blood.
1982;59:43-51
3.
Greipp PR, Katzmann JA, O'Fallon WM, Kyle RA.
Value of beta 2-microglobulin level and plasma cell labeling indices as prognostic factors in patients with newly diagnosed myeloma.
Blood.
1988;72:219-223
4.
Latreille J, Barlogie B, Dosik G, Johnston DA, Drewinko B, Alexanian R.
Cellular DNA content as a marker of human multiple myeloma.
Blood.
1980;55:403-408 5. San Miguel JF, Garcia-Sanz R, Gonzalez M, Orfao A. Immunophenotype and DNA cell content in myeloma. Baillieres Clin Haematol. 1995;8:735-759[Medline] [Order article via Infotrieve]. 6. Flactif M, Zandecki M, Lai JL, et al. Interphase fluorescence in situ hybridization (FISH) as a powerful tool for the detection of aneuploidy in multiple myeloma. Leukemia. 1995;9:2109-2114[Medline] [Order article via Infotrieve].
7.
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;66:380-390
8.
Gould J, Alexanian R, Goodacre A, Pathak S, Hecht B, Barlogie B.
Plasma cell karyotype in multiple myeloma.
Blood.
1988;71:453-456 9. Sawyer JR, Waldron JA, Jagannath S, Barlogie B. Cytogenetic findings in 200 patients with multiple myeloma. Cancer Genet Cytogenet. 1995;82:41-49[Medline] [Order article via Infotrieve]. 10. Gauwerky CE, Croce CM. Chromosomal translocations in leukemia. Semin Cancer Biol. 1993;4:333-340[Medline] [Order article via Infotrieve]. 11. Korsmeyer SJ. Chromosomal translocations in lymphoid malignancies reveal novel proto-oncogenes. Annu Rev Immunol. 1992;10:785-807[Medline] [Order article via Infotrieve].
12.
Bergsagel PL, Chesi M, Nardini E, Brents LA, Kirby SL, Kuehl WM.
Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma.
Proc Natl Acad Sci U S A.
1996;93:13931-13936 13. Chesi M, Nardini E, Brents LA, et al. Frequent translocation t(4;14)(p163;q323) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet. 1997;16:260-264[Medline] [Order article via Infotrieve]. 14. Lida S, Rao PH, Butler M, et al. Deregulation of MUM1/IRF4 by chromosomal translocation in multiple myeloma. Nat Genet. 1997;17:226-230[Medline] [Order article via Infotrieve].
15.
Chesi M, Bergsagel PL, Shonukan OO, et al.
Frequent dysregulation of the c-maf proto-oncogene at 16q23 by translocation to an Ig locus in multiple myeloma.
Blood.
1998;91:4457-4463
16.
Stec I, Wright TJ, van Ommen GJB, et al.
WHSC1, a 90 kb SET domain-containing gene, expressed in early development and homologous to a Drosophila dysmorphy gene maps in the Wolf-Hirschhorn syndrome critical region and is fused to IgH in t(4;14) multiple myeloma.
Hum Mol Genet.
1998;7:1071-1082
17.
Tricot G, Barlogie B, Jagannath S, et al.
Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotypes abnormalities.
Blood.
1995;86:4250-4256 18. Barlogie B, Sawyer J, Ayers D, et al. Chromosome 13 myeloma is a distinct entity with poor prognosis despite tandem autotransplants [abstract]. Blood. 1998;92:258a. 19. Shaughnessy J, Barlogie B. Chromosome 13 deletion in myeloma. In Melchers F, Potter M, eds. Current Topics Microbiology and Immunology. 1999;246:199-203. 20. Seong C, Delasalle K, Hayes K, et al. Prognostic value of cytogenetics in multiple myeloma. Br J Haematol. 1998;101:189-194[Medline] [Order article via Infotrieve].
21.
Perez-Simon JA, Garcia-Sanz R, Tabernero MD, et al.
Prognostic value of numerical chromosome aberrations in multiple myeloma: a FISH analysis of 15 different chromosomes.
Blood.
1998;91:3366-3371
22.
Zojer N, Konigsberg R, Ackermann J, et al.
Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization.
Blood.
2000;95:1925-1930
23.
Tricot G, Sawyer JR, Jagannath S, et al.
Unique role of cytogenetics in the prognosis of patients with myeloma receiving high-dose therapy and autotransplants.
J Clin Oncol.
1997;15:2659-2666 24. Kalachikov S, Migliazza A, Cayanis E, et al. Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia. Genomics. 1997;42:369-377[Medline] [Order article via Infotrieve]. 25. Ahmann GJ, Jalal SM, Juneau AL, et al. A novel three-color, clone-specific fluorescence in situ hybridization procedure for monoclonal gammopathies. Cancer Genet Cytogenet. 1998;101:7-11[Medline] [Order article via Infotrieve]. 26. Desikan R, Sawyer J, Munshi N, et al. Durable complete remissions in multiple myeloma with high dose therapy: experience in 1000 patients. Blood. In press. 27. Juliusson G, Oscier DG, Fitchett M, et al. Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med. 1990;323:720-724[Abstract]. 28. Dohner H, Stilgenbauer S, Fischer K, Bentz M, Lichter P. Cytogenetic and molecular cytogenetic analysis of B cell chronic lymphocytic leukemia: specific chromosome aberrations identify prognostic subgroups of patients and point to loci of candidate genes. Leukemia. 1997;2:S19-24. 29. Fais F, Ghiotto F, Hashimoto S, et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest. 1998;102:1515-1525[Medline] [Order article via Infotrieve].
30.
Shen HM, Peters A, Baron B, Zhu X, Storb U.
Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes.
Science.
1980;280:1750-1752
31.
Avet-Loiseau H, Li JY, Morineau N, et al.
Monosomy 13 is associated with the transition of monoclonal gammopathy of undetermined significance to multiple myeloma: Intergroupe Francophone du Myelome.
Blood.
1999;94:2583-2589
32.
Damle RN, Wasil T, Fais F, et al.
Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.
Blood.
1999;94:1840-1847
33.
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK.
Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.
Blood.
1999;94:1848-1854
34.
Sawyer JR, Lukacs J, Munshi N, et al.
Identification of new nonrandom translocations in multiple myeloma with multicolor spectral karyotyping.
Blood.
1998;92:4269-4278
© 2000 by The American Society of Hematology.
| ||||||||||
 |
|
 |
|
  M. Lerner, M. Corcoran, D. Cepeda, M. L. Nielsen, R. Zubarev, F. Ponten, M. Uhlen, S. Hober, D. Grander, and O. Sangfelt The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD Mol. Biol. Cell, May 1, 2007; 18(5): 1670 - 1682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Jakob, K. Egerer, P. Liebisch, S. Turkmen, I. Zavrski, U. Kuckelkorn, U. Heider, M. Kaiser, C. Fleissner, J. Sterz, et al. Circulating proteasome levels are an independent prognostic factor for survival in multiple myeloma Blood, March 1, 2007; 109(5): 2100 - 2105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Agnelli, S. Bicciato, S. Fabris, L. Baldini, F. Morabito, D. Intini, D. Verdelli, A. Callegaro, F. Bertoni, G. Lambertenghi-Deliliers, et al. Integrative genomic analysis reveals distinct transcriptional and genetic features associated with chromosome 13 deletion in multiple myeloma Haematologica, January 1, 2007; 92(1): 56 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Walker, P. E. Leone, M. W. Jenner, C. Li, D. Gonzalez, D. C. Johnson, F. M. Ross, F. E. Davies, and G. J. Morgan Integration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma Blood, September 1, 2006; 108(5): 1733 - 1743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Hanamura, J. P. Stewart, Y. Huang, F. Zhan, M. Santra, J. R. Sawyer, K. Hollmig, M. Zangarri, M. Pineda-Roman, F. van Rhee, et al. Frequent gain of chromosome band 1q21 in plasma-cell dyscrasias detected by fluorescence in situ hybridization: incidence increases from MGUS to relapsed myeloma and is related to prognosis and disease progression following tandem stem-cell transplantation Blood, September 1, 2006; 108(5): 1724 - 1732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Chen, B. Issa, S. Huang, E. Aston, J. Xu, M. Yu, A. R. Brothman, and M. Glenn A Practical Approach to the Detection of Prognostically Significant Genomic Aberrations in Multiple Myeloma J. Mol. Diagn., November 1, 2005; 7(5): 560 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
Top
Abstract
Introduction
or -
light chain antibody
conjugated with 7-amino-4-methylcourmarin-3-acitic acid (AMCA) (Vector
Laboratories, Burlingame, CA) for 30 minutes in a humidified chamber.
After incubation, the slides were washed 2 times in PBD. To enhance the
signal, the slides was incubated with 100 µL of a 1:40 dilution of
AMCA labeled rabbit-antigoat IgG antibody and incubated for 30 minutes
at room temperature in a humidified chamber. Slides were washed 2 times
in 1 × PBD, and Vectashield (Vector Laboratories) antifade and
coverslips added. At least 50 AMCA-positive plasma cells were scored
for the presence of 2 green and 2 red signals using an Olympus BX60 epifluorescence microscope equipped with appropriate filters for detecting fluoroisothiocyanate (FITC), Texas red, and DAPI. The triple
color clone-specific images were captured using a Quips XL genetic
workstation (Vysis).
entities with
different prognoses)