Methylation-associated dysregulation of the suppressor of cytokine ...

RESEARCH PAPER

RESEARCH PAPER

Epigenetics 6:8, 1047-1052; August 2011; © 2011 Landes Bioscience

Methylation-associated dysregulation of the suppressor of cytokine signaling-3 gene in multiple myeloma Stefan Wilop,1 Thomas B. van Gemmeren,1 Marjolein H.F.M. Lentjes,2 Manon van Engeland,2 James G. Herman,3 Tim H. Brümmendorf,1 Edgar Jost1,† and Oliver Galm1,†,* Medizinische Klinik IV; Universitätsklinikum Aachen; RWTH Aachen, Germany; 2Department of Pathology; Maastricht University Medical Center; Maastricht, The Netherlands; 3 The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; Baltimore, MD USA

1

These authors contributed equally to this work.

†

Key words: multiple myeloma, hypermethylation, cytokine signaling, biomarker, JAK/STAT pathway

The family of suppressor of cytokine signaling (SOCS) proteins negatively regulates cytokine signaling in different cellular pathways including interleukin-6 (IL-6). Since IL-6 plays an essential role in regulating growth and survival of multiple myeloma (MM) cells, methylation-associated dysregulation of SOCS3 may contribute to the malignant phenotype of MM cells. We used methylation-specific PCR (MSP) to assess the methylation status of the SOCS3 CpG island in five MM cell lines and 70 patient samples. Additional bisulfite sequencing and RNA expression analysis using reverse transcriptase polymerase chain reaction was performed in two cell lines. We identified aberrant SOCS3 methylation in 3/5 MM cell lines. Methylation of SOCS3 in cell lines was associated with transcriptional downregulation. Treatment of OPM-2 cells, which carry a methylated SOCS3 gene, with the demethylating agent 5-aza-2’-deoxycytidine restored SOCS3 expression in association with partial demethylation. In patient samples with malignant plasma cell disorders, SOCS3 was methylated in 5/70 (7.1 %) cases, while there was no aberrant SOCS3 methylation in normal peripheral blood and non-malignant bone marrow cells. We found an association of SOCS3 methylation with extramedullary manifestations (p = 0.03), plasma cell leukemia (p = 0.003), elevated LDH (p = 0.001), increased creatinine ( p = 0.01) and remarkably shortened survival (6.9 vs. 56.1 months, HR 5.9, p = 0.0007). Our findings reveal a novel epigenetic event possibly implicated in the pathogenesis of MM and representing a potential prognostic biomarker. Epigenetic dysregulation of the SOCS3 gene may interfere with the cellular response to the complex cytokine network thus supporting survival and expansion of MM cells.

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction

Multiple myeloma (MM) is a malignant B-cell disorder characterized by the proliferation of monoclonal plasma cells.1 In addition to several prognostic clinical parameters, a number of genetic aberrations characterizing subtypes of the disease at molecular level have been identified.2 Besides other factors, interleukin (IL)-6 plays an essential role in the malignant progression of MM by regulating growth and survival of MM cells.3 The family of suppressor of cytokine signaling (SOCS) proteins negatively regulates magnitude and duration of cytokine signaling in various cellular pathways. Cytokine stimulation results in upregulation of several members of the SOCS family.4 SOCS3 preferentially binds to the SHP-2-binding site on the shared cytokine receptor subunit gp1305 and inhibits signaling by multiple cytokines such as IL-2, IL-3, IL-4, growth hormone, prolactin, erythropoietin, leukemia inhibitory factor, insulin-like growth factor-1, interferon (IFN)γ, IFNα and others (reviewed in ref. 4).

Additionally, SOCS3 negatively regulates IL-6 signaling in vivo.6 Thus, decreased expression of SOCS3 may contribute to growth and malignant transformation of MM cells. SOCS-1, another member of the SOCS family, was previously shown to be silenced in association with CpG island hypermethylation in MM.7 Methylation of CpG islands near gene promoter regions may be a cause of transcriptional downregulation and represents, in addition to genetic aberrations, an important epigenetic mechanism of gene silencing in the pathogenesis of human cancer.8-10 In this study, we examined the methylation status of the SOCS3 gene in MM cell lines and samples from patients with malignant plasma cell disorders. Results MSP analysis of the SOCS3 methylation status in nonmalignant controls. There was no SOCS3 methylation in 20 non-malignant BM samples and five PB samples from healthy volunteers.

*Correspondence to: Oliver Galm; Email: [email protected] Submitted: 04/20/11; Accepted: 06/14/11 DOI: 10.4161/epi.6.8.16167 www.landesbioscience.com

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Figure 1. Representative MSP results for SOCS3 in patient samples and cell lines. Methylation was found in XG1 (not shown), RPMI-8226 and OPM-2 cell lines, whereas U266 and LP-1 show no aberrant methylation. Peripheral blood (PB), in vitro methylated DNA (IVD) and water served as controls.

patients. Table 1 shows clinical and laboratory parameters of our patient cohort according to the methylation status of SOCS3. We found an association of SOCS3 methylation with extramedullary manifestations (p = 0.03, Fisher’s exact test), plasma cell leukemia (p = 0.003, Fisher’s exact test), elevated LDH (p = 0.001, t-test) and increased creatinine (p = 0.01, t-test). Interestingly, SOCS-3 methylation occurred in stage III MM only. As shown in Figure 4, overall survival was remarkably shortened in patients harboring a methylated SOCS3 gene (6.9 vs. 56.1 months, HR 5.9, p = 0.0007). However, entering the variables stage, plasma cell leukemia, extramedullary manifestations, age, WBC, hemoglobin, platelets, LDH, ß2-microglobuline, albumine, creatinine, calcium and SOCS3 methylation status into multivariate Cox analysis for survival, this significance was lost. Methylation of the SOCS1 CpG island was found in 31/70 (44.3%) of our patient samples. There was no correlation between methylation of SOCS1 and SOCS3. Discussion The SOCS family members, mainly SOCS1 and SOCS3, negatively regulate the JAK/STAT pathway and exhibit profound effects on the regulation of immunity and inflammation by affecting the activation, development and homeostatic functions of all lineages involved in immune and inflammatory responses.16 STAT proteins are involved in inducing and maintaining a proinflammatory environment in cancer initiation and progression.17 Recently, methylation of the SOCS3 CpG island was found in head an neck cancer18 and lung cancer cells.19 In the latter, restoration of SOCS3 resulted in induction of apoptosis and growth suppression, thus indicating a tumor suppressor function for SOCS3.19 For the first time, we here report a role for SOCS3 methylation in malignant plasma cell disorders. SOCS1 and SOCS3 transcripts in normal cells are mostly present at only undetectable levels but are rapidly induced by a variety of cytokines.4 Whereas normal BM and PB seemed to be completely unmethylated, we found a substantial proportion of MM cell lines and samples from patients with malignant plasma cell disorders methylated for at least one of these genes. Our findings reveal a novel epigenetic event that may be implicated in the pathogenesis of MM. Patients with SOCS3 methylation had a higher proportion of extramedullary manifestations, more plasma cell leukemias, higher LDH levels and increased creatinine values at the time of sampling. Furthermore, the group of patients with SOCS3 methylation showed a remarkably shorter survival. We have previously shown that SOCS-1, another member of the SOCS family, is commonly hypermethylated in MM.7 In malignant plasma cell disorders, aberrant methylation of SOCS3 appeared to occur less frequently than methylation of SOCS1. SOCS proteins are important for the negative regulation of JAK/ STAT signaling and their inactivation may interfere with the cellular response to the complex cytokine network controlling immune function, cellular growth, differentiation and hematopoiesis.20 Among various other cytokines, IL-6 is considered

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 2. SOCS3 RT-PCR in MM cell lines before and after treatment with DAC (1 μM) for 4 days. The presence or absence of reverse transcriptase is indicated by RT+ or RT-, respectively. RT-PCR for GAPDH was performed for all samples to ensure the integrity of the reverse transcription reactions.

MSP analysis of the SOCS3 methylation status in MM cell lines. We then analyzed the methylation status of the SOCS3 CpG island by MSP in five MM cell lines. SOCS3 methylation was found in XG-1, RPMI-8226 and OPM-2 cells, while the cell lines U266 and LP-1 did not show aberrant methylation in this region (Fig. 1). SOCS3 expression in MM cell lines. Next, we examined SOCS3 expression in the OPM-2 and U266 cells by RT-PCR. As shown in Figure 2, aberrant methylation of SOCS3 was associated with transcriptional silencing in OPM-2 cells, while U266 cells showed expression of SOCS3. Treatment of OPM-2 cells with the demethylating agent DAC for four days resulted in reexpression of SOCS3, while there was no influence on SOCS3 transcription levels in U266 cells. Bisulfite sequencing of the SOCS3 CpG island. We then examined the methylation patterns of the SOCS3 CpG island in normal PB and the cell lines OPM-2 and LP-1 by sodium bisulfite sequencing of individual alleles (Fig. 3A–C). Normal PB and LP-1 cells were mostly unmethylated, while almost every allele in OPM-2 cells harbored dense methylation throughout the entire region surrounding the translation start site. Incubation of OPM-2 cells with the demethylating agent DAC (1.0 μM) for four days resulted in partial demethylation of the SOCS3 CpG island (Fig. 3C and D). SOCS3 methylation in patient samples. Methylation analysis of samples from patients with malignant plasma cell disorders by MSP revealed hypermethylation of SOCS3 in 5/70 (7.1%)

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Figure 3. Bisulfite sequencing of the SOCS3 CpG island. Each circle indicates a CpG dinucleotide (open circles, unmethylated sites; closed circles, methylated sites) in the primary sequence, and each line of circles represents analysis of a single cloned allele. The x-axis labeling indicates the position relative to the SOCS3 translation start site. Table 1. Association of clinical and laboratory parameters with the SOCS3 methylation status SOCS3 unmethylated n = 65

SOCS3 methylated n=5

IgG

44/65 (67.7%)

2/5 (40.0%)

IgA

12/65 (18.4%)

1/5 (20.0%)

Parameter

p value


Heavy chain

n.s.

IgM

1/65 (1.5%)

0/5 (0.0%)

none

8/65 (12.3%)

2/5 (20.0%)

k

49/65 (75.4%)

2/5 (40.0%)

l

12/5 (18.4%)

2/5 (40.0%)

none

4/65 (6.2%)

1/5 (20.0%)

Light chain

n.s.

Stage

#

n.s.

MGUS

3/65 (4.6%)

0/5 (0.0%)

I

7/65 (10.8%)

0/5 (0.0%)

II

9/65 (13.9%)

0/5 (0.0%)

III

46/65 (70.1%)

5/5 (100.0%)

Plasma cell leukemia

3/65 (4.6%)

3/5 (60.0%)

p = 0.0033#

Extramedullary manifestations

8/65 (12.3%)

3/5 (60.0%)

p = 0.0250#

Age (mean in years)

64.6

66.8

n.s.

WBC (mean in G/L)

7.9

8.8

n.s.

Hemoglobin (mean in g/L)

114.1

88.8

n.s.

Platelets (mean in G/L)

230

147

n.s.

LDH (mean in U/L)

203.2

375.2

p = 0.0011+

β2-microglobuline (mg/dL)

4.32

6.70

n.s.

Albumine (mean in g/L)

38.2

39.0

n.s.

Creatinine (mean in mg/dL)

1.21

2.18

p = 0.0128+

Fisher’s exact test, +t-test.

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of SOCS3 in the cell line OPM-2 as shown using RT-PCR. Conversely, as expected, there was no influence on the SOCS3 transcription levels in the unmethylated U266 cell line. Demethylating agents such as 5-aza-2'-deoxycytidine and 5-azacytidine have proven efficacy in therapy of myelodysplastic syndromes and acute myeloid leukemia. In the context of the increasing evidence for the role of DNA methylation changes in the pathogenesis of MM, demethylating agents may be regarded as novel therapeutic approaches in the future. Additional prospective studies are warranted to better clarify the consequences of epigenetic dysregulation of SOCS3 on the development of the phenotype in malignant plasma cell disorders as well as the potential role as a biomarker. Figure 4. Overall survival from time of sample according to the methylation status of the SOCS3 promoter. Survival is shorter in cases with methylated SOCS3 (6.9 vs. 56.1 months, HR 5.9, p = 0.0007).

Materials and Methods

Human tissue samples. Bone marrow (BM) and peripheral blood (PB) specimens were obtained during routine clinical assessment of 67 patients with MM and three patients with monoclonal gammopathy of undetermined significance (MGUS), who presented at the University Hospital Aachen, Germany, between 1995 and 2005. MM diagnosis and staging classification were made in accordance with standard criteria.11 Twenty BM aspirates were obtained from patients with non-metastatic solid tumors or malignant lymphoma without BM infiltration or hematopoietic dysfunction as part of the routine staging procedure. PB samples were collected from five healthy volunteers. The collection of patient samples for analysis of genetic and epigenetic changes was approved by the University hospital ethics committee and patients gave informed consent in accordance with the Declaration of Helsinki. Some of these patients have been included in previously published studies in references 7 and 12. Mononuclear cells from BM and PB were separated by density gradient centrifugation prior to further analysis. Cell culture and drug treatment. We obtained the MM cell lines U266, OPM-2, RPMI8226 and LP-1 from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). XG-1 cells were kindly provided by P.C. Heinrich (RWTH Aachen, Germany). All cell lines except LP-1 were routinely cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10–20% fetal calf serum (FCS; Gemini Bio-Products, Woodland, CA). LP-1 cells were routinely cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Rockville, MD) with 10% FCS. For gene reexpression and demethylation studies, cell lines were treated with a final concentration of 1.0 μM DAC (Sigma, St. Louis, MO) for 96 h, harvested and subjected to RNA and DNA extraction. Methylation-specific polymerase chain reaction (MSP). MSP was performed as initially described by Herman et al.13


to be the essential factor for MM cells supporting survival and expansion of MM cells by stimulating cell division and preventing apoptosis.3 All of our patients with aberrant SOCS3 methylation and therefore presumably SOCS3 downregulation had MM stage III, and 3 out of 5 MM cell lines harbored SOCS3 methylation. In accordance, using a microarray approach, Broyl et al. recently identified SOCS3 as being overexpressed in a proportion of MM samples predominantly comprising patients in early stages (67% ISS stage I).21 We previously reported hypermethylation of the related SOCS1 gene in approx 45% of MM patient samples, but we did not find any correlation with clinical or laboratory parameters or survival.7 In our current cohort of 70 patients, there was no association between SOCS1 and SOCS3 methylation. Therefore, our data suggest that methylation of SOCS1 may be a rather early event during malignant transformation whereas methylation of SOCS3 occurs mainly at more advanced disease stages and may serve as a marker for aggressive disease. However, it remains yet unknown, whether detection of SOCS3 methylation would have implications for the guidance of treatment. For non-malignant BM or PB, all samples revealed no hypermethylation of the SOCS1 and SOCS3 CpG island. Owing to the high sensitivity of MSP of 0.1%,13 periodic analyses of the methylation status of these genes during follow up may provide early evidence for progression or relapse of the malignant plasma cell clone. Our results show that CpG island methylation was associated with transcriptional silencing in OPM-2 cells, whereas in unmethylated U266 cells, SOCS3 expression was present. Bisulfite sequencing revealed a marked decrease in methylation after treatment with the demethylating agent DAC resulting in reexpression

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Primers for SOCS3 methylation analysis have been published previously in reference 14. Briefly, genomic DNA was isolated from cell lines and primary tissues using standard methods. A purification of plasma cells prior to further analysis was not performed owing to the high sensitivity of the MSP technique with a detection limit of approximately 0.1% of methylated DNA present in an otherwise unmethylated sample. Approximately 1 μg of DNA was sodium bisulfite-modified and subjected to MSP as described previously. MSP primers that specifically recognized the unmethylated SOCS3 sequence were 5'-GTT GGA GGG TTT TGG GTA TTT AAT GT-3' (sense) and 5'-TAA ATA ACC ATA ACA CAC AAA ACC AAC A-3' (antisense); primers specific for the methylated SOCS3 sequence were 5'-TGG AGG GTT TCG GGT ATT TAA CGC-3' (sense) and 5'-ATA ACC ATA ACG CAC GAA ACC AAC G-3' (antisense). The primers for the unmethylated sequence cover the bases -158 to -133 and -36 to -10, and the primers for the methylated sequence cover the bases -156 to -133 and -36 to -13 (relative to the translation start site). Reactions were hot-started at 95°C for 5 min and held at 80°C before addition of 0.625 U of Taq polymerase (Sigma, St. Louis, MO). Temperature conditions for thermocycling were as follows: 35 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by 1 cycle of 72°C for 5 min. Normal DNA from PB was treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) in order to generate in vitro methylated DNA (IVD) that served as a universally positive control for methylated alleles. PCR products were separated on 2.5% agarose gels and visualized by ethidium bromide staining. MSP analysis of the SOCS1 CpG island was performed in our extended patient cohort as described previously in reference 7. Reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was isolated using a commercially available kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Approximately 3 μg RNA per sample were reverse transcribed with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). For PCR, 1 μl cDNA was amplified using SOCS3 primers 5'-CTC AAG ACC TTC AGC TCC AA-3' (sense) and 5'-TTC TCA TAG GAG TCC AGG TG-3' (antisense) and GAPDH primers 5'-GAC CAC AGT CCA TGC CAT CAC3' (sense) and 5'-GTC CAC CAC CCT GTT GCT GTA-3' (antisense) as previously published by Niemand et al. Reactions were hot-started at 95°C for 5 min and held at 80°C before addition of 1.25 U of Taq polymerase (Invitrogen, Carlsbad, CA). Temperature conditions for SOCS3 were 35 cycles of 95°C for 1 min, 58°C for 1 min and 72°C for 1 min, followed by 1 cycle

of 72°C for 5 min, and for GAPDH 23 cycles of 95°C for 1 min, 63°C for 1 min and 72°C for 1 min, followed by 1 cycle of 72°C for 5 min. PCR products were separated on 2.5% agarose gels and visualized by ethidium bromide staining. Bisulfite sequencing. Sodium bisulfite-modified DNA was amplified with primers 5'-GTT TTA GGG GTA GTG GGT GTT TTA GT-3' (sense) and 5'-ATA CTC TCT CTT AAA ACT AAA AAT CTT AAA A-3' (antisense). Reactions were hot-started at 95°C for 5 min and held at 80°C before addition of 1.25 U of Taq polymerase (Sigma, St. Louis, MO). Temperature conditions for PCR were as follows: 35 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, followed by a final extension of 72°C for 5 min. The 247 bp PCR products were purified using a commercially available kit (Qiagen, Valencia, CA), cloned into the TA vector pCR2.1 TOPO (Invitrogen, Carlsbad, CA) and transformed into E. coli according to the manufacturer’s recommendations. Plasmid DNA from isolated clones containing the insert was purified and subjected to automated sequence analysis (ABI automated sequencing). Statistical analysis. Categorical data was compared using the Fisher’s exact test. Associations between the methylation status and laboratory parameters were tested using the independent t-test. Survival data were analyzed using the Kaplan-Meier method and compared using the log-rank test as well as the Cox proportional hazard method. Survival according to SOCS3 methylation was calculated from the date of obtaining the sample until the patient’s death or last visit. All statistical tests were two-sided and were performed using the SAS software package version 9.2 (SAS Institute Inc., Cary, NC). For all tests, p < 0.05 was used as the level of significance.


Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We thank Sandra Mellen, Claudia Schubert, Lucia Vankann and Ingeborg Wiegand for expert technical assistance. This work was supported by grants from the Deutsche Krebshilfe and the Rheinisch-Westfälische Technische Hochschule Aachen (START program). Financial Support

J.G. Herman is a paid consultant to and receives research support from MDxHealth. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflicts of interest policies.

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9. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349:2042-54. 10. Esteller M. Epigenetics in cancer. N Engl J Med 2008; 358:1148-59. 11. Durie BG. Staging and kinetics of multiple myeloma. Semin Oncol 1986; 13:300-9. 12. Jost E, Gezer D, Wilop S, Suzuki H, Herman JG, Osieka R, et al. Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma. Cancer Lett 2009; 281:24-31. 13. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93:9821-6. 14. Middeldorf I, Galm O, Osieka R, Jost E, Herman JG, Wilop S. Sequence of administration and methylation of SOCS3 may govern response to gemtuzumab ozogamicin in combination with conventional chemotherapy in patients with refractory or relapsed acute myelogenous leukemia (AML). Am J Hematol 2010; 85:477-81. 15. Niemand C, Nimmesgern A, Haan S, Fischer P, Schaper F, Rossaint R, et al. Activation of STAT3 by IL-6 and IL-10 in primary human macrophages is differentially modulated by suppressor of cytokine signaling 3. J Immunol 2003; 170:3263-72.

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