Impact of Global and Gene-specific DNA
Methylation Pattern in Relapsed Multiple
Myeloma Patients Treated with Bortezomib
Carlos Fernández de Larrea1, Beatriz Martín1, María Teresa Cibeira1,
Alfons Navarro2, Natalia Tovar1, Tania Díaz2, Laura Rosiñol1,
Mariano Monzó2, Alvaro Urbano-Ispizua1 and Joan Bladé1
1Department of Hematology. Hospital Clínic; 2Molecular Oncology and
Embryology Laboratory, Human Anatomy Unit. School of Medicine.
University of Barcelona. IDIBAPS. Barcelona, Spain.
Introduction
EPIGENETICS
DNA modification ...
DNA methylation
Protein modification ...
Chromatin, Histones
Posttranscriptional regulation...
miRNAs
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Introduction
DNA Methylation and Neoplasia
Esteller M. Nat Rev Genet 2007.
Introduction
DNA
Methylation
and Multiple
Myeloma
Global Hypomethylation
Gene-specific Hypermethylation
Palumbo A, Anderson K. N Engl J Med 2011.
Esteller M. N Eng J Med 2008.
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Objectives
· To examine the global DNA methylation pattern as
well as the specific methylation status of 30 genes in
bone marrow plasma cells of patients with refractory
or relapsed multiple myeloma.
· To correlate these findings with response rate,
progression-free and overall survival after treatment
with bortezomib.
Methods
Patient Characteristics
Variable
N=75 patients
Median age, yrs (range)
65 (29-80)
Gender (M/F)
37/38
M-protein type (%)
IgG
48.7
IgA
23.7
Light chains
21.1
IgD
3.9
Oligosecretory
1.3
Light chain subtype (%)
kappa
61.8
lambda
36.8
Median BM plasma cells (%)
46
Extramedullary involvement (%)
13.2
Median time from diagnosis, yrs
2.9 (0.5-13)
(range)
Median no. of previous lines of
2(1-6)
therapy (range)
Previous bortezomib exposure (%)
8
Previous ASCT (%)
52
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Genomic DNA Extraction
Methods
24 hours each one
Xylol
100
96
70
H
%
2O
%
%
Ethanol
7-14 days
RNase
DNA extraction
Qiagen ®
[DNA] measurement
Methods
Global DNA Methylation
(n=75 patients)
ELISA
Epigentek ®
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Methods
Gene-specific PCR Methylation
(n=42 patients)
Cytokine Network
Immune Response
Transcriptional coactivators
CXCR4, CXCL12, IL6R,
CD40, MIF
EP300, CBP
IL17RA, TGFB1, TGFBR2
Apoptosis
Tumor suppression
Transcription Factors
TNFRSF13C, TNFRSF21,
TP53, BRCA1, DAPK1,
TNFRSF25, BCL2L11
CDH1, RASD1
NFKB1, NFKBIB, IRF4
Qiagen®
Cellular cycle
Cytokine Stimulus
Efflux transporter
CCNB1, CCND1, CCNA2,
Response
ABCG2
CCNE1, CDKN2A, CDKN1A
SOCS3
RESULTS
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Results
Response to Bortezomib
Overall response (OR)
62%
Complete remission
7%
Partial response
44%
Minimal response
10%
No response
12%
Progressive disease
27%
Results
OS of Relapsed MM Patients Treated with
Bortezomib according to DNA Global Methylation
IONT
METHYLA
GLOBAL
DNA
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Results
PFS of Relapsed MM Patients Treated with Bortezomib
according to High Methylation Rate in CXCR4 Promoter Gene
TION
GENE-METHYLA
SPECIFIC
Results
OS of Relapsed MM Patients Treated with Bortezomib
according to High Methylation Rate in NFkB Promoter Gene
TION
GENE-METHYLA
SPECIFIC
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Results
Specific Subset of Patients with Worse Prognosis
Low methylated global genome (<3.95%)
and
high NFkB methylation status (6.7%)
Number of
Median OS
factors
(years)
07.6
(p=0.005)
12.7
21.1
Results
Multivariate Analysis
OS
Variable
p
ORR (95% CI)
Age (65 yrs)
0.201
-
Bone marrow plasma cells (50%)
0.823
-
Global DNA methylation (3.95%)
0.018
0.46 (0.24-0.88)
Variable
p
ORR (95% CI)
Age (65 yrs)
0.684
-
Bone marrow plasma cells (50%)
0.240
-
NFkB methylation status (6.7%)
0.018
3.79 (1.25-11.44)
PFS
Variable
p
ORR (95% CI)
Number of previous treatment lines
0.029
2.49 (1.10-5.64)
Age (65 yrs)
0.247
-
Bone marrow plasma cells (50%)
0.250
-
CXCR4 methylation status (3.97%)
0.073
2.15 (0.93-4.95)
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Conclusions
1. A relative high methylation status in the global genome (>3.95%)
was associated with longer OS after bortezomib therapy in patients
with relapsed or refractory myeloma.
2. A lower methylation status of NFkB (6.7%) and CXCR4 (3.97%)
genes were associated with longer OS and PFS, respectively.
3. Finally, a subset of patients with an ominous prognosis was
identified by DNA methylation pattern at relapse (global genome
<3.95% and NFkB 6.7%), in spite of bortezomib treatment.
Acknowledgments
Laboratory of Hematology
Beatriz Martín
David Rodríguez
Damià Romero
Montserrat Arroyes
Department of Hematology
Joan Bladé
Alvaro Urbano-Ispizua
Mª Teresa Cibeira
Laura Rosiñol
Natalia Tovar
Laboratory of Molecular
Esther Bladé
Oncology and Embryology
Alfons Navarro
Unit of Hematopathology
Tania Díaz
María Rozman
Mariano Monzó
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