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Blood, 1 July 2001, Vol. 98, No. 1, pp. 244-246
BRIEF REPORT
p16INK4a and
p15INK4b gene methylations in plasma cells from
monoclonal gammopathy of undetermined significance
Gaëlle Guillerm,
Emmanuel Gyan,
Darius Wolowiec,
Thierry Facon,
Hervé Avet-Loiseau,
Kazimierz Kuliczkowski,
Francis Bauters,
Pierre Fenaux, and
Bruno Quesnel
From the Unité Institut National de la
Santé et de la Recherche Médicale 524, Institut de
Recherche sur le Cancer de Lille (IRCL), and the Service des Maladies
du Sang, Centre Hospitalier et Universitaire (CHU) Lille, Lille,
France; the Department of Hematology, Wroclaw Medical University,
Wroclaw, Poland; and the Laboratoire d'Hématologie, CHU Nantes,
Nantes, France.
 |
Abstract |
p15INK4b and p16INK4a proteins are
cell cycle regulators involved in the inhibition of G1 phase
progression. High frequency of methylation of both genes has been
reported in multiple myeloma (MM), but it remains to be determined how
and when these alterations contribute to tumorigenesis. Monoclonal
gammopathy of undetermined significance (MGUS) represents an early
disease stage in a fraction of MMs. Plasma cells from 33 patients with
MGUS and 33 patients with MM were isolated and analyzed for
p15INK4b and p16INK4a
methylation by methylation-specific polymerase chain reaction. Selective methylation was found in 19% for
p16INK4a, 36% for
p15INK4b, and 6.5% for both genes in MGUS, and
frequencies were similar in MM suggesting that methylation of these
genes is an early event, not associated with transition from MGUS to
MM. p15INK4b and
p16INK4a gene methylation might contribute to
immortalization of plasma cells rather than malignant transformation in
the natural history of MM.
(Blood. 2001;98:244-246)
© 2001 by The American Society of Hematology.
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Introduction |
Multiple myeloma (MM) has been related to
monoclonal gammopathy of undetermined significance (MGUS). Patients
with MGUS show a significant risk of progression to MM with an annual
actuarial risk of malignant transformation of 0.8%, and up to 33% of
newly diagnosed MM patients had a previous history of
MGUS.1,2 Genes involved in the occurrence of MGUS and
those promoting malignant transformation from MGUS to MM have not been
identified.3 One of the most frequent gene alterations in
MM is methylation of the p15INK4b and
p16INK4a genes in the 5' upstream
region.4-10 p15INK4b and p16INK4a
proteins are cell cycle regulators involved in the inhibition of G1
phase progression. Both proteins associate with cyclin-dependent kinases 4 and 6 (CDK4, CDK6) and cyclin D-CDK4/6 complexes, and inhibit
their kinase activities. The p16INK4a and
p15INK4b genes can be inactivated by homozygous
deletion, point mutation, or methylation in various tumor types.
Frequencies of p16INK4a or
p15INK4b gene methylation up to 75% have been
reported in MM and in myeloma-derived cell lines.4,10
However, it is not known if p16INK4a,
p15INK4b, or both genes are already methylated
in some MGUS or if methylation occurs during malignant transformation.
The number of clonal plasma cells in the bone marrow from MGUS appears
to be very low and Zandecki et al showed that, in contrast to myeloma
cells, which are usually really monoclonal, several cytogenetic
subclones may coexist within MGUS, suggesting that detection of
p16INK4a and p15INK4b
gene methylation would need a sensitive method of
analysis.11,12 In order to investigate if methylation of
the p16INK4a and p15INK4b
genes occurs early in MGUS or later in MM, we selected plasma cells
from patients with MGUS and patients with MM and analyzed p16INK4a and p15INK4b
gene methylation using the methylation-specific polymerase chain reaction (MS-PCR).13
| |
Study design |
Bone marrow mononuclear cells from patients with MGUS
and patients with MM were isolated by Ficoll Hypaque sedimentation and plasma cells were purified using the anti-CD138 plasma cell isolation system (Miltenyi-Biotec, Bergisch Gladbach, Germany) according to the
manufacturer's recommendations. Purities of positive and negative
fractions were analyzed in 6 MGUS samples and 6 MM samples by flow
cytometry analysis using a phycoerythrin (PE)-conjugated mouse
antihuman CD138 monoclonal antibody (Beckman Coulter, Miami, FL) or
control isotype added simultaneously to cell separation. Morphologic
evaluation of the positive fraction was also performed on stained
cytocentrifuge slides. Purified plasma cells were frozen and stored in
liquid nitrogen before use.
DNA extracted from CD138-selected cells was modified for MS-PCR by
bisulfite using the CpGenome DNA Modification Kit (Intergen, Purchase,
NY) and p16INK4a and
p15INK4b gene-promoter regions were amplified
with DNA-methylated and -unmethylated specific primers using CpG WIZ
amplification kits (Intergen) according to the manufacturer's
recommendations. Reactions were hot-started using AmpliTaq Gold (Perkin
Elmer, Foster City, CA). A first step of denaturation at 95°C
for 10 minutes was followed by 35 cycles of amplification (30 seconds
at 95°C, 30 seconds at 60°C, 30 seconds at 72°C), and by a final
10-minute extension at 72°C. Controls without DNA were performed for
each set of PCR reactions. PCR products (10 µL) were loaded on 2%
agarose gels stained with GelStar nucleic acid gel stain (BioWhittaker,
Walkersville, MD) and visualized under ultraviolet (UV)
illumination directly and with a gel scan software analysis system
(Bio-Print, Marne la Vallée, France). DNA from the HeLa cell
line, which has been reported to be unmethylated for
p15INK4b and p16INK4a,
was used as negative control. DNA from the Raji cell line, previously reported to have p15INK4b and
p16INK4a extensive methylation, and from the
RPMI-8226 MM cell line, which is methylated for the
p16INK4a but not the
p15INK4b gene, were used as positive
controls.4,14
Deletion of chromosome 13 was also searched in 21 MGUS samples and 17 MM samples by fluorescence in situ hybridization (FISH) using the
D13S319-probe (Vysis, Downers Grove, IL) mapping at 13q14 as
previously reported.15
| |
Results and discussion |
Thirty-three samples of plasma cells isolated from patients with
MGUS and 33 samples from patients with MM were analyzed by MS-PCR.
Purity of the positive fraction was more than 91% in the 6 MGUS
samples and 6 MM samples. Examinations of cytocentrifuge slides showed
plasma cell morphology in all cells. CD138+ cells in all
negative fractions were less than 1%. Patient characteristics are
presented in Table 1.
In agreement with previous reports, both the RPMI-8226 and Raji cell
lines were methylated for the p16INK4a
gene.4 Results of MS-PCR in MGUS and MM samples are
summarized in Table 1. Selective methylation was found in 19% for
p16INK4a, 36% for
p15INK4b, and 6.5% for both genes in MGUS. No
correlation could be made between methylation and gender, age, isotype,
or level of M-component. MS-PCR showed in all MGUS and MM samples the
154-bp and 162-bp bands corresponding to respective amplifications of
unmethylated p16INK4a and
p15INK4b genes (Figure
1). It has been previously shown that
p16INK4a and p15INK4b
gene methylation in acute leukemia is heterogeneous and that, even in
heavily methylated cell lines, unmethylated
p16INK4a and p15INK4b
gene DNA could be detected.16 As several subclones may
coexist in MGUS, it is also possible that the
p16INK4a and p15INK4b
genes were methylated in only a fraction of clonal
cells.11,12 Although we cannot rule out that, especially
in MGUS, MS-PCR detected unmethylated DNA in non-clonal cells. Analysis
of the CD138 fraction of 16 MGUS samples and 9 MM samples
showed that among 8 MGUS samples and 4 MM samples methylated for the
p15INK4b gene, and 5 MGUS samples and 3 MM
samples methylated for the p16INK4a gene in
CD138-selected cells, one MGUS sample and one MM sample were also
methylated for p16INK4a, and one MM sample was
methylated for p15INK4b in the
CD138 fraction. Flow cytometry analysis of this MGUS
sample showed that 0.3% of cells remained CD138+ after
separation in the negative fraction. No methylation of either the
p16INK4a or p15INK4b gene
was detected in the CD138 fraction of MGUS and MM samples
unmethylated in CD138+-selected cells. These data suggest
that methylation occurs preferentially in CD138+ cells.
However, given the fact that few CD138 cells persisted in negative
fractions, we cannot rule out that other bone marrow cells were
methylated in few patients.
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| Figure 1.
Methylation-specific PCR of the
p15INK4b and p16INK4a
genes in selected plasma cells from patients with MGUS.
MS-PCR was performed with specific primers for unmethylated (U) and
methylated (M) p15INK4b and p16INK4a alleles in
Raji and RPMI-8226 cell lines as control and in CD138-selected MGUS
plasma cells (samples 76, 77, 78, and 79). PCR product of appropriate
molecular weight indicates the presence of unmethylated and/or
methylated (A) p15INK4b (162 bp and 154 bp for U and M,
respectively) and (B) p16INK4a alleles (154 bp and 145 bp
for U and M, respectively) in that sample.
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|
Frequencies of methylation of the p15INK4b and
p16INK4a genes were not significantly different
among MGUS samples and MM samples (P = .44 and .88, respectively; chi square test), and also between stage I/II and stage
III/relapsed or refractory patients with MM (P = .9 and
.637, respectively; Fisher exact test). Results were obtained on
purified CD138 cells, thus limiting possible variations of results
related to variable plasma cell marrow infiltration. Among 21 patients
with MGUS analyzed by FISH in our experiments, 2 of them showed
deletion of chromosome 13 in plasma cells and both were methylated for
the p15INK4b gene without methylation of
p16INK4a. Deletion of chromosome 13 was found in
7 of 17 patients with MM; 2 of them showed exclusive methylation of
p15INK4b gene and one showed exclusive
methylation of p16INK4a. Monosomy 13 has been
associated with the transition from MGUS to MM and to poor prognosis of
MM.15,17 These findings suggest that
p16INK4a and p15INK4b
gene methylations and acquisition of monosomy 13 are distinct events in
the evolution of MGUS. Ng et al reported similar incidence of
p15INK4b and p16INK4a
gene methylation in pretreated and posttreated patients with MM.10 Inactivation of p16INK4a gene
has been reported in benign tumors like adenomas and in premalignant
lesions.18-20 Loss of p16INK4a gene
expression by methylation occurs early in the establishment of cell
lines from primary culture. These data suggest that
p15INK4b and p16INK4a
gene methylations are not associated with malignant transformation from
MGUS to MM but rather might contribute to immortalization of plasma cells.
Fourteen MGUS samples (12 of them without methylation of
p16INK4a) had p15INK4b
gene methylation. Frequent methylation of the
p15INK4b gene has only been described in MM,
Burkitt lymphoma, acute leukemia, and myelodysplastic syndromes
(MDS).14,21 p15INK4b protein is one of the
effectors of regulatory effect of transforming growth factor-
(TGF-). TGF- can antagonize in vitro the effect of interleukin 6 (IL-6) in normal B cells, but in contrast can also trigger IL-6
secretion by malignant plasma cells and does not alter pRb
phosphorylation in these cells.22 Mutation of the TGF-
receptor and alteration of TGF- signal transduction have been
described in various human and murine tumors, and TGF- is a key
regulator of bone marrow stem cells.23 Methylation of the
p15INK4b gene might be a mechanism for plasma
cells in MGUS to escape to TGF- inhibitory effect.
Ploidy, immunophenotype, and cytokine expression profiles have been
proposed as possible specific characteristics of high-risk MGUS.
However, because it takes decades to evaluate malignant transformation
rates, the only known risk factors of transformation of MGUS toward
hematologic malignancies remain the level and kinetics of the increase
of the M-component. Given the low number of patients analyzed and the
short follow-up, we are not currently able to know whether methylation
of the p15INK4b and
p16INK4a genes might define a subset of patients
with MGUS who are likely to develop MM. Moreover, we do not know yet
extensively which other molecular events occur during the
transformation of MGUS toward MM. Such information is needed to design
experiments comparing properties of methylated and unmethylated clones
isolated from MGUS.
In conclusion, we showed that p15INK4b and
p16INK4a gene methylations are present at
similar incidences in patients with MGUS and patients with MM,
supporting the idea that alteration of the regulation of G1 phase of
the cycle is a very early event in the history of MM.
| |
Footnotes |
Submitted November 15, 2000; accepted March 9, 2001.
Supported by the Ligue Contre le Cancer (Comité du Nord and
Comité du Pas de Calais).
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: Bruno Quesnel, Service des Maladies du Sang, CHU
Lille, 1 Place de Verdun, 59037 Lille, France; e-mail:
bquesnel{at}nordnet.fr.
| |
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Abstract
Introduction
