p53 gene mutations in multiple myeloma.
ABSTRACT To assess whether p53 gene mutation is important in the pathogenesis and progression of multiple myeloma.
Thirty eight DNA samples (derived predominantly from bone marrow) obtained from 31 patients with multiple myeloma were examined for mutations in p53 exons 5-9 by polymerase chain reaction single strand conformation polymorphism. Twenty three samples were analysed at the time of diagnosis (one patient had plasma cell leukaemia), three in plateau phase, and 12 at relapse (one plasma cell leukaemia and one extramedullary relapse).
One p53 mutation was detected in this group of patients (3.2%). This was seen in the diagnostic bone marrow sample of a 35 year old man with stage IIA disease and occurred in exon 6 as a result of a silent A to G transition at codon 213 (CGA-->CGG), a polymorphism that has been reported in about 3% of breast and lung tumours.
p53 gene mutations are rare events in multiple myeloma and would seem to be of limited value as a prognostic factor.
- SourceAvailable from: Brian Walker[Show abstract] [Hide abstract]
ABSTRACT: Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P < 0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group.Genes Chromosomes and Cancer 10/2011; 50(10):765-74. · 3.55 Impact Factor
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ABSTRACT: TP53 is a tumor suppressor gene that functions as transcriptional regulator influencing cellular responses to DNA damage. Here we explored the clinical and transcriptional effects of TP53 expression in multiple myeloma (MM). We found that low expression of TP53, seen in approximately 10% of newly diagnosed patients, is highly correlated with TP53 deletion, an inferior clinical outcome, and represents an independent risk factor. Analysis of the expression of 122 known TP53 target genes in TP53-high vs -low MM cells from 351 newly diagnosed cases, revealed that only a few were highly correlated with TP53 expression. To elucidate TP53 regulatory networks in MM, we overexpressed TP53 in 4 MM cell lines. Gene expression profiling of these cell lines detected 85 significantly differentially expressed genes, with 50 up-regulated and 35 down-regulated. Unsupervised hierarchical clustering of myeloma samples from 351 newly diagnosed and 90 relapsed patients using the 85 putative TP53 target genes revealed 2 major subgroups showing a strong correlation with TP53 expression and survival. These data suggest that loss of TP53 expression in MM confers high risk and probably results in the deregulation of a novel set of MM-specific TP53-target genes. TP53 target gene specificity may be unique to different cell lineages.Blood 04/2008; 112(10):4235-46. · 9.78 Impact Factor
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ABSTRACT: Multiple myeloma, a neoplasm of plasma cells, accounts for approximately approximately 15% of lymphatohematopoietic cancers (LHC) and 2% of all cancers in the US. Incidence rates increase with age, particularly after age 40, and are higher in men, particularly African American men. The etiology is unknown with no established lifestyle, occupational or environmental risk factors. Although several factors have been implicated as potentially etiologic, findings are inconsistent. We reviewed epidemiologic studies that evaluated lifestyle, dietary, occupational and environmental factors; immune function, family history and genetic factors; and the hypothesized precursor, monoclonal gammopathies of undetermined significance (MGUS). Because multiple myeloma is an uncommon disease, etiologic assessments can be difficult because of small numbers of cases in occupational cohort studies, and few subjects reporting exposure to specific agents in case-control studies. Elevated risks have been reported consistently among persons with a positive family history of LHC. A few studies have reported a relationship between obesity and multiple myeloma, and this may be a promising area of research. Factors underlying higher incidence rates of multiple myeloma in African Americans are not understood. The progression from MGUS to multiple myeloma has been reported in several studies; however, there are no established risk factors for MGUS. To improve our understanding of the causes of multiple myeloma, future research efforts should seek the causes of MGUS. More research is also needed on the genetic factors of multiple myeloma, given the strong familial clustering of the disease.International Journal of Cancer 01/2007; 120 Suppl 12:40-61. · 6.20 Impact Factor
ClinPathol:Mol Pathol 1997;50:18-20
p53 gene mutations in multiple myeloma
R G Owen, S A A Davis, J Randerson, A C Rawstron, F Davies, J A Child, A S Jack,
G J Morgan
Aim-To assess whether p53 gene muta-
tion is important in the pathogenesis and
progression ofmultiple myeloma.
Methods-Thirty eight DNA samples (de-
rived predominantly from bone marrow)
obtained from 31 patients with multiple
myeloma were examined for mutations in
p53 exons 5-9 by polymerase chain reac-
tion single strand conformation polymor-
analysed at the time of diagnosis (one
patient had plasma cell leukaemia), three
in plateau phase, and 12 at relapse (one
plasma cell leukaemia and one extramed-
Results-One p53 mutation was detected
in this group of patients (3.2%). This was
sample ofa 35 year old man with stage IIA
disease and occurred in exon 6 as a result
of a silent A to G transition at codon 213
(CGA -< CGG), a polymorphism that has
been reported in about 3% of breast and
Conclusions-pS3 gene mutations are rare
events in multiple myeloma and would
seem to be oflimited value as a prognostic
(7 Clin Pathol: Mol Pathol 1997;50: 18-20)
Keywords: p53; multiple myeloma.
Oncology, Institute of
General Infirmary at
Leeds, Great George
Street, Leeds LS1 3EX
Dr GJ Morgan.
Accepted for publication
12 December 1996
p53 is a nuclear phosphoprotein encoded by a
gene located on the short arm of chromosome
17 (17p13) which is capable ofblocking cells at
the Gl/S transition, thereby halting the prolif-
eration of DNA damaged cells and promoting
apoptosis. Point mutations of the p53 gene
have been demonstrated in a wide variety of
solid tumours1-5 where they are usually associ-
ated with loss of the other allele. The resultant
loss of p53 function mediates resistance to
chemotherapy induced apoptosis,
often associated with a poor clinical outcome.
A vast number of p53 mutations has been
described. They occur throughout the genomic
sequence but most seem to be clustered in the
coded for by exons 5-9.6
The incidence of p53 mutation in haemato-
logical malignancy is variable: a relatively high
incidence has been reported in high grade
lymphoma, Hodgkin disease, and acute my-
eloid leukaemia but mutations are rare events
in myelodysplastic syndrome, acute lympho-
blastic leukaemia (ALL), chronic lymphocytic
leukaemia (CLL), and chronic myeloid leukae-
mia (CML).7 p53 mutations are, however, fre-
core of the molecule
quently associated with progressive disease
with a poor prognosis, such as CML blast cri-
sis, Richter syndrome, and transformation of
low grade lymphoma.7
The role of p53 gene mutations in the
pathogenesis of multiple myeloma and their
potential use as a prognostic indicator remain
uncertain. Therefore, we have used the widely
accepted technique of polymerase chain reac-
tion single strand conformation polymorphism
(PCR-SSCP) analysis to detect p53 mutations
in tumour samples obtained from 31 patients
with multiple myeloma.
The study population comprised 31 patients
(median age 56.5 years) with multiple my-
eloma (15 IgG, 1 1 IgA, one IgD, and four light
chain) as defined by standard criteria. Twenty
three patients were studied at diagnosis: 20 had
quent chemotherapy, two patients had plasma
cell leukaemia, and the remaining patient had
smouldering myeloma.8 Bone marrow aspirates
containing a minimum of 10% plasma cells
were obtained in each case. A further three
bone marrow samples were obtained from
three patients in plateau phase. Twelve samples
were also obtained from 10 patients at time of
relapse; these consisted of bone marrow in 10
cases, peripheral blood in a case of plasma cell
leukaemia, and a skin biopsy specimen from a
disease. In each case, the tumour specimen
contained a minimum of 10% plasma cells.
PREPARATION OF DNA
High molecular weight DNA was obtained
from tumour samples by proteinase K diges-
tion, phenol-chloroform extraction and cold
The oligonucleotide primers used to amplify
p53 exons 5-9 are listed in table 1. PCR reac-
tions were performed in 50glvolumes with
20-50 pg DNA, 50 pmoles sense and antisense
(NH0)2SO4,75 mM Tris-HCl (pH 9.0), 0.01%
1.5 mM MgCl2, and 50 mM each
dNTP. The reaction mixtures were held at
95°C prior to the addition of 0.5 units of Taq
polymerase (Thermoprime; Advanced Bio-
technologies, Surrey, UK). PCR amplification
conditions for exons 5, 6, 8, and 9 were 950C
for 40 seconds, 62°C for 40 seconds, and 72°C
for 40 seconds for 35 cycles followed by a fur-
ther 90 second extension at 720C. PCR ampli-
fication of exon 7 differed slightly in that the
annealing temperature was increased to 650C.
group.bmj.com on July 15, 2011 - Published by mp.bmj.com Downloaded from
p53 mutation in myeloma
Primer sequences usedfor the PCR amplification ofp53 gene exons 5-9
Exon (base pairs)
Primer sequence (5'-39
F=forward primer; R =reverse primer.
For SSCP analysis, 1.5-3 pl each PCR reac-
tion product was added to 10 jl loading buffer
and denatured at 80°C for 10 minutes before
quenching on ice for several minutes. The
denatured samples were then loaded onto
FMC Mutation Enhancement Gel (FMC Bio-
Products; Rockland, Maine, USA) and electro-
phoresed at a constant 10 W for two to six
hours depending on the fragment size. The gels
were then fixed and the PCR fragments stained
DIRECT SEQUENCING OF PCR PRODUCTS
Abnormal bands were excised from the gel,
100 jl distilled H,O, and 2 ,ul
aliquots were then reamplified with identical
primers and conditions. This PCRproduct was
run on 2%
Qiaquick gel extraction kit (Qiagen GmBH,
Hilden, Germany) and then sequenced in both
directions using an Applied Biosystems (War-
rington, UK) fluorescence ddNTP sequencing
kit according to the manufacturer's instruc-
tions. The products ofthe sequencing reactions
were then analysed using an Applied Biosys-
tems DNA sequencer (Model 373A), equipped
with sequence collection and analysis software,
and compared with the known p53 sequence.
gel, purified with
Thirty eight samples of genomic DNA were
obtained from 31 patients with confirmed
multiple myeloma and analysed for p53 muta-
tions by PCR-SSCP for exons 5-9. In each
case, the tumour sample contained a minimum
of 10% plasma cells. Normal SSCP patterns
were observed in 37/38 samples, whereas an
abnormal exon 6 band was observed in the
remaining sample. This was a diagnostic bone
marrow sample obtained from a 35 year old
man with stage IIA disease who is currently
alive and in complete remission 42 months fol-
lowing autologous bone marrow transplanta-
tion. Direct sequencing demonstrated a silent
A to G transition at codon 213 (CGA -e CGG
both coding for arginine) which was also dem-
onstrated in somatic DNA samples (data not
The aim of this study was to investigate the
degree to which p53 gene mutation contributes
to the pathogenesis and progression ofmultiple
myeloma, and whether their detection should
be included in routine prognostic factor assess-
ment. We studied 38 DNA samples obtained
from 31 patients with PCR-SSCP for p53
exons 5-9 and found a single mutant band.
This was demonstrated in the diagnostic bone
marrow samples of a 35 year old man with
stage IIA disease. Direct sequencing demon-
strated this to be caused by a silent A to G
transition at codon 213. This polymorphism
has been described previously in about 3% of
lung and breast tumours,9 and was
demonstrated in the somatic DNA in our
patient. This finding clearly emphasises the
importance of sequencing abnormal SSCP
bands, particularly as this polymorphism re-
sults in the loss of a Taql restriction site.
This low frequency ofmutation is likely to be
a real phenomenon as mutations occurring
outside exons 5-9 are rare in other tumour
types,6 and all tumour specimens analysed in
this study contained a sufficient proportion
plasma cells-that is, a minimum of 10%. It is
possible, however, that we missed some mutant
bands whose mobilities were similar to that of
wild type p53. Our results are in accordance
with other PCR-SSCP based studies which
Interestingly, germline mutations
have not been demonstrated in individuals with
two or more relatives with multiple myeloma."
PCR-SSCP, however, is not sufficiently sen-
sitive to demonstrate mutations occurring in
minor subclones which can subsequently be
responsible for relapse. This phenomenon has
leukaemia" but is unlikely to be major factor in
as we were unable
demonstrate any mutant bands in the 12 sam-
ples obtained at relapse. p53 gene mutation
proportion of patients; Mazars et al'4 demon-
myeloma cell lines derived from patients in the
terminal phases of their disease, although, of
course, these may have been acquired in vitro.
Analysis of clinically derived material with
PCR-SSCP and DNA sequencing have also
demonstrated mutations in up to 40% of
patients with end stage disease and plasma cell
leukaemia.'5-'7 We were, however, unable to
demonstrate mutations in three patients with
plasma cell leukaemia and a further patient
with end stage extramedullary disease.
We conclude that p53 mutation is a rare
event in multiple myeloma and would seem to
be of limited value as a prognostic indicator.
When detected, mutations seem to be confined
to patients with end stage and leukaemic forms
of the disease, a situation analogous to CML
blast crisis, Richter syndrome, and transforma-
tion of follicular lymphoma.
of 10 human
This work was supported by the Leukaemia Research Fund and
the Yorkshire Cancer Research Campaign.
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R G Owen, S A Davis, J Randerson, et al.
p53 gene mutations in multiple
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