1. Art and Diseño

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Mutational Activation of N- and K-ras Oncogenes in Plasma Cell Dyscrasias
By Paolo Corradini, Marco Ladetto, Claudia Voena, Antonio Palumbo, Giorgio Inghirami, Daniel M. Knowles,
Mario Boccadoro, and Alessandro Pileri
The frequency of N- and K-ras oncogene mutations was
investigatedin plasma cell dyscrasias. Genomic DNAs from
1 2 8 patients were selected for this study: 30 monoclonal
gammopathies of undetermined significance, 8 solitary
plasmacytomas, 77 multiple myelomas (MM), and 1 3
plasma cell leukemias (PCL). A two-step experimental approach was devised. All samples were screened for mutations by single-strand conformation polymorphism analysis. DNA fragments displaying an altered electrophoretic
mobility were further studied by direct sequencing to confirm and characterize the nature of the mutations. Ras mu-
tations are not randomly distributed because they are detectable only in MM (9%)and PCL (30.7%). N-ras codons
12,13, and 6 1 and K-ras codon 1 2 were found to be mutated, but N-ras codon 61 mutation was the most frequent
finding (63.6%). In conclusion, ras mutations were found
in PCL, and in a subset of MM characterized by advancedstage disease and adverse prognostic parameters. Furthermore, based on our findings, it is possible to speculate
that ras mutations represent a late molecular lesion in the
process of multistep carcinogenesis.
0 1993 by The American Society of Hematology.
P
kemia (CML)."-15 However, among more differentiated
lymphoid tumors, such as chronic lymphocytic leukemia
(CLL), hairy cell leukemia (HCL), and non-Hodgkin's lymphoma (NHL), only MM was found to harbor activated rus
genes.l 6
Several reasons prompted us to investigate ras oncogenes
in plasma cell dyscrasias. First, the incidence of rus mutations
has been reported with some dis~repancies.'.~Second, we
wanted to correlate its incidence with several clinical parameters in a large panel of MM cases. Third, the presence of
these mutations has never been evaluated in MGUS, SP, or
PCL. Finally, because these oncogenes have been already
detected in some human precancerous lesions (eg, adenomas
and myelodysplasias), indicating that they participate in the
early stages of tumorigenesis, we wanted to determine the
temporal relationship between rus activation and the onset
of an aggressive plasma cell dyscrasia.
LASMA CELL DYSCRASIAS are a group of diseases
characterized by the expansion of plasma cells producing monoclonal Igs. Monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (SP),
multiple myeloma (MM), and plasma cell leukemia (PCL)
are the clinical entities forming this group. MGUS is a benign
disorder even if, in a large series with 20 years of follow-up,
24.5% of patients developed MM.' SP is characterized by
indolent behavior, with a median survival that can exceed
10 years, whereas MM and PCL follow an aggressive clinical
course.*
The pathogenesis of these diseases is still unknown, and
the role of some dominantly acting proto-oncogenes, such
as bcl- 1, bcl-2, c-myc, Moloney leukemia virus integration4 (MLVI-4), N-rus, K-rus, and H-rus, has been investigated
only in MM.3-6So far, the molecular lesions more frequently
detected in MM patients are activating point mutations involving the N- and K-rus oncogene^.'.^
The family of rus oncogenes consists of three related genes:
H-, K-, and N-rus. They encode proteins of 2 1 Kd (p2 1) with
GTPase activity that are located at the inner surface of the
cell membrane. These oncogenes acquire their transforming
potential when point mutations occur at the codons 12, 13,
or 6 1, resulting in single amino acid substitution." Activated
rus genes have been identified in several human cancers, including some hematologic malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL),
myelodysplasias, and blast crisis of chronic myelogenous leu-
From the Department of Medicine and Experimental Oncology,
Division ofHematology, University of Torino, Torino, Italy; and the
Department of Pathology, College of Physicians and Surgeons, Coliimbia University, New York, NY.
Submitted November 2, 1992; accepted December 30, 1992.
Supported by the Associazione Italiana Ricerca SUI Cancro (AIRC,
hfiluno Italy), and by Consiglio Nazionale Ricerche (Progetto Finalizzato ACRO. N. 9202242.PF39). C. V. is a fellow of Comitato
Piemontese Gigi Ghirotti.
Address reprint requests to Paolo Corradini, MD, Divisione Universitaria di Ematologia, Via Genova 3, 10126 Torino, Italy.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1993 by The American Society ofHemuto1og.v.
0006-4971/93/81IO-O019$3.00/0
2708
MATERIALS AND METHODS
Patients and nucleic acid extraction. We selected for this study
128 patients with various forms of plasma cell dyscrasias. These patients included 77 patients with MM, 13 with PCL, 30 with MGUS,
and 8 with SP. All the patients were studied at diagnosis, except for
24 with MM that were in the relapse phase (Table 1). Bone marrow
or tissue samples (plasmacytomas) were collected during standard
diagnostic procedures. Genomic DNA was purified by proteinase K
digestion, phenol/chloroform extraction, and ethanol precipitation.
Oligonucleotideprimers. All the oligonucleotides used for polymerase chain reaction (PCR) amplification and direct sequencing
were chemically synthesized using a 39 1 PCR-MATE EP, DNA Synthesizer (Applied Biosystems, Foster City, CA) on a 0.2 pmol/L scale,
according to the users' manual. The oligonucleotide primers for Nand K-rus genes are the same as reported previously.'6Js
Single-strandconformationpolymorphism (SSCP) analysis. SSCP
analysiswas performed according to an adapted version of a previously
published neth hod.'^.^' Briefly, 100 ng of genomic DNA was amplified
in a 10 p L reaction, adding I O pmol of each primer, 0.5 U of Taq
DNA polymerase (Promega, Madison, WI), I pCi of [a-32P]dCTP
(specific activity, 3,000 Ci/mmol), 2.5 pmol/L dNTPs (Pharmacia
LKB Biotechnology, Uppsala, Sweden), I O mmol/L Tris-HC1 (pH
8.8), 50 mmol/L KC1, I mmol/L MgC12, 0.01% gelatin, final concentration. Thirty cycles of denaturation (94"C), annealing (56°C to
60°C depending on primer melting temperature), and extension
(72°C) were performed on an automated heat-block (DNA thermal
cycler; MJ Research, Boston, MA), followed by a 7-minute final extension at 72'C. Two microliters of the I O pL reaction mixture was
diluted I : I8 in 0.1% NaDodSO,/ I O mmol/L EDTA and further mixed
1:l with a sequencing stop solution containing 20 mmol/L NaOH.
Blood, Vol81, No 10(May 15). 1993: pp2708-2713
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RAS
2709
MUTATIONS IN MULTIPLE MYELOMA
PCL
MM
N-Ras
12/13
MM
PCL
N-Ras
61
Fig 1. SSCP analysis of Nand K-ras oncogene mutations
in plasma cell dyscrasias. PCRamplified fragments corresponding to codons 12,13, and
6 1 were amplified from genomic
DNA, denatured by heat, and
run on a 6% acrylamide gel containing 10% glycerol. Representative cases of MM and PCL are
shown. Samples were scored
positive for mutations when
bands different from the normal
control (N) were detectable. Arrowheads point to bands of the
mutated samples.
PCL
MM
K-Ras
12/13
Samples were heated at 95°C for S minutes. chilled on ice for 3
minutes. and then 3 pL was loaded onto a 6% acrylamide/TBE gel
containing I070 (vol/vol) glycerol. Gels were run at 8 W for 12 to IS
hours at room temperature. Autoradiography films were exposed at
-70°C with intensifying screens for varying periods of time.
D i r m seqtccwcing sl' PCR prodrrcLs. Amplifications were performed using 100 ng of genomic DNA. 20 pmol of each primer, and
2.5 U of Taq DNA polymerase (Promega) in a SO p L final volume
reaction. The number of cycles. thermocycling conditions, and reaction buffer were as described above. The PCR products were electrophoresed through a 27n low melting point (LMP) agarose (GIBCO
BRL. Gaithersburg. MD). the LMP slice was melted at 68°C. and
DNA was phenol extracted and ethanol precipitated. N- and K-rar.
5' and 3' primers. were labeled with [y-3'P]ATP using T4 polynucle-
otide kinase (Promega). Direct sequencing reactions of both strands
were performed using the Promega fmol sequencing system according
to the manufacturer's instructions. The sequencing reactions were
performed in a thermal cycler at 58°C annealing temperature for 9
cycles. Reaction products were run on a 6% acrylamide/urea gel.
fixed in I0q0 acetic acid. and then exposed at -70°C with intensifying
screens for varying periods of time.
RESULTS
Genomic DNAs from 128 patients with plasma cell dyscrasias were analyzed for mutations of the N- and K-rrrs on-
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CORRADlNl ET AL
2710
Table 2.
Table 1. Frequency of Res Oncogene Mutations
in Plasma Cell Dyscrasias
Diagnosis
MGUS
SP
MM
PCL
Sample
Positiveflested
Abbreviation: SP, solitary plasmacytoma.
cogenes (Table I). All DNAs extracted from these specimens
were amplified using pairs of oligonucleotide primers flanking
the genomic regions spanning codons 12, 13, or 6 1 of the Nrus and K-rus genes.
A two-step experimental approach was devised. All samples
were screened for mutations within the N-rus and K-rus genes
by SSCP analysis. DNA fragments displaying an altered electrophoretic mobility by SSCP analysis (Fig 1) were subsequently reamplified and studied by direct sequencing to confirm and characterize the nature of the mutations. The SSCP
Mutations in Plasma Cell Dyscrasias
Diagnosis
Res Gene
Codon
Mutation
Amino Acid
Substitution
MM
MM
PCL
MM
MM
MM
MM
PCL
MM
PCL
PCL
N
N
N
N
N
N
N
N
N
N
12
12
13
61
61
61
61
61
61
61
12
GGT - G A T
GGT -.GCT
GGT - T G T
CAA-CGA
CAA- AAA
CAA -.CAT
CAA-CTA
C A A - r AAA
CAA
CTA
CAA
CAT
GGT
CGT
Gly-r Asp
Gly
Ala
Gly-r Cys
Gln- Arg
Gln-r Lys
Gln
His
Gln-r Leu
Gln-Lys
Gln -,
Leu
Gln
His
Gly-r Arg
M22
0130
018
7/77
4/13
R8S
M93
M27
M28
M9 1
M19
M89
M6 1
M90
M117
M88
K
--.
-
-
-
technique can detect sequence changes affecting one or more
nucleotides in a defined stretch of DNA. This method, for
150- to 200-bp fragments. has a sensitivity level of at least
A dilution experiment was performed to confirm the
N-Ras 61
G A T C
='
G A T C
G A T C
~-
c
G A T C
-- -
Gq
A
C '
C
T
61[p
I
C
r
T
3'
b
,"
C
B
Normal
M28
M91
M89
G A T C
G A T C
G A T C
G A T C
i
M19
M90
M61
M117
Fig 2. N-res codon 61 mutations detected by direct sequencing of PCR-amplfied fragments. Mutated samples are matched to a control
DNA. Noncoding strands are shown. Arrows point to bands correspondingto mutated base pairs.
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ms MUTATIONS IN MULTIPLE MYELOMA
271 1
method's sensitivity under our experimental conditions (data
not shown).
The results of SSCP and sequencing analysis are summarized in Table 2. The overall frequency of ras mutations
among plasma cell dyscrasias is 8.6% ( 1 1 of 128). They are
not randomly distributed because they are detectable only in
PCL (30.7%) and MM (9%). Mutations were found in 5 of
53 (9.4%) MM cases at diagnosis and 2 of 24 (8.3%) cases at
relapse. N-rus codons 12, 13, and 61 and K-rus codon 12
were found to be mutated, although N-ras codon 6 I was the
most frequent finding (7 of 1 1 [63.6%]).The lack of detectable
mutations in the negative case was not due to insufficient
representation of tumor cells, because all the samples contained more than 5% malignant cells, whereas the threshold
of sensitivity of the method is 1%. Moreover, to evaluate the
presence of SSCP false-negative samples, 40 negative cases
were analyzed by direct sequencing (8 PCL, 4 SP, and 28
MM), showing a 100%concordance with SSCP results.
To confirm the presence and determine the nature of the
mutations, the nucleotide sequence of the involved exons
was determined by direct sequencing of both strands. A het-
Table 3. Correlation Between RBSMutations and Prognostic
Parameters in MM
Patient
~ 2 2
M93
M28
M91
M19
M89
M90
Stage
Albumin
WL)
(%I
CRP
(mg/L)
,92-Microglobulin
(mg/L)
IllA
Ill5
IllA
Ill5
IllA
lllB
IllA
29
47
31
34
25
50
34
0.5
ND
4
0.8
ND
2.7
1.2
117
ND
13
50
121.9
6
10.7
5.5
ND
7.9
41.9
13
11.5
3.8
LI
Adverse prognostic factors were albumin lessthan 30 g/L, U greater than
2%. CRP greater than 6 mg/L, and i32-microglobutingreater than 6 mg/L.
Abbreviations: U, labeling index; CRP, C-reactive protein; ND, not determined.
erogeneous pattern of mutation was observed including C
-W A, G + C, G + T, and A + T transversions, and G +
A transitions (Figs 2 and 3). Purine-pyrimidine transversions
were the most frequent finding (9 of I 1 [81.8%]). These
N-Ras 12/13
G A T C
G A T C
G A T C
G A T C
Normal
M22
M27
M93
C
3'
A
A
c
C
K-Ras 12/13
G A T C
G A T C
Normal
M88
:a
3'
G
A
T
G
13[
--
121 b
G
T
c
5'
,"
Fig 3. N- and K-res codon 12/13 mutations detected by direct sequencing of PCR-amplifiedfragments. Mutated samples are matched
to a control DNA. Noncoding strands are shown for cases M22, M27, and M93; coding strand is shown for case M88. Arrows point to
bands corresponding to mutated base pairs.
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CORRADlNl ET AL
2712
Table 4. Clinical Parameters and Ras Mutations in MM
Parameter
PositiveITested’
Stage
I
II
111
Bone marrow plasma cells
019
0117
7/37
1/26
2/20
4/18
<30%
>30% and 150%
>50%
Protein type
3/40
2/30
215
OJ2
IgG
IgA
Light chain
Nonsecretory
Albumin (g/L)
2/10
<30
230
5/62
Labelling index
3/50
211 1
<2%
>2%
Status
Diagnosis
RelaDse
5/53
2124
The clinical parameters were not available for all patients.
changes led to the replacement of glycine (N- and K-rus codons 12 and 13) and glutamine (N-rus codon 61) residues
with several different amino acids (Table 2).
The relatively small percentage of rus mutations in MM
did not allow a survival analysis. Rus mutations were detected
only in stage 111 MM (7 of 37 [ 19%]).*’In addition, a prognostic evaluation was performed in all but 1 patient, in whom
data were not available (M93). All the examined patients,
except M90, had two or more concordant adverse prognostic
parameters, as shown in Table 3.22-24Patients M19, M22,
and M28 were already dead when the analysis was performed.
The median follow-up for censored patients was 20 months
(range, 17 to 46 months).
Mutations were frequently detected in PCL that are characterized by an extremely high proliferative activity and a
short survival. Our panel included all primary PCL except
for M27 patient, who presented the leukemic transformation
of an MM. The latter, together with 3 primary PCL, were
found to harbor rus mutations.
DISCUSSION
We have investigated the mutational activation of N- and
K-rus oncogenes and its correlation with clinical parameters
in plasma cell dyscrasias. Recent studies have suggested that
these oncogenes may play a role in myeloma pathogenesis.
N- and K-rus oncogenes were found to be mutated in previous
s t ~ d i e s . ~In. ~addition, the introduction of activated N-rus
genes into Epstein-Barr virus (EBV)-immortalized human B
lymphoblasts resulted in their malignant transformation and
terminal differentiation into clonal plasma cells.25
Our study indicates that rus mutations are peculiar to aggressive forms of plasma cell dyscrasias. The relatively low
incidence of mutations in MM is confirmed. whereas the
high incidence in PCL represents a novel finding. It should
be noted that, although the SSCP technique might slightly
underestimate the frequency of mutations, our findings are
in agreement with the percentage found by Paquette et al’
in a smaller panel of cases.
The presence of activated rus genes appears to be characteristic of immature lymphoid neoplasms, and the discovery
of rus mutations in MM and PCL apparently contradicts this
finding. However, a possible explanation can derive from the
studies indicating that human myeloma originates from the
B- or even the pre-B-lymphocyte ~ o m p a r t m e n t . ’ ~ *The
~-~~
mechanism of occurrence of such mutations remains unknown, and the considerable proportion of different amino
acid substitutions may suggest that B cells are exposed to a
variety of molecular events capable of inducing many different rus mutations. However, the relatively low frequency of
rus mutations in MM argues against a general pathogenetic
role for this oncogene in such a disease. Moreover, the low
incidence at relapse rules out the possible mutagenic effect
played by chemotherapy treatment.
Correlations between rus mutations and several clinical
parameters were considered in MM only (Table 4),because
PCL have a well-defined unfavorable clinical outcome. Our
panel included 26 stage I and I1 cases, but mutations were
detected only in stage 111 patients. In addition, although the
small number of cases did not allow survival analysis, the
presence of rus mutations was associated with adverse prognostic parameters, as shown in Table 3. These mutations
identify a subset of myeloma patients characterized by an
advanced stage and the simultaneous presence of adverse
prognostic parameters.
Plasma cell dyscrasias represent a model of multistep carcinogenesis, and they can be divided according to different
tumor progression steps corresponding to defined clinical entities, ranging from the benign gammopathy to the aggressive
leukemic form. In breast and colon adenomas as well as in
myelodysplasias, rus genes are frequently altered, leading to
the general concept that such oncogenes act early in the multistep c a r c i n o g e n e ~ i s . ~This
~ - ~role
~ appears not to apply to
plasma cell dyscrasias. The absence of mutations in MGUS
indicates that they cannot be considered “precancerous lesions” similar to adenomas or myelodysplasias. Moreover,
the detection of such mutations only in stage 111 MM and
PCL suggests that rus activation most likely plays a more
important role in tumor progression rather than in tumor
initiation. The high frequency of rus mutations in PCL led
us to speculate that these mutations probably represent a late
molecular lesion conferring additional proliferative advantage
to tumor cells. However, to better define the role and timing
of rus gene activation, longitudinal studies on individual patients are required.
In conclusion, our work indicates that rus oncogene activation is not a general pathogenetic event for plasma cell
dyscrasias, and that its presence is typical of advanced-stage
MM and PCL.
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MUTATIONS IN MULTIPLE MYELOMA
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1993 81: 2708-2713
Mutational activation of N- and K-ras oncogenes in plasma cell
dyscrasias
P Corradini, M Ladetto, C Voena, A Palumbo, G Inghirami, DM Knowles, M Boccadoro and A Pileri
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