Supplementary Methods
Screen for RAS mutation using conformation sensitive gel electrophoresis
Polymerase chain reaction (PCR) primers (see table below) were designed to amplify
DNA segments for a total of 4 PCR reactions.
Primers for RAS mutation screen
Gene target
K-ras
codon 61
N-ras
codons 12-13
K-ras
codon 12-13
N-ras
codons 61
Amplicon Size
408 bp
308 bp
258 bp
181 bp
Sequence (written 5' to 3')
TTT GGA GCA GGA ACA ATG TCT
TTA AAT ATT ATA TGC ATG GC
ATG GAA GGT CAC ACT AGG GTT
TCC TTT AAT ACA GAA TAT GGG
TAC TGG TGG AGT ATT TGA TAG
TGT ATC AAA GAA TGG TCC T
ACC CCC AGG ATT CTT ACA GA
AGA GGT TAA TAT CCG CAA ATG
Direction
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
These reactions were optimized in a multiplex PCR reaction with amplicon sizes ranging
from 181 to 408 base pairs. A standard PCR mixture containing genomic DNA; a mixture
of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP),
deoxyguanosine triphosphate (dGTP), and 20% deoxycytidine triphosphate (dCTP);
multiplexed primer mix; 33P dCTP, Taq GOLD DNA polymerase (Applied Biosystems,
Foster City, CA); and sterile water for a total reaction volume of 25 L. The radiolabeled
amplicons were run through 15% mild-denaturing 0.4 mm polyacrylamide gel for 4 hours
at 40 watts. The gel was dried and placed on a phosphorimager screen for analysis.
Abnormal banding patterns were subsequently directly sequenced (see figure below).
A representative CSGE gel image. The RAS codons corresponding to each band are
highlighted and the lanes (samples) with abnormal bands are indicated by the arrows.
These were subsequently found to have RAS mutations on sequencing.
Supplemental Table 1. Prevalence of RAS mutations in current and previous studies in
newly diagnosed MM
Study
Neri et al (1989)
Paquette et al (1990)
Matozaki et al (1991)
Tanaka et al (1992)
Portier et al (1992)
Corradini et al (1993)
Millar et al (1995)
Liu et al (1996)
Bezieau et al (2001)
Kalakonda et al (2001)
Rasmussen et al
(2005)
Intini et al (2006)
Current – ECOG
KRAS
mutation
(%)
NRAS
mutation
(%)
Total RAS
mutation
(%)
CD138
selection
2/43 (7)
0/17 (0)
0/15 (0)
0/10 (0)
2/13 (15)
0/77 (0)
0/18 (0)
23/139 (17)
18/33 (55)
1/34 (3)
11/43 (26)
2/17 (11)
4/15 (26)
5/10 (50)
2/13 (15)
7/77 (9)
0/18 (0)
29/129 (23)
7/30 (23)
34/34 (100)
13/43 (33)
2/17 (11)
4/15 (26)
5/10 (50)
4/13 (30)
7/77 (9)
0/18 (0)
52/139 (37)
25/33 (76)
34/34 (100)
No
No
No
No
No
No
No
No
Yes
Yes
PCR/Oligonucleotide hybridization
PCR/Oligonucleotide hybridization
PCR/Oligonucleotide hybridization
PCR/Oligonucleotide hybridization
SSCP
SSCP
Dot blot hybridization
Dot blot hybridization and SSCP
ARMS
PCR-RFLP
11/58 (19)
5/81 (6)
28/439 (6)
7/58 (12)
11/81 (14)
74/439 (17)
18/58 (31)
16/81 (20)
102/439 (23)
Yes
Yes
No
PCR and sequence
PCR and sequence
CGSE
Method
Current – Mayo
4/60 (7)
11/60 (18)
15/60 (25)
Yes
CGSE
Abbreviation: SSCP = Single-strand conformation polymorphism; ARMS = Allele-specific amplification methods;
CGSE = Conformation gel sensitive electrophoresis
Supplemental Table 2. Patient Characteristics at Baseline by RAS Status (Continuous)
Normal RAS
RAS Mutation
Wilcoxon
Parameter
N
N Missing
Median (range)
N
N Missing
Median (Range)
p-value
BM PC %
327
10
40 (2-99)
102
0
54 (4-95)
<0.001
Creatinine
337
0
1.2 (0.4-4.9)
102
0
1.2 (0.6-4.7)
0.288
CRP
331
6
0.3 (0.1-22.5)
99
3
0.3 (0.1-9.1)
0.105
Hemaglobin
337
0
10.9 (5.1-16.7)
102
0
10.6 (6.2-14.6)
0.037
WBC
337
0
6.0 (1.6-56.0)
102
0
5.7 (2.4-13.9)
0.113
Platelets
337
0
245 (68-736)
102
0
241 (54-540)
0.555
M-Spike
271
66
3.7 (1.0-10.0)
87
15
4.4 (1.1-9.6)
0.007
PCLI
317
20
0.4 (0.0-13.2)
95
7
0.4 (0.0-20.9
0.291