Microsoft PowerPoint - H24 SNP ASH 2005.ppt

Influence of genetic single nucleotide
polymorphisms in CYP3A4, CYP3A5*3,
GSTP1, GSTM1, GSTT1 and MDR1 genes on
survival and treatment-related toxicity in
patients with Multiple Myeloma treated in the
HOVON 24 trial .
P. Sonneveld, C. Schilthuizen, B. van der
Holt, H. Lokhorst, E. Vellenga, R,
Raijmakers, Y de Knegt, E. Kamst
on behalf of
the Dutch-Belgian HOVON group

Disclosures
In compliance with ACCME policy, ASH requires the following disclosures to the
activity audience:
Research support/P.I.
N/A
Employee
N/A
orgia
Ge
Consultant
N/A
Atlanta,
Major stockholder
N/A
eeting
Speakers' bureau
N/A
M
Scientific advisory board
Janssen Cilag Orthobiotech
Annual
ASHth47
NA = not applicable (no conflicts listed)
Presentation includes discussion of the following off-label use of a drug or
medical device: N/A

Multiple factors contributing to variations in drug responses

Goal of the study
· To analyse the effect of genetic polymorphisms of
relevant xenobiotic genes on the outcome of
response, toxicity and survival parameters in a
pospective phase III myeloma trial
· To identify whether subgroups of patients can be
recognized that may benefit from individual drug
dosing in future trials

MM stage II/III A/B < 66 yr: Single vs Double intensive treatment
3-4 VAD push
Allo-SCT if HLA-id
sibling
Randomize
Cyclophosphamide
Cyclophosphamide
4g/m2 + G-CSF
4g/ m2 + G-CSF
Melphalan
Melphalan
140mg/m2 (2 x 70)
140mg/m2 (2 x 70)
Interferon 3*106 IU
Cy/TBI + ASCT
s.c 3x/week
Interferon 3* 106 IU
s.c. 3x/week

Survival by treatment arm
Overall survival
Event free survival
Treatment arm randomised
Treatment arm randomised
100
100
Single
age
75
age
75
ent
ent
Double
perc
perc
e
50
e
50
iv
iv
Double
p=0.014
mulat
mulat
u
25
u
25
C
N
F
C
N
F
IFN-a
148
86
IFN-a
148
137
PB/AuBMT
155
95
PB/AuBMT
155
126
Single
Logrank P=.39
Logrank P=.01
0
0
0
24
48
72
months 96
0
24
48
72
months 96
At risk:
At risk:
IFN-a 148
117
80
23
4
IFN-a 148
60
20
5
0
PB/AuBMT 155
108
73
26
4
PB/AuBMT 155
73
37
13
2
Time to progression from randomization, [mo] - 2
Progression free survival
Treatment arm randomised
Treatment arm randomised
100
N
F
100
IFN-a
148
125
PB/AuBMT
155
102
Single
Logrank P<.001
p=0.032
age
75
age
75
ent
ent
Double
perce
50
perce
50
iv
iv
p=0.001
Double
mulatu
mulat
25
u
25
C
C
N
F
IFN-a
127
116
Single
PB/AuBMT
140
111
Logrank P=.03
0
0
0
24
48
72
months 96
0
24
48
72
months 96
At risk:
At risk:
IFN-a 148
71
23
7
0
IFN-a 127
59
20
4
0
PB/AuBMT 155
84
43
13
2
PB/AuBMT 140
71
36
13
1
Median follow up 68 months
Sonneveld et al, MMWS
156/453 patients alive
2005
Analysis 17th March 2005

Experimental approach
· Retrospective analysis of samples from HOVON 24
trial (single vs double intensive treatment in MM)
· RT-PCR or duplex PCR on BM or PB (germline)
samples
· Multivariate analysis of pharmacogenetic variables
· Multivariate analysis with clinical variables B2M and
Salmon Durie stage

Analysis of genetic polymorphisms:
xenobiotic genes and functions
gene
potential drug substrates in HOVON 24
GSTP1
alkylating agents (melphalan, cyclophosphamide)
GSTT1
id
GSTM1
id
CYP3A4-V
anthracyclins
CYP3A5*3
anthracyclins, cyclophosphamide (?)
MDR1
anthracyclins, vincristine

Analysis of genetic polymorphisms:
xenobiotic genes and functions
gene
site
mutation
function
GSTP1
exon 5
IleVal
reduced GSH/JUN
GSTT1
deletion
no activity
GSTM1
deletion
no activity
CYP3A4-V
promotor
290 AG
increased activity
CYP3A5*3
SNP exon 3,
alt. Splicing, A6986G
no activity
MDR1
Membrane loop C3435T
Reduced activity

RT-PCR
WW
MM
WM
P
LL
WW
W
MM
WM
W
P
334
33
293
29
A-290G SNP
A6986G
220
22
168
199
16
199
148
14
SNP in
125
12
in CYP3A4
CYP3A5*3
81
Figure
r 1a.
Fi
F gu
Fi
r
gu e
r 1b.
1b
T0 / M0
WW
MM
WM
P
LL
M+
T+
T
M+T+
M+T
488
48
451
45
A1578G SNP
Multiplex-
296
29
222
22
251
25
in GSTP1
PCR of
118
11
75
GSTT1 and
74
Figure
r 1c.
Figure
r 1d.
GSTM1
L
WW
MM
WM
W
P
340
34
264
26
C3435T SNP
172
17
in MDR-1
90
78
Figu
g re
r 1e.
e

Samples analysed and haplotype frequency (%)
Single
Double
Patients
148
155
Samples for PhG
100
111
Gene
Haplotype
Total (%)
GSTP1
ww/mm/wm/missing
28/23/43/6
CYP 3A4
ww/mm/wm/missing
83/1/2/14
CYP 3A5
ww/mm/wm/missing
1/85/11/3
GSTT1
absent/present/
26/71/3
GSTM1
absent/present/
51/47/2
MDR1
ww/mm/wm/missing
10/43/43/4

Results: overall toxicity WHO 3 (%)
Gene haplotype
ww
mm
wm
P
CYP 3A4
nananana
CYP 3A5*3
na76600.09
GSTP1
8067740.31*
MDR1
67
73
77
ns
absent
present
GSTT1
75
74
ns
GSTM1
69
79
ns
* After HDM p=0.03

Partial or complete response on protocol
treatment (%)
Gene haplotype
ww
mm
wm
p
CYP 3A4
82
100
-
ns
CYP 3A5*3
848181ns
GSTP1
9281710.02
MDR1
86
75
88
0.07
absent
present
GSTT1
75
85
ns
GSTM1
79
85
ns

GSTP1
Figure 3. Unadjusted
Event free survival
Kaplan-Meier plots of EFS, PFS, TTP and OS for GSTP1 Ile105
Progression Val
free survival
polymorphism.
100
100
75
I1e/I1e
75
age
age
Val/I1e
Val/I1e
Val/Val
percent
50
percent
50
I1e/I1e
ive
ive
Val/Val
Cumulat
25
N
e
Cumulat
25
N
p
Dr. D.
R. Biesma
rouwer
I1e/I1e
60
54
I1e/I1e
55
49
Val/I1e
89
78
Val/I1e
72
61
Val/Val
48
44
Val/Val
34
30
Logrank P=.16
Logrank P=.32
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months 60
At risk:
At risk:
I1e/I1e 60
51
32
23
13
7
I1e/I1e 55
46
27
20
11
6
Val/I1e 89
66
51
34
23
17
Val/I1e 72
60
46
30
22
13
Val/Val 48
32
20
12
6
4
Val/Val 34
27
18
10
6
4
Time to progression
Overall survival
100
100
N
p
I1e/I1e
60
40
Val/I1e
89
63
Val/Val
48
38
Logrank P=.07
75
75
I1e/I1e
age
age
Val/Val
Val/I1e
percent
50
I1e/I1e
percent
50
ive
ive
Val/Val
Val/I1e
Cumulat
25
Cumulat
25
N
d
I1e/I1e
60
39
Val/I1e
89
55
Val/Val
48
34
Logrank P=.28
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months 60
At risk:
At risk:
I1e/I1e 60
54
34
23
13
7
I1e/I1e 60
57
45
42
33
21
Val/I1e 89
79
63
43
27
17
Val/I1e 89
81
69
60
51
35
Val/Val 48
37
23
15
9
6
Val/Val 48
39
32
25
23
13
Kaplan-Meier curves for Genotype GSTP1

GSTT1
Event free survival
Progression free survival
100
100
75
75
percentage
50
percentage
50
ve
ve
ti
ti
la
la
u
u
absent
absent
Cum
25
Cum
25
N
e
N
p
absent
55
48
present
present
absent
41
34
present
149
134
present
126
111
Logrank P=.56
Logrank P=.19
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
absent 55
38
27
21
15
11
absent 41
33
26
20
14
9
present 149
114
81
51
28
18
present 126
104
69
42
26
15
Time to progression
Overall survival
100
100
N
p
absent
55
38
present
149
106
Logrank P=.24
present
75
75
absent
absent
percentage
50
percentage
50
ve
ve
ti
ti
present
la
la
u
u
Cum
25
Cum
25
N
d
absent
55
34
present
149
97
Logrank P=.62
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
absent 55
47
35
25
19
12
absent 55
48
40
36
32
23
present 149
127
91
59
31
19
present 149
133
111
97
79
48
Kaplan-Meier curves for Genotype GSTT1

GSTM1
Event free survival
Progression free survival
100
100
75
75
percentage
50
percentage
50
ve
ve
ti
ti
la
la
u
u
Cum
25
25
absent
absent
Cum
N
e
N
p
present
absent
107
94
present
absent
84
71
present
100
91
present
85
76
Logrank P=.86
Logrank P=.42
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
absent 107
79
56
41
22
14
absent 84
70
50
34
20
12
present 100
75
52
32
21
15
present 85
68
45
29
20
12
Time to progression
Overall survival
100
100
N
p
absent
107
75
present
100
71
Logrank P=.78
absent
75
75
present
absent
percentage
50
percentage
50
ve
ve
ti
ti
la
la
u
u
present
Cum
25
Cum
25
N
d
absent
107
65
present
100
69
Logrank P=.29
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
absent 107
92
68
47
25
16
absent 107
95
80
72
63
38
present 100
85
58
38
25
15
present 100
89
71
61
48
34
Kaplan-Meier curves for Genotype GSTM1

MDR-1
Event free survival
Progression free survival
100
100
75
75
age
age
CC
ent
CC
ent
TT
perc
perc
e
50
TT
e
50
iv
iv
CT
lat
lat
u
CT
u
m
m
u
u
C
25
N
e
C
25
N
p
CC
21
20
CC
18
17
CT
91
85
CT
68
62
TT
90
76
TT
79
65
Logrank P=.10
Logrank P=.41
0
0
0
24
48
months 72
0
24
48
months 72
At risk:
At risk:
CC 21
12
4
2
CC 18
12
3
2
CT 91
43
15
4
CT 68
35
14
4
TT 90
50
24
11
TT 79
45
24
9
Time to progression
Overall survival
100
100
N
p
CC
21
18
CT
91
67
TT
90
60
Logrank P=.35
75
75
age
age
ent
ent
CC
perc
CT
perc
e
50
e
50
TT
iv
iv
lat
lat
u
TT
u
CT
m
m
u
u
C
25
CC
C
25
N
d
CC
21
12
CT
91
65
TT
90
56
Logrank P=.15
0
0
0
24
48
months 72
0
24
48
months 72
At risk:
At risk:
CC 21
14
5
2
CC 21
18
12
8
CT 91
54
19
4
CT 91
63
42
19
TT 90
55
26
11
TT 90
68
58
23
Kaplan-Meier curves for MDR-1 C3435T

CYP3A5*3
Event free survival
Progression free survival
100
100
75
75
percentage
50
percentage
50
ve
ve
ti
ti
la
la
u
u
mm
Cum
25
mm
Cum
25
N
e
N
p
ww/wm
25
24
ww/wm
ww/wm
21
20
ww/wm
mm
179
157
mm
145
123
Logrank P=.12
Logrank P=.04
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
ww/wm 25
19
13
4
2
2
ww/wm 21
17
10
2
2
1
mm 179
134
92
66
41
28
mm 145
120
82
59
38
24
Time to progression
Overall survival
100
100
N
p
ww/wm
25
18
ww/wm
mm
179
125
Logrank P=.03
75
75
mm
mm
percentage
50
percentage
50
ve
ve
ti
ti
la
la
u
u
Cum
25
Cum
25
ww/wm
N
d
ww/wm
25
21
mm
179
110
Logrank P=.01
0
0
0
12
24
36
48
months 60
0
12
24
36
48
months
60
At risk:
At risk:
ww/wm 25
19
13
4
2
2
ww/wm 25
21
15
14
10
4
mm 179
157
110
78
48
30
mm 179
161
135
117
100
68
Kaplan-Meier curves for Genotype Cyp3A5*3 (RFLP-PCR)
Inactivated CYP3A5*3 is associated with improved TTP,
EFS and OS

Conclusions (1)
· Pharmacogenomic analysis reveals that patients with
homozygous mutations Cyp3A5*3 have a significantly
better overall survival and more toxicity
· Cyp 3A4-V mutations are rare and do not have an
effect on survival outcome

Conclusions (2)
· Patients with GSTP1 Val/Val homozygous mutations
have less toxicity and a lower response rate, and no
significant difference of survival
· No influence of MDR1, GSTT1 or GSTM1 mutations
· Multivariate analyses of genetic polymorphisms in
validation trial ongoing

Acknowledgements
Clinicians
Laboratory
· P Sonneveld
·
B. Beverloo
· CM Segeren
·E. Kamst
· E Vellenga
·
Y de Knegt
· AJ Croockewit
· GEG Verhoef
·
R van Schaik
· JJ Cornelissen
· MR Schaafsma
· MHJ van Oers
Datamanagement & Statistics
· PW Weijermans
· WE Fibbe
· S Wittebol
·
PHM Westveer
· HC Schouten
·
MMC Steijaert
· M van Marwijk Kooy
·
B van der Holt
· DH Biesma
· JW Baars
· HM Lokhorst