Projet Colohybritest Programme Emergence Bio 2008

206
A multiplex promoter hypermethylation DNA test used as biomarker for
blood-based colorectal cancer detection. Jean-Pierre Roperch, Floriane Bard, Solène Forbin,
Iradj Sobhani (coordinateur). Service de Gastroentérologie, CIC, IFR, CHU Henri Mondor, 94000 Créteil
Colorectal cancer (CRC) remains a major medical problem in the world.
Sooner is it detected better are chances to cure it. Fecal occult blood
test (FOBT) is a current noninvasive screening method for CRC
detection. However, it detects less than 50% asymptomatic cancers
and lacks of specificity. In this context, new specific CRC biomarkers
for
diagnosis
of
early
stage
tumours
are
needed
.
Aberrant
DNA
Projet Colohybritest
Programme Emergence Bio 2008
BIOLOGIE & SANTÉ 2011
Background
At the optimal threshold value in QM-MSP, three candidate genes
(MK1, MK2, MK3), were tested in DNA serum as either single or
combined marker. The sensitivity and specificity for detecting CRC
of the combined multiplex hypermethylated DNA test were 78.1%
and 89.6% respectively with MK1 showing 59.4% and 90.1% and
MK
2
56
.
3
%
and
89
.
6
%
and
MK
3
,
56
.
3
%
and
90
.
1
%
,
respectively
as
Results
for
diagnosis
of
early
stage
tumours
are
needed
.
Aberrant
DNA
hypermethyation is recognized as playing a crucial role during CRC
development. The main goal of the study was to develop and
validate a CRC-specific methylated DNA test in serum.
Methods
MK
2
56
.
3
%
and
89
.
6
%
and
MK
3
,
56
.
3
%
and
90
.
1
%
,
respectively
as
a single gene marker.
Schematic representation of the study design clinical
Tissues n= 72
CRC n= 16
Normal n= 16
Stool
CRC n= 10
Normal
n= 10
DNA methylation
profiling
Initially, the GoldenGate Methylation Cancer Panel I containing 1,505
CpG loci within 807 cancer-related genes was used to study
methylation patterns in CRC patients. DNA methylation microarray-
based assays was performed on 32 tissues (16 CRC and 16 normal)
and effluents (20 blood and 20 stool samples). Each effluent includes
10 patients with cancer and 10 healthy subjects as assessed by
colonoscopy and pathology findings. Three candidate genes referring
to the highest level of methylation were included in a relative
quantitative multiplex methylation-specific PCR (QM-MSP). These
three genes were evaluated on a additional series of 30 tissues
samples (15 CRC and 15 homologous normal tissues). Finally, this test
was validated in 224 serum samples (32 CRC and 192 normal).
Quantitative multiplex methylation-specific PCR in serum samples of normal
and colorectal cancer DNA
Methylation
Specificity Sensitivity
Threshold
(%)
Ratio Value (%) Ratio Value (%)
MK1 0.38 173/192 90.1% 19/32 59.4%
0.92
183/192
95.3%
17/32
53.1%
MK2
0.05
172/192
89.6%
18/32
56.3%
0.24
183/192
9
5
.
3
%
14/32
43.8%
MK3
0.6
3
173
/192
90.1
%
18
/32
56.3
%
0.93
1
83
/192
95.3
%
14
/32
43
.
8
%
Comparison of paired carcinoma and homologous normal colorectal tissue
0
50
100
150
200
250
300
350
400
123456789101112131415123456789101112131415Ctrl
Relative M ethylation
Homologous normal tissues (H) Tumor tissues (T)
MK1
MK2
MK3
0
50
100
150
200
250
300
Relative Methylation
p
< 0.0001
H T
0
20
40
60
80
100
120
140
1 I / II III / IV
P > 0.1
Cumulative Mean
CONTACT :
[e-mail]
[e-mail]
Colorectal tissues n= 30
Carcinomas n= 15
Controls* n= 15
Training set n= 50
CRC n= 9
Normal n= 41
Evaluation of
candidate genes
*, homologous normal tissues, Na= Not applicable
Normal
n= 10
Serum
CRC n= 10
Normal n= 10
Validation set n= 174
CRC n= 23
Normal n= 151
Serum samples for validation
Grand total n= 224
CRC Normal
n= 32 n= 192
Sex
Male 21 104
Female 11 88
Age
Mean 67.9 60.4
Range 36-94 18-94
Stage
I/II 6 (2/4) Na
III/IV 26 (7/19) Na
0.93
1
83
/192
95.3
%
14
/32
43
.
8
%
CMI: 3 genes
0.61
0.94
154/192
172/192
80.2%
89.6%
28/32
25/32
87.5%
78.1%
2.05
18
3/192
95.3%
19/32
59.4%
Sensibility is calculated as the number of true positives divided by the number of true positives plus false
negatives.
Specificity is calculated as the number of true negatives divided by the number of true negatives plus false
positives. CMI is the cumulative methylation index.
PPV = Positive Predictive Value and NPV = Negative Predictive Value
Symptoms + Test
Threshold PPV NPV
0.61% 42% 96.8%
2.05% 67.8% 93.3%
Conclusion
Aberrantly methylated DNA multiplex including MK1, MK2 and MK3 is proposed as
epigenetic combined biomarkers in a simple blood test for CRC detection.
In progress
1- In screening program schedule including
1000 asymptomatic individuals (50-74 years)
(VATNIMAD, PHRC 2009).
2- In symptomatic patients referring for colonoscopy
including 500 patients (ANR Biotecs 2010)
Valorisation
• Creation of Profilome S.A.S
• Patented biomarkers
• Article in progress