HPV Selbstuntersuchung - Jena 2002

Epigenetic signature as molecular
marker for the detection of HPVinduced severe dysplasia and
cervical carcinoma
A new trend in cancer diagnostics?
A. Hansel, M. Dürst
MBD
Gynäkologische Molekularbiologie,
Universitäts-Frauenklinik, Jena
HMT
Me
Ac
HPV Infection and Cervical Carcinoma
•Cervical cancer (CxCa) is 2nd most common cancer in
women worldwide
•500,000 new cases, 270,000 deaths / year worldwide
•hr-HPV infection is a prerequisite for cancer
•HPV is transmitted sexually
>50% of people get infected (cumulative)
in most cases infections clear within a year
HPV Infection and Cervical Carcinoma
Wheeler, 2007; Nat Clin Pract Oncol 4
early detection of precancerous lesions → almost 100%
curable
Detection of CIN2+ using Pap-based cytology
Uneven distribution of cellular material associated with the conventional Pap smear (A) may
be avoided by ThinPrep technology (B). Nonetheless the success depends mainly upon the
experience of the cytologist.
Detection of CIN2+ using Pap-based cytology
(Cuzick et al., Int J Cancer (2006) 119:1095-1101)
Sensitivity Pap-Test 53%, positive
predictive value 20.3% (n = 60.000)
→ reliable markers for the detection of
precancerous lesions are required
Detection of CIN2+ using Pap-based cytology
and HPV test
(Cuzick et al., Int J Cancer (2006) 119:1095-1101)
Sensitivity HPV-Test 96.1%
but PPV only 15.5%
Sensitivity Pap-Test 53%, positive
predictive value (PPV) 20.3% (n = 60.000)
→ reliable markers for the detection of
precancerous lesions are required
Promoter CpG island hypermethylation
may contribute to carcinogenesis
CpG-island methylation → carcinogenesis
•hypomethylation of repetitive elements
•hypermethylation of CpG islands
•hypermethylation may occur early in carcinogenesis
→ inactivation of genes: repair, cell cycle,
apoptosis, control of gene expression, cell adhesion
CpG island methylation may be
tumor-specific
tumor entity
gene
Esteller, M. Hum. Mol. Genet. 2007 16:50-59
→ epigenetic signatures may be useful as
carcinogenesis markers?
Search for candidate CpG islands
•Gene expression profiles:
normal vs CxCa tissue
•CpG island arrays:
HPV-positive cervical
scrapes vs. CxCa tissue
sections
Search for candidate genes
CpG island
microarrays:
•27,800 CpG islands
covering 21 MB
•237,220 probes in
or within 95bp of
CpG islands
AGACCATGTAAGAGACAATGTCTTGTCGAAATTGTCATTGTTTTTTGATTCCAGACAAATGGGGTGTGGGGAACAG
AAAGGCGATTTGGACTAGTTTCCCCGGACGGGATGAGGCAGCGCCGGAAGTATGACGCTCATTTCTTTAAGCCACT
AGAAGCTCACCGTGAGTCCATTCACAGGGCCAGAGTTCCAAGTGTGCGGCGTGGGCGGGGCCTGCAGAAAGGGACG
GGGCCTGCAGAAAGGGACGGGGCTGGCAAAGGCCTGGTCTACCTCACCGATCCGGGTCGCGGGGGCTCCTAGGGGG
TTCCTTCCACAGCTGGTGCGCGTGCGCAAGAGTGCGTCGAGGTTGTTCCGCGGCAATTTATGAAACTCGGAGCCGG
CAGTGAGTTGAGGGTTCGGCGAGGTCAGGGATTCCTAGTTGCAGCGTGATTACCAACGCCAGCTCCCTTCACTGTC
CAAGACAGCGATTCCGCGGCTCCTCTGAGAACTGGCGCTTGTGAGTGACTGAACGGGCCACTGGAGAGGAGCGGTG
CTCACGGGTATCGTTCTCGTCAGCGGGCGGTGGGGACGCGGGGTCTTGGAATTGGGAATCACGCTTGAGGGCGTCC
ATGCGGAACGTGCAGCTTACCTCAGCTTGCTTGCAGTTACCCAGTTTCCACGCTGCCCTCCAAGGTCTGTGCCGGA
AAACCTGGAGGAGGGCGCGAGCTGACGGGAGGGATGGAAACGTGGGCTGCGGAAGTAAGACAAGCGTTGAGAGCAG
CGACAGAGGGCGAGGGGGTGGGCGGCGGGACGGCTGGGGTCAGGATCCAGCTCCGCTAAAGCAAGGATTTAACCAC
ACTCCTGTCGCCTCTTCCACTCTGCAATAGGGGCAGTCTCCAGACGCGCAGAGTGG
→ putative marker candidates
Experimental workflow
CpG islands in candidate genes:
Microarray results give hints for
primer positions
Design and test MS-PCR primers
with in vitro CpG-methylated DNA
Isolate DNA from tumor and
control samples
Bisulfite treatment of DNA
Realtime MS-PCR
Methylated DNA
genomic
Bisulfite-converted
MS-PCR primer
Unmethylated DNA
genomic
Bisulfite-converted
MS-PCR primer
CGCGTACGTGCACTCCCCCGGCGGGATCATCGTCCTCGG…
CGCGTACGTGTATTTTTTCGGCGGGATTATCGTTTTCGG…
CGCGTACGTGTATTTTTTCGGC
CGCGTACGTGCACTCCCCCGGCGGGATCATCGTCCTCGG…
TGTGTATGTGTATTTTTTTGGTGGGATTATTGTTTTTGG…
CGCGTACGTGTATTTTTTCGGC
Sensitivity and specificity of methylationspecific PCRs
Methylated DNA
genomic
Bisulfite-converted
MS-PCR primer
Unmethylated DNA
genomic
Bisulfite-converted
MS-PCR primer
1pg
100fg 10fg
CGCGTACGTGCACTCCCCCGGCGGGATCATCGTCCTCGG…
CGCGTACGTGTATTTTTTCGGCGGGATTATCGTTTTCGG…
CGCGTACGTGTATTTTTTCGGC
CGCGTACGTGCACTCCCCCGGCGGGATCATCGTCCTCGG…
TGTGTATGTGTATTTTTTTGGTGGGATTATTGTTTTTGG…
CGCGTACGTGTATTTTTTCGGC
1fg
→ primers are highly specific for the
methylated and bisulfite-treated DNA region
AHRR is a bad methylation signature marker
AHRR
TM2 TM3 TM4 TM5 TM6 KM1 KM2 Kmeth
AHRR:
Test of candidate genes with DNA pools
TM = DNA-Pools from CxCa tissue sections
KM = pooled DNA from cervical scrapes from
HPV-positive, inconspicious women
Kmeth = in vitro-methylated genomic DNA
•Reported to be nonmethylated in inconspicious,
and increasingly methylated in lesions → CxCa
Gene 4: a good methylation signature
marker
Gen 4
TM1 TM2 TM3 TM4 TM5 TM6 KM1 KM2 KM3 Kmeth
Gene 4:
•Downregulated: CIN → CxCa (cDNA array)
Test of candidate genes with DNA pools
TM = DNA-Pools from CxCa tissue sections
KM = pooled DNA from cervical scrapes from
HPV-positive, inconspicious women
Kmeth = in vitro-methylated genomic DNA
•Methylated: inconspicious → CxCa (CpG array)
A signature of six methylation markers....
Methylation in cervical scrapes
100
% methylated samples
90
80
70
HPV-negative (77)
60
50
HPV-positive (90)
CIN3 (48)
40
CxCa (65)
30
20
10
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Sensitivity for CIN3+ >90%, positive predictive value ca. 90%
Epigenetic signature for the detection of CIN2+
Professor Dürst‘s research group
„gynecologic molecular biology“
•Daniel Steinbach
•Norman Häfner
•Matthias Dürst
special thanks to: