1. Discovery of Novel Methylated Genes in Cervical Carcinogenesis: Methylation of GREM1 and NID2 is Detected in the Majority of Invasive Cervical Cancers and Defines a Subset of HSILs KS Gustafson, AR Davis, L Van Neste, BM Ronnett, JG Herman The Johns Hopkins Hospital, Baltimore, MD

2. Conflict of Interest  No relevant financial relationships with commercial interests

3.  “Heritable changes in the pattern of gene expression that do not involve changes to the underlying DNA sequence.”  Covalent modifications (e.g. methylation, acetylation) that alter chromatin structure  DNA: CpG sites  Histone proteins Luger et al, Nature 1997 Nucleosome

4. Epigenetic Changes and Gene Expression  Critical for normal development and differentiation  Aberrant changes occur during tumorigenesis  Diagnostic/prognostic biomarkers  Therapeutic targets ▪ Demethylating agents ▪ Histone deacetylase inhibitors

5. Normal versus Cancer Epigenome Ting et al, Genes & Dev, 2006

6. GeneFrequency (%)Function TP7339Apoptosis TNFRSF10C100Apoptosis PTEN58WNT-pathway CDH128-80.5WNT-pathway MGMT5-81DNA repair DAPK145-100Metastasis/cell death IGSF4/CADM158-65Cell adhesion HIC118-45Transcription factor RARB33-66Cell differentiation Candidate Tumor Suppressor Genes Hypermethylated in Cervical Cancer Adapted from Duenas-Gonzalez et al, Molecular Cancer (2005)

7. Gene Methylation is Detected More Frequently in HSIL Pap Tests Gene Methylation (Percentage of Samples) p=.0081 p=.0027 p=.0015 p=.0066 p<.0001 (Fisher exact) (n=60) (n=39) Kahn et al, Cancer 2008

8. Specific Aims  Discover novel cancer-specific hypermethylated genes in cervical cancer  Determine if these novel hypermethylated genes can serve as molecular biomarkers of HSIL

9. Study Design  Gene expression microarray analysis  Agilent 44K human genome arrays  SiHa and CaSki cervical cancer cell lines  Pharmacologic treatment ▪ Demethylating agent: 5-aza-2’-deoxycytidine (DAC) ▪ Histone deacetylase inhibitor: Trichostatin A (TSA)  Selection of CpG island-containing candidate genes ▪ Increased expression with DAC and no change with TSA ▪ No basal expression in mock-treated cells

10. Study Design  Validation studies  Gene expression and methylation status ▪ SiHa and CaSki cell lines ▪ Primary human foreskin keratinocytes (HFK)  Methylation status in cervical tissues ▪ Normal cervix (n=20) ▪ Invasive cervical cancer (n=20)  Biomarker analysis in precursor lesions  Methylation in HSIL vs LSIL/NILM Pap Tests ▪ HSIL (n=36), LSIL (n=29), NILM (n=28)

11. Gene Expression Analysis in Cervical Cancer Cell Lines

12. Candidate Hypermethylated Genes  85 shared CpG island- containing genes  36 genes (in TT of both)  31 genes (in TT of one)  39 genes selected for validation Hypermethylome spikeSiHaCaSki All genesCGIAll genesCGI Top Tier (≥2-fold change)236126234122 Next Tier (>1.4- and <2-fold change) 256138292159 Total candidate genes492264526281 85 SiHa CaSki 179196

13. Cell Line Validation: Gene Expression by RT-PCR

14. Cell Line Validation: DNA Methylation by MSP

15. GREM1 and NID2 are Cancer-Specific Hypermethylated Genes in Cervical Tissues Fisher exactP<.0001 P=1.000P=0.3203

16. Relative Level of Gene Methylation in Cervical Tissues by qMSP Mann-Whitney Cervical Tissues: Normal (n=20) Invasive Cervical Cancer (ICC; n=20): GREM1 Mean = 22.65% Median = 21.10% NID2 Mean = 6.03% Median = 5.24% P<.0001

17. Can methylation of GREM1 and NID2 serve as a biomarker of HSIL in Pap tests?

18. GREM1 and NID2 Methylation is Detected in HSIL Pap Tests Pap Tests: NILM/LSIL (n=57); HSIL (n=36) Fisher exactP<.0001

19. GREM1 and NID2 Methylation Clusters in a Subset of HSIL Pap Tests

20. HSILs with Similar Morphologies Differ in Methylation Status Negative for MethylationPositive for Methylation

21. Determinants of Methylation in HSIL Pap Tests are Unknown  Relative level of methylation in HSIL Pap tests does not correlate with estimated number of lesional cells  Methylation status of HSIL Pap tests is not associated with higher grade of cervical intraepithelial neoplasia (CIN)  CIN 2 versus CIN 3

22. Summary  GREM1 and NID2 are novel cervical cancer- specific methylated genes.  GREM1 and NID2 methylation represents a molecular signature that defines a subset of HSILs.  The biologic determinants of gene methylation and behavior of this subset of HSILs are unknown.  Gene methylation may serve as a prognostic molecular biomarker of HSIL.

23. Acknowledgements  Funding for research:  Johns Hopkins University Cervical Cancer SPORE (KSG)  NIH/NCI CA123612 (KSG)