OBJECTIVE

Testing for high risk human papillomavirus (HR-HPV) is increasing; however due to limitations in specificity there remains a need for better triage tests. Research efforts have focused recently on methylation of human genes which show promise as diagnostic classifiers.

METHODS

Methylation of 26 genes: APC, CADM1, CCND2, CDH13, CDKN2A, CTNNB1, DAPK1, DPYS, EDNRB, EPB41L3, ESR1, GSTP1, HIN1, JAM3, LMX1, MAL, MDR1, PAX1, PTGS2, RARB, RASSF1, SLIT2, SOX1, SPARC, TERT and TWIST1 was measured by pyrosequencing in cytology specimens from a pilot set of women with normal or cervical intraepithelial neoplasia grade 3 (CIN3) histology. Six genes were selected for testing in Predictors 1, a colposcopy referral study comprising 799 women. The three genes EPB41L3, DPYS and MAL were further tested in a second colposcopy referral study, Predictors 2, comprising 884 women.

RESULTS

The six genes selected from the pilot: EPB41L3, EDNRB, LMX1, DPYS, MAL and CADM1 showed significantly elevated methylation in CIN2 and CIN3 (CIN2/3) versus ≤CIN1 in Predictors 1 (p<0.01). Highest methylation was observed in cancer tissues. EPB41L3 methylation was the best single classifier of CIN2/3 in both HR-HPV positive (p<0.0001) and negative samples (p=0.02). Logistic regression modeling showed that other genes did not add significantly to EPB41L3 and in Predictors 2, its classifier value was validated with AUC 0.69 (95% CI 0.65-0.73).

CONCLUSION

Several methylated genes show promise for detecting CIN2/3 of which EPB41L3 seems the best. Methylated human gene biomarkers used in combination may be clinically useful for triage of women with HR-HPV infections.