Cervical dysplasia: assessing methylation status (Methylight) of CCNA1, DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy.
OBJECTIVE
Diagnosis of cervical neoplasia hinges upon microscopic inspection of cervical samples. This has inherent operator-dependent variability. Testing for high-risk human papilloma virus (HPV) may help to triage patients with pre-invasive disease in determining clinical intervention and follow-up. However, HPV presence/absence does not reflect the cervical epithelial cell's molecular status. Epigenetic modifications, e.g. DNA methylation, have been observed in the early stages of neoplastic change, preceding gene mutations. Here, we assess the correlation between cytologic/histologic results and combined DNA methylation data of 5 genes in different grades of cervical dysplasia.


METHODS
Cervical specimens collected via the liquid-based cytology system were each microscopically examined. Residual cells were subjected to DNA methylation analysis (Methylight) of gene loci CCNA1, PAX1, HS3ST2, DAPK1 and TFPI2. Methylation data were compared with cytologic/histologic reports. Statistical methods were applied to assess the ability of DNA methylation status to subtype the cervical neoplastic lesions according to their corresponding cytologic/histologic reports.


RESULTS
A total of 165 subjects provided cytologically proven 63 HSIL, 49 LSIL and 53 normal samples. All patients with HSIL and LSIL underwent colposcopic examination. Patients with LSIL were all found to be CIN1; patients with HSIL were subsequently subdivided into 10 squamous cell carcinoma (SCC), 31 CIN3, 10 CIN2 and 12 CIN1. For each gene, there was increasing frequency of methylation from normal and LSIL (CIN1), through HSIL (CIN2 and CIN3), to SCC. Methylation of ≥1 of genes investigated was observed in 88% of combined HSIL (CIN2 and CIN3) and SCC cases. All genes showed significant increase in methylation level (PMR value) with increasing disease grade (p<0.005). CCNA1 was the only gene that was able to distinguish CIN2 from CIN3 specimens (p=0.016). Based on receiver operating characteristic (ROC) analysis, HS3ST2 was the most significant candidate in segregating HSIL/SCC from normal/LSIL cases (p<0.0001); at an optimal cutoff value, sensitivity and specificity between 70% and 80% were obtained.


CONCLUSIONS
Development of DNA methylation status of a gene panel to improve diagnostic accuracy in cervical neoplasia is warranted.
Lim
Elaine H
EH
Oncology Research Institute, 28 Medical Drive, 117456, Singapore. elainelim77@ymail.com
Ng
Shi Len
SL
Li
Jia Liang
JL
Chang
Alexander R
AR
Ng
Joseph
J
Ilancheran
A
A
Low
Jeffrey
J
Quek
Swee Chong
SC
Tay
Eng Hseon
EH
D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology; D056750:Cyclin A1 / Q000235:genetics; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases; D005260:Female; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D016015:Logistic Models; D051761:Paired Box Transcription Factors / Q000235:genetics; D012307:Risk Factors; D015238:Sulfotransferases / Q000235:genetics; D002578:Uterine Cervical Dysplasia / Q000235:genetics* / Q000473:pathology; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000473:pathology
D051017:Apoptosis Regulatory Proteins; C503745:CCNA1 protein, human; D056750:Cyclin A1; D006023:Glycoproteins; D051761:Paired Box Transcription Factors; C021240:tissue-factor-pathway inhibitor 2; C070835:PAX1 transcription factor; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015238:Sulfotransferases; C021350:heparitin sulfotransferase