Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

<div><p>Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (<i>APC</i>, <i>CCNA</i>, <i>CDH1</i>, <i>CDH13</i>, <i>WIF1</i>, <i>TIMP3</i>, <i>DAPK1</i>, <i>RARB</i>, <i>FHIT</i>, and <i>SLIT2</i>). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of <i>DAPK1</i>, <i>SLIT2</i>, <i>WIF1</i> and <i>RARB</i> with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, <i>p-value</i> = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as <i>DAPK1</i>, <i>RARB</i>, <i>WIF1</i>, and <i>SLIT2</i>, may also occur early in cervical carcinogenesis and should be evaluated.</p></div

Distribution of DNA methylation Index among ten tumor suppressor genes in cervical cancer cases and controls.

<p>a. Number of cases and controls per gene vary due to exhausted DNA and/or inability to generate pyrosequencing data</p><p>b. Summary statistics with a nonparametric Mann-Whitney test comparing cases and controls for each gene</p><p>Distribution of DNA methylation Index among ten tumor suppressor genes in cervical cancer cases and controls.</p

The proportion of cancers correctly classified (true positives) and the proportion of normal tissues incorrectly classified (false positives) using an <i>a priori</i> threshold ≥15% methylation.

<p>a. AUC = Area under the curve from Receiver Operator Curve (ROC); FP = False positive rate; PPV = Positive predictive value; NPV = negative predictive value; Sensitivity = true positive rate</p><p>The proportion of cancers correctly classified (true positives) and the proportion of normal tissues incorrectly classified (false positives) using an <i>a priori</i> threshold ≥15% methylation.</p

Demographics and clinical characteristics of women with invasive cervical cancer and frequency matched controls.

<p>a. Numbers may not add up to total due to missing data.</p><p>b. Differences between cases and controls determined using the Fisher exact Chi2 test or T-test.</p><p>Demographics and clinical characteristics of women with invasive cervical cancer and frequency matched controls.</p

Receiver Operator Curve (ROC) Analysis of DNA Methylation Index and HPV.

<p>ROC of predicted sensitivity and 1-specificity when DNA MI for <i>DAPK1</i>, <i>RARB</i>, <i>SLIT2</i> and <i>WIF1</i> genes was added to a model with HPV status and age. The test of the equality between AUC for HPV and age (AUC = 79%, dashed line) compared to methylation index for <i>DAPK1</i>, <i>RARB</i>, <i>SLIT2</i> and <i>WIF1</i>, HPV, and age (AUC = 98%, solid line), <i>p-value</i> = 0.0002.</p

Box-plot of methylation indices for each candidate gene analyzed by pyrosequencing.

<p>Genes are ordered by Mann-Whitney <i>p-value (</i>** p-value<0.0001 and * p-value<0.05). Methylation index (MI) of each gene is presented for normal cervical sample (N) and cancer (T) as a boxplot. Whiskers of the boxplot mark the 5^th and 95^th percentiles, the box marks the 25^th (low boundary of box), median, and 75th (upper boundary of box) percentiles, and extreme values (●).</p