Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay

Abstract

Background: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/principal findings: Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was approximately 20%.

Conclusions/significance: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

Figure 1. Sample processing workflow.

The diagram depicts the major sample processing and laboratory workflow steps. It shows where process control samples are introduced into the workflow and what assays were used to measure output of each process step. Grey boxes indicate test set specific workflow steps.

Figure 2. Concentrations of methylated SEPT9 DNA in plasma.

Box-percentile plots of training set methylated SEPT9 DNA concentrations in plasma are shown for colonoscopy-verified normal patients (Normal) and patients with colorectal cancer (CRC). Median DNA concentrations are red horizontal lines; 25th and 75th percentiles are blue horizontal lines. The width of the box-percentile plot at any given height is proportional to the percent of observations that are more extreme in the direction leading away from the median. Individual measurement values are plotted as grey circles. B) Box-percentile plots of testing set methylated SEPT9 DNA concentrations in plasma.

Figure 3. Correlation between standard and diluted SEPT9 measurement in testing set.

X-axis – concentration of SEPT9 in standard test set samples (mean of three standard replicates) Y-axis – concentration of SEPT9 in 1∶10 diluted test set samples (single replicate). mDNA – methylated DNA. Group 1 – samples with the same SEPT9 amplification in standard and diluted concentration, group 2 – samples with SEPT9 amplification only in standard concentration, group 3 – samples with SEPT9 amplification only in diluted concentration. Green triangles – healthy colon, red circles – colorectal cancer, black squares – polyps.