Methylated SEPTIN9 plasma test for colorectal cancer detection may be applicable to Lynch syndrome

Abstract

Objective: The plasma-based methylated SEPTIN9 (mSEPT9) is a colorectal cancer (CRC) screening test for adults aged 50-75 years who are at average risk for CRC and have refused colonoscopy or faecal-based screening tests. The applicability of mSEPT9 for high-risk persons with Lynch syndrome (LS), the most common hereditary CRC condition, has not been assessed. This study sought preliminary evidence for the utility of mSEPT9 for CRC detection in LS.

Design: Firstly, SEPT9 methylation was measured in LS-associated CRC, advanced adenoma, and subject-matched normal colorectal mucosa tissues by pyrosequencing. Secondly, to detect mSEPT9 as circulating tumor DNA, the plasma-based mSEPT9 test was retrospectively evaluated in LS subjects using the Epi proColon 2.0 CE assay adapted for 1mL plasma using the "1/1 algorithm". LS case groups included 20 peri-surgical cases with acolonoscopy-based diagnosis of CRC (stages I-IV), 13 post-surgical metastatic CRC, and 17 pre-diagnosis cases. The control group comprised 31 cancer-free LS subjects.

Results: Differential hypermethylation was found in 97.3% (36/37) of primary CRC and 90.0% (18/20) of advanced adenomas, showing LS-associated neoplasia frequently produce the mSEPT9 biomarker. Sensitivity of plasma mSEPT9 to detect CRC was 70.0% (95% CI, 48%-88%)in cases with a colonoscopy-based CRC diagnosis and 92.3% (95% CI, 64%-100%) inpost-surgical metastatic cases. In pre-diagnosis cases, plasma mSEPT9 was detected within two months prior to colonoscopy-based CRC diagnosis in 3/5 cases. Specificity in controls was 100% (95% CI 89%-100%).

Conclusion: These preliminary findings suggest mSEPT9 may demonstrate similar diagnostic performance characteristics in LS as in the average-risk population, warranting a well-powered prospective case-control study.

Keywords: SEPTIN9; circulating tumor dna; colorectal cancer; lynch syndrome; plasma.

Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1

Levels and prevalence of SEPT9 methylation in colon and rectal adenocarcinoma (COADREAD) compared with adjacent normal tissue in Infinium Human Methylation 450K Array data from The Cancer Genome Atlas (TCGA). (A) Dot plot of differential methylation levels (tumour-normal; y-axis) by genomic location (x-axis) according to the GRCh37/hg19 human genome assembly at each of the SEPT9 probes contained within the 450K array data from the TCGA COADREAD data set. The closest probe to the plasma-based Epi proColon 2.0 CE assay, cg2027558, located at Chr17: 75369484 is indicated. (B) Manhattan plot of the −log10 p values (Wilcoxon rank-sum test; y-axis) for a difference in methylation between tumour and normal tissues by genomic location (x-axis). Probe cg2027558, indicated, showed the most significant difference in tumour versus normal tissue. (C) Spaghetti plot of normalised levels of methylation (β values) at probe cg2027558 in normal tissue and COADREAD tumour tissue. Subject-matched sample pairs are linked by lines. Median, IQRs and 95% CI are indicated. Green=tumours showing hypermethylation, red=tumours with methylation levels in the normal range. (D) Histogram of the frequency of SEPT9 hypermethylation at cg2027558 in colorectal cancer (CRC) from COADREAD stratified by tumour stage.

Figure 2

Measurement of SEPTIN9 methylation levels by quantitative CpG pyrosequencing in archival Lynch syndrome (LS)-associated colorectal cancer (CRC), advanced adenoma, and paired normal colorectal mucosa (NCM) tissues. (A) Assay design. Sequence coordinates GRCh37/hg19 assembly Chr17: 75369414–75369587 are shown as bisulfite converted. YG indicates CpG sites where the cytosine may be methylated (CG) or unmethylated (TG). PCR primer-binding sites are italicised and indicated by black arrows. Pyrosequencing primer-binding site is italicised and indicated by a red arrow. The sequence analysed (red text) contains five CpG sites, of which the first corresponds to Infinium probe cg20275528, located 92 bp upstream of the fluorescent probe-binding sequence within the plasma-based Epi proColon 2.0 CE assay (blue text). (B) Simulated pyrosequencing assay. Grey shading shows the five CpG sites interrogated. Yellow shading shows the single cytosine control for bisulfite conversion efficiency. (C) Analytical sensitivity and linearity of the quantitative CpG pyrosequencing assay to measure methylation levels at SEPTIN9. The titration curve shows the observed (±1 SD) versus expected methylation values for the hypermethylated RKO CRC cell line DNA (expected value 100% methylated) diluted into the peripheral blood DNA from a healthy control (0% expected methylation value) in specific proportions. (D) Illustrative pyrograms obtained from formalin-fixed paraffin-embedded (FFPE) samples of primary CRC, a liver metastasis, and paired NCM from a Lynch syndrome patient with an MSH2 mutation.

Figure 3

Frequent hypermethylation of SEPT9 in Lynch syndrome (LS)-associated colorectal cancer (CRC) and advanced adenomas. (A) Spaghetti plot showing actual values of SEPT9 methylation measured by pyrosequencing at five CpG sites, including cg20275528, in CRC, advanced adenomas, and normal colorectal mucosa (NCM) from patients with LS (each black dot represents a single tissue sample and subject-matched samples are linked by lines), overlaid with box and whisker plot indicating the median value (horizontal black line) and IQRs (boxes). Dotted horizontal lines indicate the threshold values of 23% and 18% for methylation-positive test results to discriminate CRC and adenoma samples from NCM samples, respectively. Accompanying clinicopathological and SEPTIN9 methylation data are provided in online supplementary tables 1 and 2. (B) Receiver operating characteristic (ROC) curve for SEPT9 methylation values in CRC versus paired NCM (left), and advanced adenomas versus paired NCM (right). The diagonal lines represent the non-informative prediction model with an area under the ROC curve (AUC) of 0.5. ROC analyses were performed for the subset of neoplasms with a subject-matched NCM sample, including 29 CRC samples with 28 unique paired NCM samples, and 13 adenoma samples with 10 unique paired NCM samples.