A novel index combining fecal immunochemical test, DNA test, and age improves detection of advanced colorectal adenoma

Abstract

Although the fecal immunochemical test for hemoglobin (FIT) is a widely used screening test for colorectal cancer, it is not sensitive enough to detect advanced colorectal adenoma. To address this issue, we performed this study to investigate whether combining the FIT and fecal DNA testing of methylated somatostatin (SST) could improve diagnostic performance for advanced colorectal adenoma. We collected feces from 79 healthy subjects with negative results on colonoscopy, 43 patients with non-advanced colorectal adenoma, 117 patients with advanced colorectal adenoma, and 126 patients with colorectal cancer. After fecal DNA was incubated with methylation-sensitive restriction enzymes, SST methylation levels were measured by droplet digital PCR. Using logistic multivariate analysis, we established a prediction formula for detecting colorectal neoplasia and named it the FAMS (FIT, age, methylated SST) index. The diagnostic performance of a single use of FIT for advanced colorectal adenoma showed a sensitivity of 29.1% (34/117) and specificity of 89.3% (109/122). In contrast, the FAMS index showed a sensitivity of 56.4% (66/117) at a similar specificity point of 91.0% (111/122). Furthermore, even at the higher specificity point of 94.3% (115/122), the sensitivity was still higher than that of FIT, reaching 42.7% (50/117). As the FAMS index showed better diagnostic performance for advanced colorectal adenoma than a single use of FIT, the FAMS index could be a promising tool for detecting advanced colorectal adenoma.

Keywords: SST; advanced colorectal adenoma; fecal DNA test; fecal immunochemical test for hemoglobin; methylation.

FIGURE 1

Flow diagram of enrollment. Of the 377 participants who provided informed consent, 12 were excluded due to failure of fecal sampling. FAMS index, multivariate analysis consisting of fecal immunochemical test for hemoglobin, age, and methylated SST; FIT, fecal immunochemical test for hemoglobin.

FIGURE 2

DNA sequences of SST and hTERT for CORD assay.

FIGURE 3

Comparison of SST methylation ratios between the tumor tissue and surrounding normal mucosa. SST methylation ratio represents the ratio of methylated SST to hTERT copy numbers. Fresh‐frozen tissue of colorectal tumor and normal mucosa adjacent to the tumor were collected from one patient with advanced colorectal adenoma and 10 patients with colorectal cancer.

FIGURE 4

Fecal DNA test. Distributions of methylated SST copy numbers (A), hTERT copy numbers (B), and ratios of methylated SST to hTERT copy numbers (C) in each group are shown. p values are only shown for comparisons with a p value less than 0.05. Each sample is indicated by a closed circle. The solid horizontal lines indicate medians, and the dotted lines indicate cutoff points. AA, advanced colorectal adenoma; CRC, colorectal cancer; NAA, non‐advanced colorectal adenoma.

FIGURE 5

Comparison of DNA test results. Simple linear regression analysis through the origin of the SST methylation ratio between the fecal and tissue samples (A). R ² is the coefficient of determination. Comparison of fecal DNA test with the SST methylation ratios in the colorectal tumor tissues (B). The criterion for a positive result of fecal DNA testing is either 67.9 or more copy numbers of methylated SST or SST methylation ratio of 0.5 or more or both. SST methylation ratio represents the ratio of methylated SST to hTERT copy numbers. Forty pairs of fecal samples and formalin‐fixed, paraffin‐embedded colorectal tumor tissue samples were collected. The negative fecal DNA test group consisted of patients with non‐advanced colorectal adenoma (n = 1), advanced colorectal adenoma (n = 18), and colorectal cancer (n = 4). The positive fecal DNA test group consisted of patients with advanced colorectal adenoma (n = 12) and colorectal cancer (n = 5).

FIGURE 6

Results of the combination test. The distribution of the results of FIT and fecal DNA testing of SST methylation in each group is shown. The criterion for a positive result of fecal DNA testing is either 67.9 or more copy numbers of methylated SST or SST methylation ratio of 0.5 or more or both. Roman numerals indicate stages of colorectal cancer. AA, advanced colorectal adenoma; both (+), both FIT and fecal DNA testing are positive; both (−), both FIT and fecal DNA testing are negative; CRC, colorectal cancer; fecal DNA alone (+), fecal DNA testing alone is positive; FIT, fecal immunochemical test for hemoglobin; FIT alone (+), FIT alone is positive; NAA, non‐advanced colorectal adenoma.

FIGURE 7

Venn diagrams. Copy number, number of subjects with copy number of methylated SST ≥ 67.9 copies/test; FIT, number of subjects with positive fecal immunochemical test for hemoglobin; Ratio, number of subjects with the ratio of methylated SST to hTERT copy numbers ≥0.5.

FIGURE 8

Diagnostic performance of the FAMS index. The distribution of the FAMS index in each group is shown (A). Each sample is indicated by a closed circle. The solid horizontal lines indicate medians. Receiver operating characteristic curve analyses between the control/NAA group and the AA/CRC group (B), and between the control/NAA group and the AA group (C) are shown. AA, advanced colorectal adenoma; AUC, area under the ROC curve; CI, confidence interval; CRC, colorectal cancer; FAMS index, multivariate analysis consisting of fecal immunochemical test for hemoglobin, age, and methylated SST; NAA, non‐advanced colorectal adenoma.