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.

Abbreviations

• AA
• advanced colorectal adenoma
• AUC
• area under the ROC curve
• CI
• confidence interval
• CRC
• colorectal cancer
• FIT
• fecal immunochemical test for hemoglobin
• hTERT
• human telomerase reverse transcriptase
• NAA
• non-advanced colorectal adenoma
• ROC
• receiver operating characteristic
• SST
• somatostatin

INTRODUCTION

Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. More than 1.9 million new cases of CRC and 935,000 deaths from CRC were estimated to occur in 2020, accounting for about one in 10 cancers and deaths.¹ Early detection and cure of CRC are important because it has been estimated that curative surgery is beneficial for more than 95% of patients with CRC if diagnosed at an early stage of cancer or premalignancy.² The fecal immunochemical test for hemoglobin (FIT) is one of the most widely used approaches to CRC screening in the world.³ Whereas FIT has a high sensitivity and specificity for diagnosing CRC, it is less sensitive for advanced colorectal adenoma (AA), which is generally considered a precancerous lesion.⁴ Indeed, the sensitivity of FIT for CRC is 65.8%–73.8%, whereas that for AA is only 23.8%–27.1%.^5,6 Furthermore, as the number of patients with AA is increasing in individuals under 50 years of age,⁷ the development of clinical laboratory tests that can efficiently predict the presence of AA is an urgent matter.

Cologuard® (Exact Sciences, Madison, WI, USA) is a CRC screening test approved by the US Food and Drug Administration in 2014 that combines FIT with fecal DNA testing including KRAS mutations, methylation of BMP3 and NDRG, and the copy number of ACTB. Its sensitivity for CRC is 92.3%, whereas that for advanced precancerous lesions including AA is only 42.4%.⁵ In addition, because Cologuard requires one whole stool per defecation as a test specimen (estimated to be about 200 g),⁵ it is difficult to perform fecal DNA testing of Cologuard in countries other than the United States because of the lack of clinical laboratory testing systems that can hygienically transport and process such a large volume of fecal sample.

To address these issues, we have developed the combined restriction digital PCR (CORD) assay, a highly sensitive DNA methylation assay that can count even one copy of a methylated gene in a small amount of DNA sample.^8,9 Among various genes that are hypermethylated, we have found that somatostatin (SST) is hypermethylated in the tissues of colorectal neoplasia (unpublished data). Therefore, we thought that methylated SST could be used as a biomarker for colorectal neoplasia and that measuring fecal SST methylation levels using the CORD assay would be useful for the detection of AA. In this study, we evaluated the diagnostic performance of combined fecal SST methylation testing and FIT to detect AA.

METHODS

Clinical materials

We collected fresh-frozen tissues of colorectal tumor and the normal mucosa adjacent to the tumor from one patient with AA and 10 patients with CRC who had undergone surgical resection at Yamaguchi University Hospital. We also collected pairs of a fecal sample and a formalin-fixed, paraffin-embedded colorectal tumor tissue sample from one patient with non-advanced colorectal adenoma (NAA), 30 patients with AA, and 9 patients with CRC who received endoscopic mucosal resection or surgical treatment at Yamaguchi University Hospital. We prospectively enrolled 377 participants, accumulated at St. Hill Hospital, Ajisu Kyoritsu Hospital, IMSUT Hospital, and Yamaguchi University Hospital from October 2007 to December 2019. Of these 377 participants, 365 had fully evaluable results, including 79 subjects with negative colonoscopy results (control group), 43 cases of NAA including 2 patients with serrated adenoma, 117 cases of AA including 8 patients with serrated adenoma, and 126 cases of CRC (Table 1, Figure 1). Participants from previous reports were included in this study.^8,10 Criteria for AA were defined as adenomas of 1 cm or larger in size, or with high-grade or severe dysplasia, or with villous components (tubulovillous or villous).^5,11,12 Non-neoplastic polyps and non-advanced adenomatous polyps other than AA were categorized as NAA.⁵ Staging was in accordance with the Union for International Cancer Control (International Union Against Cancer).¹³ Clinicopathologic characteristics of the subjects are shown in Table 1.

TABLE 1 Clinicopathologic characteristics.

	Control n = 79	NAA n = 43	AA n = 117	CRCa n = 126
Age, years, median (range)	50 (33–83)	58 (37–78)	69 (36–91)	69 (33–91)
Sex
Men	34	27	77	65
Women	45	16	40	61
Tumor location
Left		20	46	84
Right		23	71	42
Tumor size, mm, median (range)		4 (2–8)	22 (5–100)	35 (6–150)

[IMAGE OMITTED. SEE PDF]

FIT

For FIT, 10 mg of fresh fecal samples were collected into an OC-Hemodia sampling container filled with 2 mL of hemoglobin stabilizing buffer using the OC-Hemodia sampling probe before bowel preparation for colonoscopy. To quantify the hemoglobin level, the latex agglutination test kit OC-Hemodia and the fecal immunochemical test analyzer instrument OC sensor IO were used (all from Eiken Chemical, Tokyo, Japan). We used a cutoff value of 20 μg hemoglobin/g feces, equivalent to 100 ng hemoglobin/mL buffer.^14,15

Methylation assay

DNA was extracted using an AllPrep DNA/RNA Mini Kit for fresh-frozen tissue samples, a QIAmp DNA FFPE tissue kit for formalin-fixed paraffin-embedded tissue samples, and a QIAamp FAST DNA Stool Mini Kit for fecal samples (all from QIAGEN, Hilden, Germany). Fecal samples for DNA testing were collected prior to bowel preparation for colonoscopy or surgical procedures and stored at −20°C. After fecal samples were thawed from −20°C, approximately 200 mg of each sample was used for DNA extraction, and each DNA was eluted in 100 μL of ATE buffer (QIAGEN). A modified CORD assay, which is simpler and allows a shorter enzyme reaction time compared with the conventional CORD assay,⁹ was used to measure the copy number of human telomerase reverse transcriptase (hTERT) and of methylated SST. The detailed procedure is described below.

First, 10 μL of eluted DNA was incubated with 6 μL of enzyme reaction mixture at 37°C for 1 h and then at 60°C for 16 h, followed by an incubation for 10 min at 98°C. The enzyme reaction mixture contained 1 μL of AmpliTaq Gold buffer II, 1 μL each of HhaI, HpaII, and ExoI (all from Thermo Fisher Scientific, Waltham, MA, USA), 1 μL of BstUI (New England Biolabs, Hitchin, UK), and 1 μL of 25 mM MgCl₂ (Thermo Fisher Scientific). BstUI, HhaI, and HpaII are methylation-sensitive restriction enzymes that recognize the DNA sequences 5′-CGCG-3′, 5′-GCGC-3′, and 5′-CCGG-3′, respectively. ExoI was added to eliminate single-stranded DNA that would evade restriction enzyme cleavage, thus preventing these fragments from being amplified by PCR.

Second, 2 μL of enzymatically treated DNA was mixed with 20 μL of PCR reaction solution containing 1 μL of primer mixture, 1 μL of probe mixture, 10 μL of 2× ddPCR Supermix for Probes, and 8 μL of H₂O (BioRad, Hercules, CA, USA). The primer mixture consisted of 10 μmol/L of each of the sense and antisense primers of the SST gene and 5 μmol/L of each of the sense and antisense primers of the hTERT gene. The probe mixture consisted of 10 μmol/L of SST probe and 5 μmol/L of hTERT probe. The DNA sequences of the primers and the probes are as shown in Figure 2 and our previous reports.^9,16 Because hTERT has no recognition site for HhaI, HpaII, and BstUI, it is always amplified by PCR when there is human DNA present in the sample.⁹ Therefore, hTERT is used as an internal control to reflect the suitability of the fecal sample. The PCR reaction solution mixture was fractioned into droplets using an automated droplet generator (BioRad), followed by PCR. PCR conditions were as follows. After preheating at 95°C for 10 min in a thermal cycler, 40 cycles of denaturation at 94°C for 30 s followed by annealing at 56°C for 60 s were repeated to simultaneously amplify the SST and the hTERT DNA. PCR was completed at 98°C for 10 min. After the PCR plate was transferred to a QX200 droplet reader, fluorescence data were acquired using QuantaSoft Analysis Pro software (all from BioRad).

[IMAGE OMITTED. SEE PDF]

Statistical analysis

The Mann–Whitney U test, Fisher's exact test, simple linear regression analysis, Dunn's test for multiple comparisons, multiple logistic regression analysis, and receiver-operating characteristic (ROC) analysis were performed. A p value <0.05 was considered statistically significant. Statistical analyses were performed with StatFlex Ver. 7 (Artec, Osaka, Japan), Microsoft Excel software in Microsoft 365 (Microsoft, Redmond, WA, USA), and GraphPad Prism Ver. 9 (GraphPad Software, La Jolla, CA, USA).

RESULTS

FIT

The sensitivity of FIT was 7.0% (3/43) for NAA, 29.1% (34/117) for AA, and 91.3% (115/126) for CRC. The specificity was 87.3% (69/79) for the control group and 89.3% (109/122) for the control/NAA group (Table 2). The sensitivity for left-sided AA/CRC was significantly higher than that for right-sided AA/CRC (70.8% vs. 50.4%, p = 0.0015, Table 2).

TABLE 2 Diagnostic performance of each test for the findings of colonoscopy.

Most advanced finding	(n)	FIT	Fecal DNA test	Combination test
Positive resultsa (n)	Sensitivity (%)	95% Cl (%)	p b	Positive resultsc (n)	Sensitivity (%)	95% Cl (%)	p b	Positive resultsd (n)	Sensitivity (%)	95% Cl (%)	p b
NAA	43	3	7.0	2.4–18.6		6	14.0	5.3–27.9		9	20.9	10.0–36.0
Left	20	2	10.0	1.2–31.7	0.5900	2	10.0	1.2–31.7	0.6688	4	20.0	5.7–43.7	1.0000
Right	23	1	4.3	0.1–21.9		4	17.4	5.0–38.8		5	21.7	7.5–43.7
AA	117	34	29.1	21.6–37.9		46	39.3	30.5–48.2		72	61.5	52.7–70.4
Left	46	13	28.3	15.2–41.3	1.0000	14	30.4	17.1–43.7	0.1256	23	50.0	35.6–64.4	0.0518
Right	71	21	29.6	19.0–40.2		32	45.1	33.5–56.6		49	69.0	58.3–79.8
CRC	126	115	91.3	85.0–95.1		38	30.2	22.1–38.2		119	94.4	88.9–97.7
Left	84	79	94.0	86.7–98.0	0.1779	31	36.9	26.6–47.2	0.0236	81	96.4	90.0–99.3	0.2206
Right	42	36	85.7	71.5–94.6		7	16.7	7.0–31.4		38	90.5	77.4–97.3
AA/CRC	243	149	61.3	55.1–67.2		84	34.6	28.6–40.5		191	78.6	73.4–83.8
Left	130	92	70.8	63.0–78.6	0.0015	45	34.6	26.4–42.8	1.0000	104	80.0	73.1–86.9	0.6388
Right	113	57	50.4	41.2–59.7		39	34.5	25.7–43.3		87	77.0	69.2–84.8
			Specificity (%)				Specificity (%)				Specificity (%)
Control	79	10	87.3	78.2–93.0		6	92.4	84.2–97.2		16	79.7	70.9–88.6
Control/NAA	122	13	89.3	82.6–93.7		12	90.2	84.9–95.4		25	79.5	72.3–86.7

Comparison of SST methylation levels between tumor tissue and adjacent normal mucosa

The SST methylation levels were significantly higher in colorectal tumor than in adjacent normal mucosa (p = 0.0029, Figure 3).

[IMAGE OMITTED. SEE PDF]

Fecal DNA test

We performed fecal DNA testing with a large sample size. The fecal DNA test consists of two evaluation elements. One is the copy number of the methylated SST, and the other is the ratio of methylated SST, calculated by dividing the absolute copy number of methylated SST by the absolute copy number of hTERT. Because the copy number of human DNA in fecal samples is affected by the form of the feces (e.g., watery or solid), we added the SST methylation ratio as an evaluation element to the fecal DNA test so that we can evaluate SST methylation levels for any fecal form. The distributions of the methylated SST copy number, the hTERT copy number, and the methylated SST ratio are shown in Figure 4A–C, respectively. The methylated SST copy number was significantly higher in the AA group than in the control group (Figure 4A) and also significantly higher in the CRC group than in the control group and in the NAA group (Figure 4A). For hTERT, the copy number was significantly higher in the CRC group compared with all other groups (Figure 4B). Interestingly, the SST methylation ratio was significantly higher in the AA group compared with all other groups (Figure 4C).

[IMAGE OMITTED. SEE PDF]

We set 67.9 copies/test as the cutoff for the methylated SST copy number (Figure 4A) and 0.50 for the SST methylation ratio (Figure 4C). Each cutoff was set to have a specificity of approximately 95%. The criterion for a positive fecal DNA test result was that either the methylated SST copy number or the SST methylation ratio exceeded the respective cutoff point, or that both were above the cutoff point. As a result, we found a sensitivity of 14.0% (6/43) for NAA, 39.3% (46/117) for AA, and 30.2% (38/126) for CRC with a specificity of 92.4% (73/79) for the control group and 90.2% (110/122) for the control/NAA group (Table 2). For AA, there was a trend toward higher sensitivity of the fecal DNA test for right-sided AA than for left-sided AA (45.1% vs. 30.4%, p = 0.1256). For CRC, however, the sensitivity was significantly lower for right-sided than for left-sided CRC (16.7% vs. 36.9%, p = 0.0236, Table 2).

There was a positive correlation of the methylated SST ratio between the fecal sample and the tumor tissue (R² = 0.5589, Figure 5A). When the SST methylation ratios in the colorectal tumor tissues were compared with the fecal DNA test results, the SST methylation ratio in the tumor tissues tended to be higher in the positive fecal DNA test group than in the negative fecal DNA test group (p = 0.0899, Figure 5B), suggesting that the fecal DNA test would, to some extent, reflect SST methylation levels in colorectal tumor tissue.

[IMAGE OMITTED. SEE PDF]

FIT/Fecal DNA testing (combination test)

The criterion for a positive result of the combination of FIT and fecal DNA test (combination test) was defined as either the FIT or fecal DNA test being positive, or both being positive. The sensitivity of the combination test was 20.9% (9/43) for NAA, 61.5% (72/117) for AA, and 94.4% (119/126) for CRC, with a specificity of 79.7% (63/79) for the control group and 79.5% (97/122) for the control/NAA group (Table 2, Figure 6). Interestingly, there was a trend toward higher sensitivity for right-sided AA than for left-sided AA (69.0% vs. 50.0%, p = 0.0518, Table 2). Of the 117 patients with AA, 72 were positive by the combination test, of which 26 were positive for FIT alone, 38 were positive for the fecal DNA test alone, and only 8 were positive for both tests (Figures 6 and 7). When focusing on the 38 patients with AA who were positive for the fecal DNA test alone, 14 were positive for the methylated SST copy number test alone, 19 were positive for the SST methylation ratio test alone, and only 5 were positive for both tests (Figure 7).

[IMAGE OMITTED. SEE PDF]

[IMAGE OMITTED. SEE PDF]

Although a single use of the FIT had a sufficient sensitivity of 80.0% (32/40) for stage I CRC, the remaining 20% (8/40) were missed by FIT. Of the 8 stage I patients with CRC with a negative FIT result, 4 were picked up by the fecal DNA test, resulting in a sensitivity of 90% for stage I CRC by the combination test (Figure 6).

Multivariate analysis and FAMS index

Multiple logistic regression analyses using the variables of fecal DNA test, FIT, age, and sex were performed to identify independent factors associated with AA/CRC. As a result, fecal DNA test, FIT, and age were the independent predictors (Table 3). We then established a prediction formula as shown below and named it the FAMS (FIT, age, methylated SST) index:$\text{FAMS index}=\frac{1}{1+e^{-X}}$$X=-6.441+1.622\times \left[\text{Fecal}\mathrm{DNA}\text{test}\right]+2.69\times \left[\mathrm{FIT}\right]+0.09609\times \mathrm{Age}$

TABLE 3 Multivariate analysis for predicting AA/CRC.

Variable	β (SE)	OR (95% CI)	p
Constant	−6.441 (0.8623)
Fecal DNA testa	1.622 (0.3971)	5.062 (2.325–11.024)	0.00004
FIT	2.69 (0.3732)	14.74 (7.091–30.622)	<0.00001
Age (years)	0.09609 (0.01354)	1.101 (1.072–1.13)	<0.00001

Each of [Fecal DNA test] and [FIT] in this formula is a binary datum in which 1 is substituted into each variable if each test result is positive, and 0 is substituted into each variable if each test result is negative. Age is a continuous variable (years). The FAMS index ranges from 0 to 1, with a value near 0 indicating a lower probability of AA/CRC and a value near 1 indicating a higher probability of AA/CRC. The distributions of the FAMS index in each group are shown in Figure 8A, in which the FAMS index was significantly higher in the AA group than in the control group and the NAA group (p < 0.0001). Additionally, the CRC group had a significantly higher FAMS index compared to all other groups (p < 0.0001).

[IMAGE OMITTED. SEE PDF]

Next, we performed ROC analyses. The area under the ROC curve (AUC) was 0.90 to discriminate between the control/NAA group and the AA/CRC group (Figure 8B) and 0.85 between the control/NAA group and the AA group (Figure 8C). At the specificity points of 80.3%, 86.1%, 91.0%, and 94.3%, the sensitivities for AA were 68.4%, 61.5%, 56.4%, and 42.7%, respectively, and those for stage I CRC were 87.5%, 82.5%, 80.0%, and 75.0%, respectively (Table 4). The sensitivity of the FAMS index at the above specificity points was not statistically different between the left-sided and right-sided tumors (Table 5).

TABLE 4 Diagnostic performance of the FAMS index.

Cutoff point	Control/NAA (n = 122)	AA/CRC (n = 243)	AA (n = 117)	CRC stage I (n = 40)	CRC stage II (n = 37)	CRC stages III–IV (n = 49)
Specificity % (n), 95% CI%	Sensitivity % (n), 95% CI%
0.64	80.3 (98)	73.3–87.4	81.9 (199)	77.1–86.7	68.4 (80)	60.0–76.8	87.5 (35)	73.2–95.8	97.3 (36)	85.8–99.9	98.0 (48)	89.1–99.9
0.74	86.1 (105)	79.9–92.2	75.7 (184)	70.3–81.1	61.5 (72)	52.7–70.4	82.5 (33)	67.2–92.7	97.3 (36)	85.8–99.9	87.8 (43)	75.2–95.4
0.79	91.0 (111)	85.9–96.1	72.0 (175)	66.4–77.7	56.4 (66)	47.4–65.4	80.0 (32)	64.4–90.9	94.6 (35)	81.8–99.3	85.7 (42)	72.8–94.1
0.87	94.3 (115)	88.5–97.7	61.7 (150)	55.6–67.8	42.7 (50)	33.8–51.7	75.0 (30)	58.8–87.3	83.8 (31)	68.0–93.8	79.6 (39)	65.7–89.8

TABLE 5 Sensitivity of the FAMS index for right- and left-sided colorectal tumors.

Cutoff point	NAA	p	AA	p	CRC	p
Left (n = 20)	Right (n = 23)	Left (n = 46)	Right (n = 71)	Left (n = 84)	Right (n = 42)
Sensitivity % (n), 95% CI%	Sensitivity % (n), 95% CI%	Sensitivity % (n), 95% CI%
0.64	20.0 (4)	5.7–43.7	30.4 (7)	13.2–52.9	0.5012	60.9 (28)	46.8–75.0	73.2 (52)	62.9–83.5	0.2218	94.0 (79)	86.7–98.0	95.2 (40)	83.8–99.4	1.0000
0.74	10.0 (2)	1.2–31.7	21.7 (5)	7.5–43.7	0.4205	56.5 (26)	42.2–70.8	64.8 (46)	53.7–75.9	0.4377	88.1 (74)	79.2–94.1	90.5 (38)	77.4–97.3	0.7728
0.79	5.0 (1)	0.1–24.9	17.4 (4)	5.0–38.8	0.3508	50.0 (23)	35.6–64.4	60.6 (43)	49.2–71.9	0.3401	86.9 (73)	79.7–94.1	85.7 (36)	71.5–94.6	1.0000
0.87	5.0 (1)	0.1–24.9	13.0 (3)	2.8–33.6	0.6105	41.3 (19)	27.1–55.5	43.7 (31)	32.1–55.2	0.8498	78.6 (66)	69.8–87.3	81.0 (34)	65.9–91.4	0.8193
Cutoff point	Stage I CRC	p	Stages II CRC	p	Stages III–IV CRC	p
Left (n = 27)	Right (n = 13)	Left (n = 24)	Right (n = 13)	Left (n = 33)	Right (n = 16)
Sensitivity % (n), 95% CI%	Sensitivity % (n), 95% CI%	Sensitivity % (n), 95% CI%
0.64	85.2 (23)	66.3–95.8	92.3 (12)	64.0–99.8	1.0000	100.0 (24)	85.8–100.0	92.3 (12)	64.0–99.8	0.3514	97.0 (32)	84.2–99.9	100.0 (16)	79.4–100.0	1.0000
0.74	77.8 (21)	57.7–91.4	92.3 (12)	64.0–99.8	0.3930	100.0 (24)	85.8–100.0	92.3 (12)	64.0–99.8	0.3514	87.9 (29)	71.8–96.6	87.5 (14)	61.7–98.4	1.0000
0.79	77.8 (21)	57.7–91.4	84.6 (11)	54.6–98.1	1.0000	100.0 (24)	85.8–100.0	84.6 (11)	54.6–98.1	0.1171	84.8 (28)	68.1–94.9	87.5 (14)	61.7–98.4	1.0000
0.87	70.4 (19)	49.8–86.2	84.6 (11)	54.6–98.1	0.4507	87.5 (21)	67.6–97.3	76.9 (10)	46.2–95.0	0.6435	78.8 (26)	61.1–91.0	81.3 (13)	54.4–96.0	1.0000

DISCUSSION

Methylated SST

In this study, the methylation level of SST was significantly higher in colorectal tumor tissue than in the adjacent normal mucosa (Figure 3). This finding corresponded with the report of other investigators.¹⁷ As shown in Figure 5A, there was a positive correlation of the methylated SST ratio between the fecal sample and the tumor tissue, but the correlation was weak (R² = 0.5589). This could be due to the small sample size (n = 40) and differences in the size, location, growth type, and tumor cell fraction in each tumor as well as to differences in the degree of normal human cell contamination in each fecal sample, which would affect the fecal SST methylation ratio. Nevertheless, we found that the positive fecal DNA test group tended toward a higher SST methylation level in the tumor tissue compared to the negative fecal DNA test group (p = 0.0899, Figure 5B). We thought that the fecal DNA test would reflect the SST methylation level in the colorectal tumor to some extent and that the fecal DNA test combined with FIT would be applicable for the detection of colorectal neoplasia. Therefore, we conducted the following studies to investigate the diagnostic performance of the combined tests.

FIT/fecal DNA testing (combination test)

Advanced colorectal adenoma

The sensitivity for AA of FIT alone and the fecal DNA test alone was 29.1% and 39.3%, respectively (Table 2). Interestingly, the two tests complemented each other, with the combination test resulting in a sensitivity of 61.5%, showing 32.4% and 22.2% higher sensitivities compared with those of FIT and the fecal DNA test, respectively (Table 2, Figure 6). These results suggest that the combination test is useful for improving the detection rate of AA.

Colorectal cancer

Of the 126 patients with CRC, the number of positives for each test was 115 (91.3%) for FIT, 36 (28.6%) for the methylated SST copy number test, and only 4 (3.2%) for the SST methylation ratio test (Figure 7). The extremely low positive rate of the SST methylation ratio test appears to be due to the relatively higher increase in hTERT copy number compared with methylated SST (Figure 4B,C). The elevated copy number of hTERT in feces from patients with CRC may indicate an increase in the amount of human DNA present in the fecal samples. Some of the human DNA may be derived from inflammatory cells and epithelial cells from CRC and the adjacent mucosa as chronic inflammation plays a role in the development and progression of CRC.¹⁸ Additionally, white blood cells from CRC oozing or bleeding may influence the increased amount of human DNA, which would result in an increase in hTERT copies.

FAMS index

As described above, the combination test showed a moderately high sensitivity (61.5%) for AA, but the specificity of approximately 80% seems insufficient for clinical use (Table 2, Figure 6). To address this issue, we performed multivariate analysis to determine predictive factors for AA/CRC. We found FIT, age, and the fecal DNA test to be the independent predictors (Table 3). We then established a prediction model, the FAMS index, which showed a considerably high AUC, reaching 0.90, to discriminate between the control/NAA group and the AA/CRC group (Figure 8B). Further, the AUC was still high (0.85) even between the control/NAA group and the AA group (Figure 8C). In addition, the sensitivity of the FAMS index was not statistically different between the left- and right-sided tumors (Table 5), suggesting that the FAMS index would be useful to detect AA and CRC regardless of their location. This is a great advantage of the FAMS index as compared to a single use of FIT, which is less sensitive for right-sided than for left-sided AA/CRC (Table 2).

Comparison of the diagnostic performance of the FAMS index to other tests showed the FAMS index to have a sensitivity of 56.4% for detecting AA at a specificity point of 91.0% (Table 4), indicating a sensitivity of approximately twice that of FIT (sensitivity 29.1%, specificity 89.3%, Table 2). The FAMS index also had better sensitivity for AA compared with the combination test: 68.4% with the FAMS index vs. 61.5% with the combination test at almost the same specificity point of about 80% (Tables 2 and 4). Compared with Cologuard, which had a sensitivity of 42.4% for advanced precancerous lesions and a specificity of 86.6%,⁵ the FAMS index had a similar sensitivity of 42.7% for AA but a higher specificity of 94.3%. This report is the first to date to demonstrate the utility of FAMS index for the detection of AA. However, in terms of diagnostic performance, we cannot directly compare the FAMS index and Cologuard because Cologuard is not available in Japan. In addition, this study included a limited sample size of both asymptomatic and symptomatic subjects, whereas the Cologuard study included a large sample size (n = 9989) of only asymptomatic subjects.⁵ To compare diagnostic performance between the FAMS index and Cologuard, clinical performance testing will need to be performed outside of Japan.

The FAMS index and Cologuard have in common that they both combine FIT and fecal DNA testing to detect colorectal tumors. For gene testing, Cologuard separately measures mutations of KRAS, methylation of BMP3 and NDRG4, and the copy number of ACTB.⁵ In contrast, the FAMS index simultaneously counts the methylated SST copy number and hTERT copy number, making the FAMS index simpler and less labor intensive. Further, Cologuard requires the entire amount of defecation per bowel movement (estimated to be approximately 200 g),⁵ whereas our fecal DNA test requires only 0.2 g, allowing for hygienic transportation and less labor in processing the fecal samples, which results in lower costs.

Limitations of this study include the following. First, because the cutoff values for the methylated SST copy number and the ratio of methylated SST to hTERT were determined using a single cohort, the sensitivity and specificity may differ to some extent from those in other cohorts. Therefore, further studies in independent cohorts are needed to confirm the diagnostic performance of the fecal DNA testing. Second, because SST hypermethylation has been reported in cancers of various organ sites,¹⁷ a positive result of fecal DNA testing of SST methylation may reflect the presence of a malignancy other than CRC. However, we could not determine which type of cancer would be detected by the fecal DNA test because we focused only on colorectal tumors in this study. Therefore, further studies are needed to address this issue.

In conclusion, the FAMS index may be a promising test for the diagnosis of AA. However, as this was a training study and a validation study has not yet been performed, a confirmatory study with a larger sample size is required to clarify its diagnostic performance.

AUTHOR CONTRIBUTIONS

Yukari Inoue: Data curation; formal analysis; investigation; writing – original draft. Akiyo Ishiguro: Data curation; formal analysis; investigation; writing – original draft. Yutaka Suehiro: Conceptualization; funding acquisition; methodology; project administration; supervision; writing – review and editing. Yuki Kunimune: Formal analysis. Yuko Yamaoka: Data curation; resources. Shinichi Hashimoto: Data curation; resources. Katsuhiko Nakamura: Data curation; resources. Atsushi Goto: Data curation; resources. Koichi Hamabe: Data curation; resources. Toshihiko Matsumoto: Data curation; resources. Shinobu Tomochika: Data curation; resources. Shingo Higaki: Data curation; resources. Ikuei Fujii: Data curation; resources. Chieko Suzuki: Data curation; resources. Michiko Koga: Data curation; resources. Takeya Tsutsumi: Data curation; resources; supervision. Lay Ahyoung Lim: Data curation; resources. Yasuo Matsubara: Data curation; resources. Hiroshi Yotsuyanagi: Resources; supervision. Hiroaki Nagano: Resources; supervision. Naoki Yamamoto: Data curation; resources. Isao Sakaida: Supervision. Taro Takami: Supervision. Mitsuaki Nishioka: Formal analysis. Takahiro Yamasaki: Supervision; writing – review and editing.

ACKNOWLEDGMENTS

We are very grateful to Ms. Tomoe Senkoji of the Institute of Medical Science, the University of Tokyo, for her invaluable help in collecting the clinical data and samples. We are also very grateful to Dr. Kiyoshi Ichihara of Yamaguchi University Graduate School of Medicine for his invaluable help in analyzing the data.

FUNDING INFORMATION

This study was supported by JSPS KAKENHI grant no. 25460687.

CONFLICT OF INTEREST STATEMENT

Yutaka Suehiro and Takahiro Yamasaki received grants from Eiken Chemical. The other authors have no conflict of interest.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: This study was conducted according to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Yamaguchi University (H29-228).

Informed Consent: Informed consent was obtained from all of the subjects.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: N/A.

References

Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209‐249.

Pawa N, Arulampalam T, Norton JD. Screening for colorectal cancer: established and emerging modalities. Nat Rev Gastroenterol Hepatol. 2011;8:711‐722.

Allison JE, Fraser CG, Halloran SP, Young GP. Population screening for colorectal cancer means getting FIT: the past, present, and future of colorectal cancer screening using the fecal immunochemical test for hemoglobin (FIT). Gut Liver. 2014;8:117‐130.

Pan Y, Zhang L, Zhang R, et al. Screening and diagnosis of colorectal cancer and advanced adenoma by bionic glycome method and machine learning. Am J Cancer Res. 2021;11:3002‐3020.

Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal‐cancer screening. N Engl J Med. 2014;370:1287‐1297.

Morikawa T, Kato J, Yamaji Y, Wada R, Mitsushima T, Shiratori Y. A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology. 2005;129:422‐428.

Penz D, Waldmann E, Hackl M, et al. Colorectal cancer and precursor lesion prevalence in adults younger than 50 years without symptoms. JAMA Netw Open. 2023;6: [eLocator: e2334757].

Suehiro Y, Hashimoto S, Goto A, et al. Fecal DNA testing of TWIST1 methylation identifies patients with advanced colorectal adenoma missed by fecal immunochemical test for hemoglobin. Clin Transl Gastroenterol. 2020;11: [eLocator: e00176].

Suehiro Y, Zhang Y, Hashimoto S, et al. Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia. Ann Clin Biochem. 2018;55:59‐68.

Yamaoka Y, Sasai M, Suehiro Y, et al. Comparison of two primer‐probe sets of Fusobacterium nucleatum using droplet digital polymerase chain reaction for the detection of colorectal neoplasia from faecal samples. Ann Clin Biochem. 2022;59:396‐403.

Regula J, Rupinski M, Kraszewska E, et al. Colonoscopy in colorectal‐cancer screening for detection of advanced neoplasia. N Engl J Med. 2006;355:1863‐1872.

Winawer SJ, Zauber AG. The advanced adenoma as the primary target of screening. Gastrointest Endosc Clin N Am. 2002;12(1–9):v‐9.

Brierley JD, Gospodarowicz MK, Wittekind C. TNM Classification of Malignant Tumours. 8th ed. Wiley‐Blackwell; 2017:xviii, 253.

Fraser CG, Allison JE, Halloran SP, Young GP. A proposal to standardize reporting units for fecal immunochemical tests for hemoglobin. J Natl Cancer Inst. 2012;104:810‐814.

Chiang TH, Chuang SL, Chen SLS, et al. Difference in performance of fecal immunochemical tests with the same hemoglobin cutoff concentration in a nationwide colorectal cancer screening program. Gastroenterology. 2014;147:1317‐1326.

Suehiro Y, Suenaga S, Kunimune Y, et al. CA19‐9 in combination with methylated HOXA1 and SST is useful to diagnose stage I pancreatic cancer. Oncology. 2022;100:674‐684.

Manoochehri M, Wu Y, Giese NA, et al. SST gene hypermethylation acts as a pan‐cancer marker for pancreatic ductal adenocarcinoma and multiple other tumors: toward its use for blood‐based diagnosis. Mol Oncol. 2020;14:1252‐1267.

Shimomura K, Hattori N, Iida N, et al. Sleeping beauty transposon mutagenesis identified genes and pathways involved in inflammation‐associated colon tumor development. Nat Commun. 2023;14:6514.