Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis

doi:10.3748/wjg.v28.i25.2920

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World Journal of Gastroenterology

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Basic Study

World J Gastroenterol. Jul 7, 2022; 28(25): 2920-2936
Published online Jul 7, 2022. doi: 10.3748/wjg.v28.i25.2920

Table 1 The reaction system

Position	Reagent
Wells 1 and 7	Proteinase K: 20 μL
Wells 1 and 7	Lysate: 200 μL
Wells 2 and 8	Buffer KCL: 750 μL
Wells 3 and 9	Buffer GW1: 750 μL
Wells 4 and 10	Buffer GW2: 750 μL
Wells 5 and 11	Buffer MW3: 750 μL
Wells 6 and 12	Buffer GE: 100 μL

Table 2 Clinical data analysis of colorectal cancer patients

Clinical feature	Stratification	Number of people (%)
Age (years old)	≥ 60	30 (60.00)
Age (years old)	< 60	20 (40.00)
Sex	Male	26 (52.00)
Sex	Female	24 (48.00)
Tumor location	Sigmoid colon + left hemicolon	11 (22.00)
	Right hemicolon	10 (20.00)
	Rectum	29 (58.00)
Tumor size (cm)	≥ 6	12 (24.00)
Tumor size (cm)	< 6	38 (76.00)
Degree of tumor differentiation	High + medium high	12 (24.00)
	Moderately	25 (50.00)
	Low medium + low	12 (24.00)
TNM classification	I + II	33 (66.00)
TNM classification	III + IV	17 (34.00)

Table 3 Summary of gene mutations in colorectal cancer patients

Gene	Tumor tissue		Preoperative feces		Postoperative stools
Gene	Total number of mutations	Mutation frequency	Total number of mutations	Mutation frequency	Total number of mutations	Mutation frequency
TP53	40	62.00% (31/50)	27	59.26% (16/27)	7	31.58% (6/19)
APC	15	24.00% (12/50)	3	11.11% (3/27)	0	0
KRAS	30	58.00% (29/50)	11	37.04% (10/27)	0	0
PIK3CA	13	22.00% (11/50)	1	3.70% (1/27)	2	10.53% (2/19)
FBXW7	5	10.00% (5/50)	1	3.70% (1/27)	0	0
GNAS	2	4.00% (2/50)	1	3.70% (1/27)	0	0
PTEN	11	22.00% (11/50)	1	3.70% (1/27)	0	0
ABL1	0	0	1	3.70% (1/27)	0	0
PDGFRA	12	24.00% (12/50)	8	29.63% (8/27)	6	31.58% (6/19)
ATM	1	2.00% (1/50)	0	0	0	0
SMAD4	1	2.00% (1/50)	0	0	0	0
BRAF	2	4.00% (2/50)	0	0	2	10.53% (2/19)
PTPN11	1	2.00% (1/50)	0	0	0	0
NRAS	1	2.00% (1/50)	0	0	0	0
CTNNB1	3	6.00% (3/50)	1	3.70% (1/27)	0	0
STK11	2	4.00% (2/50)	1	3.70% (1/27)	0	0
AKT1	1	2.00% (1/50)	1	3.70% (1/27)	0	0
CDKN2A	3	6.00% (3/50)	4	7.41% (2/27)	3	5.26% (1/19)
HRAS	21	42.00% (21/50)	12	40.74% (11/27)	8	42.11% (8/19)
EGFR	2	4.00% (2/50)	2	7.41% (2/27)	1	5.26% (1/19)
IDH1	2	4.00% (2/50)	2	7.41% (2/27)	1	5.26% (1/19)
NOTCH1	2	4.00% (2/50)	0	0	2	5.26% (1/19)
VHL	0	0	1	3.70% (1/27)	0	0
KIT	7	14.00% (7/50)	2	7.41% (2/27)	4	21.05% (4/19)
MET	9	18.00% (9/50)	5	18.52% (5/27)	4	21.05% (4/19)
MLH1	3	6.00% (3/50)	2	7.41% (2/27)	2	10.53% (2/19)
MPL	0	0	0	0	1	5.26% (1/19)

Total number of mutations: The overall number of mutations of any gene in various samples. Mutation frequency: The number of people with mutations in various samples/total number of people.

Table 4 Fecal gene mutation results in the control groups

Group	Genes	Location	Amino acid mutation	Mutation frequency
Normal control	PDGFRA	Exon 19	V824V	2
	KIT	Exon 10	K546K	1
	HRAS	Exon 2	H27H	1
Intestinal benign disease	PDGFRA	Exon 19	V824V	3
	HRAS	Exon 2	H27H	1
	KIT	Exon 10	M541L	1
	STK11	Exon 8	F354L	1

Table 5 Comparison of pathogenic mutation sites in preoperative stools vs tumor tissues

Gene	Positive rate of pathogenic gene mutation sites
Gene	Preoperative feces	Tumor tissues
TP53	37.04% (10/27)	46.00% (23/50)
APC	11.11% (3/27)	18.00% (9/50)
KRAS	25.93% (7/27)	54.00% (27/50)
PIK3CA	3.70% (1/27)	22.00% (11/50)
FBXW7	3.70% (1/27)	6.00% (3/50)
GNAS	3.70% (1/27)	4.00% (2/50)
PTEN	3.70% (1/27)	0
ABL1	3.70% (1/27)	0
PDGFRA	3.70% (1/27)	0
ATM	0	2.00% (1/50)
SMAD4	0	2.00% (1/50)
BRAF	0	2.00% (1/50)
PTPN11	0	2.00% (1/50)
NRAS	0	2.00% (1/50)
CTNNB1	0	2.00% (1/50)
VHL	3.70% (1/27)	0

Positive rate of pathogenic gene mutation site: The number of people with mutations corresponding to the pathogenic mutation sites of any gene/total number of samples.

Table 6 Comparison of pathogenic mutation sites in preoperative stools vs normal control stools

Gene	Positive rate of pathogenic gene mutation sites		χ²	P value
Gene	Preoperative feces	Normal control group	χ²	P value
TP53	37.04% (10/27)	0	7.328	0.007
APC	11.11% (3/27)	0	0.878	0.349
KRAS	25.93% (7/27)	0	4.219	0.040
PIK3CA	3.70% (1/27)	0	Fisher exact test	1
FBXW7	3.70% (1/27)	0	Fisher exact test	1
GNAS	3.70% (1/27)	0	Fisher exact test	1
PTEN	3.70% (1/27)	0	Fisher exact test	1
ABL1	3.70% (1/27)	0	Fisher exact test	1
PDGFRA	3.70% (1/27)	0	Fisher exact test	1

Positive rate of pathogenic gene mutation site: The number of people with mutations corresponding to the pathogenic mutation sites of any gene/total number of samples.

Table 7 Comparison of pathogenic mutation sites in preoperative stools vs benign control group

Gene	Positive rate of pathogenic gene mutation sites		χ²	P value
Gene	Preoperative feces	Benign control group	χ²	P value
TP53	37.04% (10/27)	0	7.328	0.007
APC	11.11% (3/27)	0	0.878	0.349
KRAS	25.93% (7/27)	0	4.219	0.040
PIK3CA	3.70% (1/27)	0	Fisher exact test	1
FBXW7	3.70% (1/27)	0	Fisher exact test	1
GNAS	3.70% (1/27)	0	Fisher exact test	1
PTEN	3.70% (1/27)	0	Fisher exact test	1
ABL1	3.70% (1/27)	0	Fisher exact test	1
PDGFRA	3.70% (1/27)	0	Fisher exact test	1

Positive rate of pathogenic gene mutation site: The number of people with mutations corresponding to the pathogenic mutation sites of any gene/total number of samples.

Table 8 Comparison of pathogenic mutation sites in preoperative stools vs postoperative stools

Gene	Positive rate of pathogenic gene mutation sites		χ²	P value
Gene	Preoperative feces	Postoperative stools	χ²	P value
TP53	37.04% (10/27)	0	6.947	0.008
APC	11.11% (3/27)	0	0.804	0.370
KRAS	25.93% (7/27)	0	3.974	0.046
PIK3CA	3.70% (1/27)	5.26% (1/19)	Fisher exact test	1
FBXW7	3.70% (1/27)	0	Fisher exact test	1
GNAS	3.70% (1/27)	0	Fisher exact test	1
PTEN	3.70% (1/27)	0	Fisher exact test	1
ABL1	3.70% (1/27)	0	Fisher exact test	1
PDGFRA	3.70% (1/27)	0	Fisher exact test	1
BRAF	0	5.26% (1/19)	Fisher exact test	1
MPL	0	5.26% (1/19)	Fisher exact test	1

Positive rate of pathogenic gene mutation site: The number of people with mutations corresponding to the pathogenic mutation sites of any gene/total number of sample.

Table 9 Analysis of pathogenic mutation sites in tumor tissues

Gene	Location	Amino acid mutation	Mutation frequency	N	M
TP53	Exon 4	R110L	1
	Exon 5	R175H	4
		Y163C	1
		C176Y	1
	Exon 6	R196¹	2
	Exon 6	Y220C	2
	Exon 7	R248Q/W	3
	Exon 7	G245S/C	2
	Exon 8	R273H/C	4
		R282W	2
		R306¹	1
	Exon 10	R342¹	1	24/40	23/31
KRAS	Exon 2	G12D/V/S	22
	Exon 2	G13D/C	4
	Exon 3	Q61H	1	27/30	27/29
APC	Exon 17	Q886¹	1
		S1483fs	1
		R876¹	2
		R1450¹	2
		E1306¹	1
		Q1294¹	1
		G1312¹	1	9/15	9/12

¹Indicates stop codon.

N: Pathogenic sites/total mutation sites; M: Number of pathogenic cases/total number of mutation cases.

Table 10 Analysis of pathogenic mutation sites in preoperative feces

Gene	Location	Amino acid mutation	Mutation frequency	N	M
TP53	Exon 5	C176Y	1
		F134V	1
	Exon 6	R196¹	1
	Exon 7	N235D	1
		R248Q	3
		G245C/S	2
	Exon 8	R273H/C	3	12/27	10/16
KRAS	Exon 2	G12D/V	5
		G13D	1
	Exon 3	Q61H	1
		A59T	1	8/11	7/10
APC	Exon 17	R1450¹	1
		Q886¹	1
		Q1294¹	1	3/3	3/3

¹Indicates stop codon.

N: Pathogenic sites/total mutation sites; M: Number of pathogenic cases/total number of mutation cases.

Table 11 Colorectal cancer diagnosis results by TP53 and KRAS mutations in preoperative stools, n (%)

Gene	TP	FP	FN	TN	Sensitivity	Specificity	PPV	NPV
TP53	16	0	11	20	59.26	100.00	100.00	64.52
KRAS	10	0	17	20	37.04	100.00	100.00	54.05
TP53 or KRAS	18	0	9	20	66.67	100.00	100.00	68.97

TP: True positive; FP: False positive; FN: False negative; TN: True negative; PPV: Positive predictive value; NPV: Negative predictive value.

Table 12 "Undetected" gene mutation sites in tumor tissues

Gene	Location	Amino acid mutation	Mutation start position	Mutation end position	Number of cases	Undetected sites/total mutation sites
TP53	Exon 4	A84G	7579436	7579436	4
	Exon 5	P152A	7578476	7578476	1
	Exon 8	L289P	7577072	7577072	1	6/40
APC	Exon 17	S1346¹	112175328	112175328	1
		K1573fs	112175953	112175954	1
		E1327fs	112175213	112175217	1	3/15
FBXW7	Exon 7	R278¹	153258983	153258983	1
FBXW7	Exon 12	R266C	153247289	153247289	1	2/5
NOTCH1	Exon 26	R1599P	139399350	139399350	2	2/2
EGFR	Exon 20	I821T	55249164	55249164	1	1/2

¹Indicates stop codon.

fs: Frameshift mutation.

Table 13 "Undetected" gene mutation sites in preoperative feces

Gene	Location	Amino acid mutation	Mutation start position	Mutation end position	Number of cases	Undetected sites/total mutation sites
TP53	Exon 4	A84G	7579436	7579436	1
	Exon 5	S166P	7578434	7578434	1
	Exon 7	N247D	7577542	7577542	3
	Exon 8	L289P	7577072	7577072	1	6/27
CDKN2A	Exon 2	V51A	21971206	21971206	2
CDKN2A	Exon 2	L63P	21971170	21971170	1	3/4
HRAS	Exon 2	G12R	534289	534289	1	1/10
EGFR	Exon 20	I821T	55249164	55249164	1	1/2
IDH1	Exon 4	R119Q	209113151	209113151	1	1/2
PDGFRA	Exon 13	G594fs	55141059	55141059	1	1/8

fs: Frameshift mutation.

Citation: He SY, Li YC, Wang Y, Peng HL, Zhou CL, Zhang CM, Chen SL, Yin JF, Lin M. Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis. World J Gastroenterol 2022; 28(25): 2920-2936
URL: https://www.wjgnet.com/1007-9327/full/v28/i25/2920.htm
DOI: https://dx.doi.org/10.3748/wjg.v28.i25.2920