Clinical Significance of Fusobacterium nucleatum and Microsatellite Instability in Evaluating Colorectal Cancer Prognosis

Figures & data

Figure 1 (A) Gram stain: Gram-negative bacteria appear red when stained. Original magnification 1000×. (B) F. nucleatum was positive by immunohistochemistry, original magnification 1000×. C-P: Immunohistochemical detection. Original magnification 400×. (C) Negative F. nucleatum expression in paracancerous tissue, (D) positive F. nucleatum expression in paracancerous tissue, (E) negative F. nucleatum expression in cancer tissue, (F) positive F. nucleatum expression in cancer tissue, (G) negative MLH-1 expression, (H) positive MLH-1 expression, (I) negative MSH2 expression, (J) positive MSH2 expression, (K) negative PMS2 expression, (L) positive PMS2 expression, (M) negative MSH6 expression, (N) positive MSH6 expression, (O) negative HER-2 expression, (P) positive HER-2 expression.

Table 1 Clinical Features of CRC Patients

Factors	No. of the Patients	Percentage,%
Sex
Male	101	54.9%
Female	83	45.1%
Age (years)
<60	85	46.2%
≥60	99	53.8%
Tumor location
Proximal colon	47	25.5%
Distal colon	62	33.7%
Rectum	75	40.8%
TNM Stages
Stage I	21	11.4%
Stage II	95	51.6%
Stage III	26	14.1%
Stage IV	42	22.9%
HER-2
Negative	68	39.8%
Positive +	67	39.2%
Positive ++	36	21.0%
CD31
Negative	25	14.5%
positive	147	85.5%
D2-40
Negative	25	14.5%
Positive	147	85.5%
Syn
Negative	155	85.6%
Positive	26	14.4%
CgA
Negative	159	87.8%
Positive	22	12.2%

Abbreviations: CRC, colorectal cancer; HER-2, human epidermal growth factor receptor-2; Syn, synaptophysin; CgA, chromogranin A; TNM, Tumor-node-metastasis stage was determined according to the latest guidelines of the Union for International Cancer Control (8th edition).

Table 2 Clinical and Molecular Features of CRC According to Microsatellite Status n (%)

Factors(n)	MSI (n=22)	MSS (n=162)	χ^2	df	P value
Sex			1.783	1	0.182
Male(101)	15 (14.9)	86 (85.1)
Female(83)	7 (8.4)	76 (91.6)
Age			0.701	1	0.402
<60 (85)	12 (14.1)	73 (85.9)
≥60 (99)	10 (10.1)	89 (89.9)
Tumor location			11.659	2	0.003**
Proximal colon(47)	12 (25.5)	35 (74.5)
Distal colon(62)	6 (9.7)	56 (90.3)
Rectum(75)	4 (5.3)	71 (94.7)
TNM Stages			8.633	3	0.035*
Stage I (21)	0 (0)	21 (100)
Stage II(95)	11 (11.6)	84 (88.4)
Stage III (26)	7 (26.9)	19 (73.1)
Stage IV(42)	4 (9.5)	38 (90.5)
HER-2			6.248	2	0.044*
Negative (68)	14 (20.6)	54 (79.4)
Positive +(67)	6 (9.0)	61 (91.0)
Positive ++(36)	2 (5.6)	34 (94.4)
CD31			14.126	1	<0.01**
Negative (25)	9 (36.0)	16 (64.0)
Positive (147)	13 (8.8)	134 (91.2)
D2-40			14.126	1	<0.01**
Negative (25)	9 (36.0)	16 (64.0)
Positive (147)	13 (8.8)	134 (91.2)
Syn			0.297	1	0.586
Negative(155)	18 (11.6)	137 (88.4)
Positive (26)	4 (15.4)	22 (84.6)
CgA			1.778	1	0.182
Negative(159)	22 (13.0)	147 (87.0)
Positive (22)	0 (0)	22 (100)

Note: *p <0.05 and **p<0.01 were considered to be statistically significant.

Abbreviations: df, degree of freedom; CRC, colorectal cancer; HER-2, human epidermal growth factor receptor-2; Syn, synaptophysin; CgA, chromogranin A; MSS, microsatellite stability; MSI, microsatellite instability.

Table 3 Comparison of the Positive Rate of F. nucleatum Infection in CRC Tissues and Adjacent Noncancerous Tissues in Patients with CRC n (%)

		Carcinoma Tissues		χ^2	df	P value
		F. nucleatum (-) 60(61.9)	F. nucleatum(+) 37(38.1)
adjacent noncancerous tissues	F. nucleatum (-) 74(76.3)	60(81.1)	14(18.9)	48.89	1	<0.01**
	F. nucleatum (+) 23(23.7)	0(0)	23(100)

Note: ** p<0.01 were considered to be statistically significant.

Abbreviations: df, degree of freedom; CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum.

Table 4 F. nucleatum Infection in CRC Tissues and Adjacent Noncancerous Tissues in CRC Patients with Different TNM Stages

	Carcinoma Tissues		df	P value	Adjacent Noncancerous Tissues		df	P value
	F. nucleatum (-)	F. nucleatum (+)			F. nucleatum(-)	F. nucleatum (+)
TNM stages			3	0.452			3	0.633
Stage I	13	5			15	3
Stage II	17	13			21	9
Stage III	13	5			15	3
Stage IV	17	14			23	8

Abbreviations: df, degree of freedom; CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum.

Table 5 Clinical and Molecular Features of CRC According to F. nucleatum Infection

Factors (n = 97)	F. nucleatum		df	X^2	P value
	Negative(n=60)	Positive(n=37)
Sex			1	1.773	0.183
Male(58)	39(67.2)	19(32.8)
Female(39)	21(53.8)	18(46.2)
Age			1	0.047	0.829
<60 (38)	23(60.5)	15(39.5)
≥60 (59)	37(62.7)	22(37.3)
Tumor location			2	0.614	0.736
Proximal colon(27)	18(66.7)	9(33.3)
Distal colon(30)	17(56.7)	13(43.3)
Rectum(40)	25(62.5)	15(37.5)
TNM stages			3	2.629	0.452
Stage I(48)	13(72.2)	5(27.8)
Stage II(48)	17(56.7)	13(43.3)
Stage III(48)	13(72.2)	5(27.8)
Stage IV(49)	17(54.8)	14(45.2)
HER-2			1	0.029	0.866
Negative(43)	27(62.8)	16(37.2)
Positive(54)	33(61.1)	21(38.9)
CD31			1	1.387	0.239
Negative (25)	13(52.0)	12(48.0)
Positive(72)	47(65.3)	25(34.7)
D2-40			1	1.387	0.239
Negative(25)	13(52.0)	12(48.0)
Positive(72)	47(65.3)	25(34.7)
Syn			1	1.403	0.236
Negative(81)	48(59.3)	33(40.7)
Positive(16)	12(75.0)	4(25.0)
CgA			1	0.736	0.391
Negative(92)	56(60.9)	36(39.1)
Positive(5)	4(80.0)	1(20.0)

Abbreviations: df, degree of freedom; CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2;Syn, synaptophysin;CgA, chromogranin A.

Table 6 Correlation Analysis of Clinical-Pathological Indices with CRC

	Sex	Age	F. nucleatum	MSI	HER-2	CD31	D2-40	TNM stages
Age	r=0.098 p=0.336
F. nucleatum	r=0.087 p=0.396	r=−0.049 p=0.630
MSI	r=−0.093 p=0.364	r=−0.120 p=0.239	r=−0.013 p=0.902
HER-2	r=0.182 p=0.075	r=0.049 p=0.631	r=−0.039 p=0.701	r=−0.21* p=0.039
CD31	r=0.051 p=0.620	r=0.107 p=0.297	r=−0.004 p=0.969	r=−0.187 p=0.066	r=0.138 p=0.175
D2-40	r=0.051 p=0.620	r=0.107 p=0.297	r=−0.004 p=0.969	r=−0.187 p=0.066	r=0.138 p=0.175	r=1.00 p<0.001**
TNM stages	r=0.001 p=0.991	r=−0.005 p=0.960	r=−0.026 p=0.781	r=−0.018 p=0.851	r=−0.196 p=0.036*	r=−0.252 p=0.007**	r=−0.252 p=0.007**
Tumor Location	r=−0.046 p=0.651	r=−0.186 p=0.069	r=−0.073 p=0.474	r=−0.254 p=0.013*	r=0.115 p=0.259	r=0.158 p=0.121	r=0.158 p=0.121	r=−0.250 p=0.007**

Notes: Age is divided into two levels: ≥60 and <60. *p <0.05 and **p<0.01 were considered to be statistically significant.

Abbreviations: CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2; MSI, microsatellite instability.

Table 7 Receiver Operating Characteristic Curve Analysis of Parameters

Variable	AUC	Std. Error	95% CI	P value	Sensitivity(%)	Specificity (%)
Age	0.711	0.053	0.607–0.815	0.001**	88.2	54.0
TNM stages	0.677	0.057	0.566–0.789	0.004**	73.5	61.9
F. nucleatum	0.591	0.061	0.471–0.711	0.139	50.0	68.3
Sex	0.530	0.062	0.409–0.651	0.626	44.1	61.9
MSI	0.529	0.062	0.407–0.651	0.637	26.5	79.4
CgA	0.506	0.506	0.384–0.582	0.928	5.9	95.2
Syn	0.464	0.061	0.35–0.582	0.555	11.8	81.0
CD31	0.449	0.062	0.327–0.571	0.412	67.6	22.2
D2-40	0.449	0.062	0.327–0.571	0.412	67.6	22.2
HER-2	0.411	0.061	0.292–0.531	0.150	44.1	38.1
Tumor location	0.364	0.058	0.250–0.477	0.027*	23.5	49.2

Notes: AUC indicates the ability of the biomarkers and combined components to analyze prognosis; *p <0.05 and **p<0.01 were considered to be statistically significant.

Abbreviations: AUC area under the curve; Std, standard; 95% CI, 95% confidence interval; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2; MSI, microsatellite instability; Syn, synaptophysin; CgA, chromogranin A.

Table 8 Cox Regression Analysis of Prognostic Factors in Patients with CRC

Clinical Variables	B	SE	Wald	df	HR	95% CI	P value
	Univariate cox analysis
Sex(male vs female)	−0.293	0.347	0.711	1	0.746	0.378–1.473	0.399
Age(≥60 vs <60)	1.889	0.534	12.513	1	0.151	0.053–0.431	<0.001**
Tumor location (Colon vs Rectum)	1.128	0.411	7.546	1	3.089	1.382–6.909	0.006**
TNM stages(Stage III-IV vs Stage I-II)	1.213	0.393	9.526	1	3.364	1.557–7.268	0.002**
MSI status(MSI vs MSS)	0.084	0.391	0.046	1	1.088	0.505–2.342	0.830
F. nucleatum (Positive vs Negative)	0.690	0.345	4.0	1	1.994	1.014–3.922	0.045*
HER-2(Positive vs Negative)	0.575	0.346	2.758	1	0.563	0.286–1.109	0.097
CD31(Positive vs Negative)	0.382	0.370	11.064	1	1.465	0.7709–3.627	0.302
D2-40(Positive vs Negative)	0.382	0.370	11.064	1	1.465	0.7709–3.627	0.302
CgA(Positive vs Negative)	0.256	0.732	0.122	1	0.774	0.184–3.251	0.122
Syn(Positive vs Negative)	0.470	0.533	0.777	1	1.600	00.563–4.548	0.378
	Multivariate Cox analysis
Sex(male vs Female)	0.105	0.418	0.063	1	1.110	0.490–2517	0.802
Age(≥60 vs <60)	2.080	0.567	13.472	1	0.125	0.041–0.379	<0.001**
Tumor location (Colon vs Rectum)	0.965	0.489	3.891	1	2.626	1.006–6.852	0.049*
TNM stages(Stage III-IV vs Stage I-II)	1.311	0.438	8.939	1	0.270	0.114–0.637	0.003**
MSI status(MSI vs MSS)	0.507	0.454	1.246	1	0.602	0.247–1.467	0.264
F. nucleatum (Positive vs Negative)	0.861	0.426	4.085	1	0.423	0.183–0.974	0.043*

Notes: *p <0.05 and **p<0.01 were considered to be statistically significant.

Abbreviations: B, beta; SE, standard error; df, degree of freedom; HR, hazard ratio;95% CI, 95% confidence interval; CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2; MSI, microsatellite instability; MSS, microsatellite stability; Syn, synaptophysin; CgA, chromogranin A.

Table 9 Univariate Analysis of the Impact of Possible Risk Factors on Disease-Free Survival in CRC Patients

Factors	DFS(Weeks±SD)	df	P value
Sex		1	0.395
Male	(86.14±10.864)
Female	(70.67±11.368)
Age		1	<0.001**
<60	(61.69±7.547)
≥60	(108.21±15.605)
Tumor location		1	0.004*
Colon	(64.07±8.409)
Rectum	(102.50±14.476)
TNM stages		1	0.001**
Stage I-II	(96.15±13.295)
Stage III-IV	(64.02±8.338)
MSI status		1	0.829
MSS	(78.16±9.088)
MSI	(85.91±16.637)
F. nucleatum		1	0.041*
Negative	(87.77±10.466)
Positive	(67.19±11.940)
HER-2		1	0.091
Negative	(70.47±10.831)
Positive	(87.91±16.637)
CD31		1	0.297
Negative	(70.52±13.744)
Positive	(83.18±9.599)
D2-40		1	0.297
Negative	(70.52±13.744)
Positive	(83.18±9.599)
CgA		1	0.371
Negative	(81.21±8.325)
Positive	(56.20±14.263)
Syn		1	0.724
Negative	(78.8±8.693)
Positive	(85.56±20.106)

Note: *p <0.05 and **p<0.01 were considered to be statistically significant.

Abbreviations: DFS, disease-free survival; df, degree of freedom; CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2; MSI, microsatellite instability; MSS, microsatellite stability; Syn, synaptophysin; CgA, chromogranin A.

Figure 2 Kaplan–Meier survival curves were used to evaluate the suitability of these molecules as prognostic factors. The results showed that the DFS in the F. nucleatum-negative group was significantly longer than that in the F. nucleatum-positive group. Older people (>60 years old) had a significantly shorter DFS than younger people (<60 years old), and patients with TNM stage III–IV had significantly a shorter DFS than those with TNM stage I–II. Patients with colon cancer had a shorter DFS than those with rectal cancer.

Table 10 Loading Scores of Variables on the First Three Significant Principal Components That Combined Components for the Analysis of Prognosis

Variable	Component 1	Component 2	Component 3	Component 4
Sex	0.383	0.034	0.797	0.116
Age	0.644	0.154	−0.195	−0.025
Tumor location	0.166	0.384	−0.081	−0.571
TNM stages	0.434	0.324	0.512	−0.327
MSI	−0.825	−0.127	0.098	−0.195
F. nucleatum	−0.012	−0.029	0.091	0.760
HER-2	0.572	0.108	0.599	−0.058
CD31	0.670	0.207	−0.346	−0.114
CgA	−0.012	0.908	−0.060	0.246
Syn	−0.431	0.767	−0.060	0.267

Notes: Bold values indicate strong and moderate loadings: Component 1 has strong positive loadings on Age, HER-2, CD31 and negative loadings on MSI; Component 2 has positive loadings on CgA, Syn; Component 3 has positive loadings on Sex,TNM stages and HER-2;Component 4 has positive loadings on F. nucleatum and negative loadings on Tumor location.

Abbreviations: CRC, colorectal cancer; F. nucleatum, Fusobacterium nucleatum; HER-2, human epidermal growth factor receptor-2; MSI, microsatellite instability; Syn, synaptophysin; CgA, chromogranin A.

Table 11 Receiver Operating Characteristic Curve Analysis of Components

Variable	AUC	Std. Error	95% CI	P value	Sensitivity(%)	Specificity(%)
Component 1	0.537	0.063	0.414–0.660	0.550	61.8	57.1
Component 2	0.613	0.057	0.500–0.725	0.068	76.5	50.8
Component 3	0.523	0.068	0.390–0.655	0.711	58.8	20.6
Component 4	0.415	0.065	0.288–0.541	0.166	58.8	14.3

Notes: AUC indicates the ability of the biomarkers and combined components to analyze prognosis.

Abbreviations: AUC, area under the curve; Std, standard; 95% CI, 95% confidence interval.