Pre-S1 Mutations as Indicated by Serum Pre-S1 Antigen Negative is Associated with an Increased Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients

Figures & data

Table 1 Association of Pre-S1 Single Test Result with Clinical Parameters

Variables	Pre-S1 Negative (N=305)	Pre-S1 Positive (N=535)	Z/χ^2/t value	P value
	No. (%); M (P25~P75)
Age (years)			7.101	0.029
<40	36(11.8%)	45(8.4%)	2.565
40~60	182(59.7%)	293(54.8%)	1.903
>60	87(28.5%)	197(36.8%)	5.977
Genders			0.368	0.544
Male	252(82.6%)	433(80.9%)
Female	53(17.4%)	102(19.1%)
HBV markers
HBsAg (COI)	1232(237~3236)	5773(3282~6984)	−15.983	<0.001
HBeAg positive/HBeAg negative	38(12.5%)/267(87.5%)	138(25.8%)/397(74.2%)	20.858	<0.001
^†HBVDNA positive/HBV DNA negative	122(42.7%)/164(57.3%)	238(46.6%)/273(53.4%)	1.137	0.286
Liver function
ALT (U/L)	31(20~49)	31(20~52)	−0.310	0.757
AST (U/L)	38(24~61)	36(25~57)	0.245	0.807
ALP (U/L)	98(73~139)	97(72~36)	0.614	0.539
GGT (U/L)	62(31~138)	63(32~147)	−0.212	0.832
ALB (g/L)	39(36~53)	41(37~44)	−2.088	0.037
Blood fat
TC (mmol/L)	3.95(3.26~4.57)	3.91(3.31~4.62)	−0.596	0.551
TG (U/L)	1.00(0.73~1.43)	0.96(0.69~1.39)	0.698	0.485
Blood routine
Hb (g/dl)	11.15(10.00~13.03)	10.95(9.35~12.5)	1.448	0.149
PLT (×10^9/L)	124(77~185)	132(82~188.5)	−1.120	0.263
Coagulation function
PT (s)	12.4 (11.6~13.9)	12.5 (11.8~13.6)	−0.552	0.581
APTT (s)	28.7 (26.8~30.8)	28.5 (27.1~30.6)	0.114	0.909
Tumor markers
AFP (ng/mL)	9.2(2.5~214.75)	13.6(3~269.2)	−1.242	0.214
CEA (ng/mL)	2.52(1.66~3.9)	2.4(1.6~3.48)	1.029	0.304
CA19-9 (ng/mL)	17.6(9.1~38.1)	18.2(10.3~39.8)	−0.764	0.445
Liver cirrhosis
With/without cirrhosis	136(44.6%)/169(55.4%)	270(50.5%)/265(49.5%)	2.687	0.101
Liver function (Child-Pugh grade)
A^#	259(84.9%)	484(90.4%)	/	0.041
B	43(14.1%)	48(9.0%)
C	3(1.0%)	3(0.6%)
HCC
With/without HCC	253(83.0%)/52(17.0%)	419(78.3%)/116(21.7%)	2.606	0.106

Notes: No.: number of patients who had single pre-S1 test with these characteristics. M (P₂₅~P₇₅) refered to medium (25th percentile~75th percentile). Non-normal distribution measurement data were analyzed by Wilcoxon rank sum test. The measurement data of ALB and Hb that conform to normal distribution after variable transformation were analyzed by t-test. Counting data of HBeAg positive and HBV DNA positive (number/total number) were analyzed by Chi-square test. The bold font indicates P-values less than 0.05. ^†The data of HBV DNA is partially missing. ^#Child-Pugh A group included CHB patients scored<=6 without liver cirrhosis.

Table 2 Association of Pre-S1 Single Test Result with Other HBV Markers

Variables	Pre-S1 Negative	Pre-S1 Positive	χ^2 value	P value
	No. (%)
HBsAg (COI)	N=305	N=535	317.963	<0.001*
<1000	140 (45.9%)	11 (2.0%)
1000~2000	50 (16.4%)	37 (6.9%)
2000~5000	70 (22.9%)	174 (32.5%)
>5000	45 (14.8%)	313 (58.5%)
^†HBV DNA (IU/mL)	N=286	N=511	15.745	<0.001*
~1E+04	259 (90.6%)	411 (80.4%)
1E+04~1E+06	21 (7.3%)	62 (12.1%)
1E+06~1E+08	6 (2.1%)	38 (7.4%)

Notes: No.: number of patients who had single pre-S1 test with these characteristics. The bold font indicates P-values less than 0.05. *P<0.001 after chi-square test and Bonferroni correction. ^†The data of HBV DNA is partially missing.

Table 3 Single and Multivariate Analysis for the Association of Clinical Factors with HCC Risk in Chronic HBV Infected Patients

Variables in HCC Patients (N=672)	Patients with Pre-S1 Test (N=840)	Single Factor Analysis		Multivariate Analysis
	No.(%)/No.(%)	χ^2	P	Z	P	OR
Pre-S1 (negative/positive)	253(30.1%)/419(49.9%)	2.606	0.106	−2.00	0.045	1.61
Cirrhosis (with/without)	313(37.3%)/359(42.7%)	4.148	0.042	−2.47	0.013	1.59
Age (≥50/<50)	475(56.5%)/197(23.5%)	5.504	0.019	2.61	0.009	1.02
Gender (male/female)	579(68.9%)/93(11.1%)	47.518	<0.001	6.70	<0.001	4.04
HBsAg (COI)	/	0.050	0.960	1.45	0.147	1.00
HBeAg (positive/negative)	137(16.3%)/535(63.7%)	0.649	0.421	−0.94	0.345	0.81
ALT (U/L)	/	−0.187	0.852	1.10	0.273	1.00
Child Pugh (A^#+B/C)	601(71.5%)/71(8.5%)	3.173	0.075	−1.81	0.071	0.64
^†HBV DNA (negative/positive)	359(42.7%)/282(33.6%)	2.036	0.154	−1.04	0.300	0.82

Notes: No.: number of HCC patients who had single pre-S1 test with these characteristics. ^#Child-Pugh A group included CHB patients without liver cirrhosis which scored <=6. The bold font indicates P-values less than 0.05. ^†The data of HBV DNA is partially missing.

Table 4 The Association Between Four Pre-S1 Follow-Up Modes with Cirrhosis and HCC, and Antiviral Treatment

Variables	Patients with Multiple Pre-S1 Tests N=143				χ^2*	P	χ^2	P
	No.(%)
	Negative-Negative (N=33)	Positive-Positive (N=94)	Positive-Negative (N=6)	Negative-Positive (N=10)
Cirrhosis	17(51.5%)	44(46.8%)	1(16.7%)	5(50.0%)	2.524	0.471
HCC	16(48.5%)	23(24.5%)	1(16.7%)	4(40.0%)	-	0.054*	6.621	0.010^#
Antiviral therapy/Total	14(42.4%)	53(56.4%)	1(16.7%)	5(50.0%)	4.895	0.180

Notes: No.: number of patients who had multiple pre-S1 tests with these characteristics. The bold font indicates P-values less than 0.05. Correlation of sustained pre-S1 serological status with HCC, *P=0.054 after chi-square test, ^#P=0.010 after Bonferroni correction, respectively.

Table 5 Single and Multivariate Analysis for the Association of Clinical Factors with HCC Risk in Chronic HBV Infected Patients with Sustained Pre-S1 Test Results

Variables in HCC Patients (N=39)	Patients with Sustained Pre-S1 Test Results (N=127)	Single Factor Analysis		Multivariate Analysis
	No.(%)/No.(%)	χ^2/Z	P	Z	P	OR
Pre-S1(sustained negative/ sustained positive)	16(12.6%)/23(18.1%)	6.621	0.010	−2.56	0.011	7.12
Cirrhosis (with/without)	28(22.0%)/11(8.7%)	12.365	<0.001	1.81	0.022	4.61
Age (≥50/<50)	26(20.5%)/13(10.2%)	8.519	0.004	1.87	0.062	1.05
Gender (male/female)	29(22.8%)/10(7.9%)	0.679	0.410	0.88	0.378	1.77
HBsAg (COI)	/	−2.112	0.035	1.83	0.068	1.00
HBeAg (positive/negative)	8(6.3%)/30(23.6%)	1.467	0.226	−0.34	0.735	0.79
ALT (U/L)	/	−0.050	0.960	/	/	/
Child Pugh (A^#+B/C)	37(29.1%)/2(1.6%)	0.199	0.655	−1.01	0.312	0.23
HBV DNA (negative/positive)	26(20.5%)/12(9.4%)	1.677	0.195	0.17	0.868	1.11
Antiviral treatment (effective/ ineffective or without treatment)	28(22.0%)/11(8.7%)	8.186	0.004	1.62	0.105	2.71

Notes: No.: number of patients who had HCC with these characteristics. ^#Child-Pugh A group included CHB patients without liver cirrhosis which scored <=6. The bold font indicates P-values less than 0.05.

Table 6 Information of Pre-S1 Sequence Results of Pre-S1 Negative and Positive, Treatment Naïve Patients

Information	Patient 1	Patient 2	Patient 3
Gender	Male	Male	Female
Age (years)	43	48	62
Liver cirrhosis	Yes	Yes	Yes
HCC	No	No	No
Family history of HCC	No	Yes	Yes
Pre-S1 status	Positive	Negative	Negative
HBsAg (COI)	7040	3000	3360
e-antigen	Negative	Positive	Negative
HBV DNA (IU/mL)	8.01E+04	2.02E+06	4.89E+06
Pre-S1 Sequences	D^†	1: C-(V90A) 2: C-frame shift mutation	1: C^† 2: C-deletion mutation (54bp) 3: C-deletion mutation (54bp)

Notes: ^†Reference sequences: type C toxic strain (has not been submitted to GeneBank) and type D toxic strain (GeneBank accession No. V01460.1). All data were obtained at presentation.

Figure 1 Pre-S1 amino acid sequences of pre-S1 positive and Pre-S1 negative treatment naïve patients. (A) The pre-S1 amino acid sequence of pre-S1 positive patient 1 (Seq_1) was consistent with D-type HBV. (B) The sample of pre-S1 negative patient 2 contained two pre-S1 sequences. Compared with C-type HBV, Seq_1 of the pre-S1 amino acid sequence had only one site mutation (V90A); another pre-S1 sequence had frame shift mutation resulting the mutation and premature termination. (C) Sequence analysis showed that the sample of pre-S1 negative patient 3 contained three pre-S1 sequences. Among them, the amino acid sequence of pre-S1 Seq_1 was identical with that of C type HBV; pre-S1 Seq_2 and Seq_3 contained deletion mutations.