Diagnostic performance of serum origin recognition complex subunit 1 protein for hepatitis B virus-related hepatocellular carcinoma

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, with hepatitis B virus (HBV) infection serving as a significant etiological factor in endemic regions. Alpha-fetoprotein (AFP), the most commonly used biomarker, has limited sensitivity, particularly in AFP-negative HCC. Recent studies have identified origin recognition complex subunit 1 (ORC1) and extra spindle pole bodies-like 1 (ESPL1) as promising serum biomarkers, both linked to HBV DNA integration, a mechanism known to drive hepatocarcinogenesis.

AIM

To assess serum ORC1’s diagnostic value for HBV-HCC and its link to S gene integration.

METHODS

In this case-control study, 479 HBV-infected patients were enrolled, including 20 with HBV S gene integration, 47 with non-S gene integration, 162 with chronic hepatitis B, 154 with HBV-related cirrhosis, and 96 with HBV-HCC. The control group comprised 73 individuals: 29 with non-HBV-HCC and 44 healthy participants. Serum ORC1 and ESPL1 were measured by enzyme-linked immunosorbent assay. HBV integration sites were identified via whole-genome sequencing. Diagnostic performance was assessed using receiver operating characteristic analysis, including in AFP-negative patients.

RESULTS

HBV integration near the ORC1 locus (chromosome 1p32.3) was detected in 71.4% of HBV-HCC tissues. Serum ORC1 levels were significantly higher in HBV-infected patients than in non-HBV-infected controls (980.11 ng/L vs 746.82 ng/L, P < 0.05) and in HBV-HCC compared with non-HBV-HCC (1077.07 ng/L vs 749.54 ng/L, P < 0.05). Serum ORC1 and ESPL1 were elevated in HBV-HCC regardless of AFP status, and detected 64.8% and 73.2% of AFP-negative cases, respectively. The combined panel of ORC1 [Area under receiver operating characteristic curve (AUC) = 0.587], ESPL1 (AUC = 0.776), and AFP (AUC = 0.844) achieved an AUC of 0.887, significantly higher than any single marker (P < 0.05), with a sensitivity of 84.44%, specificity of 84.19%, and a negative predictive value of 94.91%.

CONCLUSION

Serum ORC1, driven by HBV integration, is a promising biomarker especially for AFP-negative HBV-HCC. Its combination with ESPL1 and AFP significantly improves early detection.

Key Words: Hepatocellular carcinoma; Hepatitis B virus; Origin recognition complex subunit 1; Diagnostic biomarker; Alpha-fetoprotein-negative; Enzyme-linked immunosorbent assay

Core Tip: This study identifies serum origin recognition complex subunit 1 (ORC1) as a novel diagnostic biomarker for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). ORC1 levels were markedly elevated in HBV-HCC patients, especially those negative for alpha-fetoprotein. Mechanistically, recurrent HBV integration near the ORC1 locus appears to drive its aberrant expression, contributing to hepatocarcinogenesis. These findings highlight ORC1 as a promising biomarker that complements alpha-fetoprotein, substantially improving early detection accuracy and offering strong potential for clinical translation in HBV-HCC screening.

INTRODUCTION

Primary liver cancer is the sixth most frequently diagnosed malignancy and the third leading cause of cancer-related death worldwide[1,2]. Hepatocellular carcinoma (HCC) accounts for nearly 90% of primary liver cancers[3]. In high-burden regions such as China, chronic hepatitis B virus (HBV) infection remains the predominant risk factor for HCC development[4]. Integration of HBV DNA into the host genome plays a critical role in HBV-HCC[5]. These events often occur early in HBV infection[6], leading to persistent genomic instability and dysregulation of host gene expression, which ultimately drives malignant transformation[7-9].

Several serum biomarkers are available for HBV-HCC detection, including alpha-fetoprotein (AFP), AFP-L3, des-gamma-carboxy prothrombin, glypican-3, and Golgi protein 73, but their diagnostic performance is limited by insufficient sensitivity and specificity[10,11]. AFP, the most widely used biomarker, has poor sensitivity (51.5%-60%) in early-stage HBV-HCC[12] and fails to detect 30%-40% of AFP-negative cases[13-15]. In our previous work, we identified a recurrent HBV-human fusion gene involving HBV S gene integration into extra spindle pole bodies-like 1 (ESPL1), which encodes a secreted cell-cycle regulator[16-18]. Serum ESPL1 levels increased with disease progression, supporting its potential as a circulating biomarker for HBV-HCC.

In our initial integration site analysis of 15 HBV-HCC tissues, we identified recurrent HBV S gene insertions near the ORC1 locus, suggesting a potential integration hotspot. While well-established hotspots such as telomerase reverse transcriptase (TERT, telomere maintenance) and mixed-lineage leukemia 4 (chromatin remodeling) have been extensively characterized in HCC[19], ORC1 has not previously been validated as a target. Located on chromosome 1p32.3, ORC1 encodes the largest subunit of the ORC1-6 complex, which initiates DNA replication licensing during the G1-S transition[20,21]. Unlike TERT or mixed-lineage leukemia 4, disruption of ORC1 may impair replication licensing. Although aberrant ORC1 expression has been reported in lung adenocarcinoma[22], glioblastoma[23], and HCC[24], most studies focused on tumor tissues, with limited evidence on serum expression or diagnostic potential. Importantly, ORC1’s secretory nature and its unexplored association with HBV-driven dysregulation suggest its promise as a noninvasive biomarker candidate for HBV-HCC.

Building on these observations, the present study aimed to: (1) Expand profiling of HBV integration sites in HCC tissues; (2) Quantify serum ORC1 protein expression across HBV-infected and control subgroups; and (3) Evaluate the diagnostic accuracy of serum ORC1 - alone and in combination with ESPL1 and AFP - for HBV-HCC, with particular focus on AFP-negative cases. Through a comprehensive case-control design integrating enzyme-linked immunosorbent assay (ELISA)-based quantification and receiver operating characteristic (ROC) analysis, this study provides novel insights into the clinical utility of ORC1 as a noninvasive biomarker for HBV-HCC detection.

MATERIALS AND METHODS

Study design and participants

This retrospective case-control study enrolled patients from a longitudinal HBV infection cohort at the First Affiliated Hospital of Guangxi Medical University between January 2002 and January 2024. A total of 552 participants who met predefined inclusion and exclusion criteria were included. To minimize bias, strict cohort definitions and standardized laboratory protocols were applied, and all analyses were performed under blinded conditions. The study population was stratified into HBV-infected groups - HBV S gene integration (HBV-S-Int), non-S gene integration (HBV-NS-Int), chronic hepatitis B (CHB), HBV-related liver cirrhosis (HBV-LC), and HBV-HCC - and non-HBV-infected controls, which included patients with non-HBV-HCC and healthy individuals. Ethical approval was granted by the Institutional Review Board of the First Affiliated Hospital of Guangxi Medical University (Approval No. 2025-E0523). All procedures complied with the Declaration of Helsinki (2024 revision).

Inclusion and exclusion criteria

Eligible participants in the HBV-infected group met the following inclusion criteria: Positivity for hepatitis B surface antigen (HBsAg) and/or detectable HBV DNA, with subgroup classification based on integration status and disease stage. Patients in the HBV-S-Int subgroup were confirmed to harbor HBV S gene integration in liver tissue, validated by Alu-polymerase chain reaction (PCR) or whole-genome sequencing[16]. Those in the HBV-NS-Int subgroup had comparable serologic profiles but lacked detectable S gene integration. CHB was defined as persistent HBsAg positivity and/or detectable HBV DNA in the absence of imaging evidence of cirrhosis or regenerative nodules[25]. HBV-LC was diagnosed in patients with compatible serology and imaging or histologic confirmation of cirrhosis[25]. The HBV-HCC subgroup included patients with HBsAg and/or HBV DNA positivity and pathologically confirmed HCC[26].

The non-HBV-infected control group comprised two subgroups: Patients with non-HBV-HCC and healthy controls. Non-HBV-HCC was defined as histologically confirmed HCC with negative HBsAg in both serum and liver tissue. Healthy controls were HBsAg-negative individuals without evidence of liver disease or systemic illness. Exclusion criteria, applied uniformly across groups, were: Age < 18 years, co-infection with human immunodeficiency virus or tuberculosis, liver failure, decompensated cirrhosis, or other malignancies. Patients with HCC of non-HBV etiology (e.g., alcohol- or drug-induced) or other viral hepatitis were also excluded. Only participants meeting all inclusion and no exclusion criteria, with complete clinical data, were included in the final analysis.

Sample collection and processing

For each participant, 5 mL of peripheral venous blood was collected into serum-separating tubes during routine clinical evaluation. Samples were centrifuged at 1500 × g for 10 minutes at 4 °C, and serum aliquots were stored at -80 °C until batch analysis. All sample processing was conducted under blinded conditions using standardized protocols to minimize variability and reduce batch effects.

Serum biomarker quantification

Serum concentrations of ORC1 and ESPL1 were measured using commercial sandwich ELISA kits (Human ORC1 ELISA Kit, Cat. No. ORC1-Hu-96T; Human ESPL1 ELISA Kit, Cat. No. ESPL1-Hu-96T; Nova Lifetech Inc., Hong Kong, China), following the manufacturer’s instructions. Both assays had a detection range of 156-10000 ng/L and were performed in duplicate. Absorbance was read at 450 nm using a microplate reader, and calibration curves (R² ≥ 0.99) were applied for quantification. Serum AFP levels were measured using a validated chemiluminescence immunoassay platform in the Department of Laboratory Medicine, First Affiliated Hospital of Guangxi Medical University.

HBV integration detection

Genomic DNA was extracted from surgically resected HBV-HCC tumor tissues using a magnetic bead-based kit (MagPure Tissue DNA Kit, Magen Biotech, Guangzhou, China). DNA quality and concentration were assessed with a NanoPhotometer (Implen) and a Qubit 3.0 Fluorometer (Thermo Fisher, Waltham, MA, United States). Library preparation included DNA fragmentation, ligation, and PCR enrichment (Annoroad Genomics, Beijing, China), followed by quality validation using an Agilent 2100 and qPCR. Sequencing was performed on the Illumina NovaSeq 6000 platform (2 × 150 bp paired-end). HBV integration sites were identified using a hybrid pipeline: Cutadapt for adapter trimming, Burrows-Wheeler Aligner-maximal exact match for alignment to human (No. GRCh37) and HBV (No. NC-003977.2) reference genomes, Picard for duplicate removal, and SeekSV for split-read detection and integration site mapping.

Statistical analysis

Statistical analyses were performed using SPSS version 26.0 (IBM Corp., NY, United States), GraphPad Prism version 8 (GraphPad Software), and MedCalc version 20.0 (MedCalc Software). Continuous variables were expressed as mean ± SD or median (interquartile range), as appropriate. Group comparisons were performed using independent t-tests or Mann-Whitney U tests. Univariate and multivariate logistic regression analyses were applied to identify determinants of elevated serum ORC1 levels. Correlations between serum ORC1 and ESPL1 were evaluated using Spearman’s rank correlation. Diagnostic performance was assessed by ROC curve analysis, with area under the curve (AUC), 95% confidence intervals (CI), sensitivity, specificity, and accuracy calculated. The maximum Youden index determined optimal cutoff values. Comparisons of AUCs were conducted using the DeLong method, and categorical data were analyzed with χ² tests. Missing data (< 10%) were imputed via multiple imputation by chained equations, generating ten datasets pooled with Rubin’s rules. Statistical significance was defined as P < 0.05.

RESULTS

General characteristics of the study population

A total of 552 participants were enrolled, including 479 HBV-infected patients and 73 non-HBV-infected controls. The HBV-infected cohort comprised five subgroups: HBV-S-Int (n = 20; mean age, 44.35 ± 8.06 years; 85.0% male), HBV-NS-Int (n = 47; 51.14 ± 9.86 years; 70.2% male), CHB (n = 162; 42.45 ± 10.07 years; 67.3% male), HBV-LC (n = 154; 47.31 ± 9.67 years; 78.6% male), and HBV-HCC (n = 96; 49.13 ± 9.44 years; 88.5% male). The control group included 29 patients with non-HBV-HCC (59.34 ± 9.98 years; 65.5% male) and 44 healthy subjects (38.18 ± 13.33 years; 47.7% male) (Table 1).

Table 1 The characteristics of study subjects, n (%)/mean ± SD.

Group	Subgroup	Number	Male	Age (year)	ALT (U/L)	AST (U/L)	ORC1 (ng/L)	ESPL1 (ng/L)	AFP (ng/mL)
HBV-infected group	HBV-S-Int	20	17 (85.0)	44.35 ± 8.06	29.70 ± 14.03	33.45 ± 8.70	1660.65 (1428.60, 2218.60)	484.58 (300.22, 553.09)	3.04 (1.70, 8.19)
	HBV-NS-Int	47	33 (70.2)	51.14 ± 9.86	25.21 ± 13.64	30.17 ± 9.03	954.75 (681.19, 1435.25)	231.41 (189.15, 281.58)	2.5 (1.69, 3.23)
	CHB	162	109 (67.3)	42.45 ± 10.07	23.00 ± 10.66	29.31 ± 16.96	914.68 (642.23, 1319.14)	198.34 (151.29, 260.22)	2.39 (1.92, 315)
	HBV-LC	154	121 (78.6)	47.31 ± 9.67	31.94 ± 19.73	37.31 ± 20.40	918.30 (638.24, 1466.50)	276.14 (220.62, 327.27)	2.45 (1.82, 3.89)
	HBV-HCC	96	85 (88.5)	49.13 ± 9.44	50.5 ± 47.13	50.69 ± 41.41	1077.07 (727.03, 1675.67)	439.00 (261.25, 641.72)	10.55 (3.61, 263.81)
Non-HBV-infected control group	Non-HBV-HCC	29	19 (65.5)	59.34 ± 9.98	24.48 ± 12.85	34.69 ± 16.81	749.54 (660.24, 1150.33)	245.62 (212.75, 438.93)	60.22 (3.62, 2842.69)
	Healthy control	44	21 (47.7)	38.18 ± 13.33	-	-	731.51 (503.39, 1043.57)	128.89 (105.02, 150.82)	-

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; AFP: Alpha-fetoprotein; HBV-S-Int: Hepatitis B virus S gene integration; HBV-NS-Int: Hepatitis B virus non-S gene integration; CHB: Chronic hepatitis B; HBV-LC: Hepatitis B virus-related liver cirrhosis; HBV-HCC: Hepatitis B virus-related hepatocellular carcinoma.

Genome-wide sequencing and HBV integration site analysis in HBV-HCC patients

Whole-genome sequencing of tumor tissues from 15 HBV-HCC patients identified HBV-host integration events in 93.3% (14/15) of cases. Among these, 71.4% (10/14) showed integration near the ORC1 locus, and 40% (4/10) specifically involved the HBV S gene. ORC1 is located on chromosome 1p32.3 (approximately 249 Mb), with integration sites clustering within a 6 Mb window (approximately 2.4%) surrounding this locus. One patient exhibited direct HBV S gene integration at the ORC1 promoter region (Figure 1).

Figure 1 Hepatitis B virus S gene integration cluster near the human origin recognition complex subunit 1 gene locus on chromosome 1 (Chr1 1p32.3). Genome-wide analysis of hepatitis B virus (HBV) integration in 15 HBV-related hepatocellular carcinoma tumor tissues revealed 14 cases with HBV-human integration. Among these, 10 (71.4%) harbored integration events clustered within a 6 Mb region centered around the origin recognition complex subunit 1 gene (indicated by orange dashed box). A representative case showed HBV S gene integration at the origin recognition complex subunit 1 promoter region. ORC1: Origin recognition complex subunit 1; HBV: Hepatitis B virus.

Serum ORC1 expression across groups

Serum ORC1 concentrations were significantly higher in HBV-infected patients compared with non-HBV controls (980.11 ng/L vs 746.82 ng/L, P < 0.05; Figure 2A). Within the HBV-infected cohort, HBV-S-Int patients had significantly higher ORC1 levels than HBV-NS-Int patients (1660.65 ng/L vs 954.75 ng/L, P < 0.05; Figure 2B). HBV-HCC patients also exhibited higher ORC1 levels compared with non-HBV-HCC patients (1077.07 ng/L vs 749.54 ng/L, P < 0.05). No significant difference was observed between non-HBV-HCC patients and healthy controls (749.54 ng/L vs 731.51 ng/L, P > 0.05; Figure 2C).

Figure 2 Comparison of serum origin recognition complex subunit 1 levels among study groups assessed by the Mann-Whitney U test. A: Comparison between hepatitis B virus (HBV)-infected patients (n = 479) and non-HBV-infected controls (n = 73); B: Comparison between HBV S gene integration (n = 20) and HBV non-S gene integration (n = 47) subgroups; C: Comparison between HBV-related hepatocellular carcinoma (n = 96), non-HBV-hepatocellular carcinoma (n = 29), and healthy controls (n = 44). ORC1: Origin recognition complex subunit 1; HBV: Hepatitis B virus; HBV-S-Int: HBV S gene integration; HBV-NS-Int: HBV non-S gene integration; HBV-HCC: HBV-related hepatocellular carcinoma; NS: Not significant.

Analysis of factors associated with serum ORC1 levels

To identify determinants of elevated serum ORC1 levels, clinical data were analyzed from 67 HBV-infected patients (10 with HBV-HCC, 8 with HBV-LC, and 49 with CHB) who underwent HBV S gene integration testing[16]. Ten clinical variables were evaluated: Gender, age, history of anti-HBV therapy, HBV DNA load, hepatitis B e antigen status, HBsAg level, alanine aminotransferase, aspartate aminotransferase, AFP level, and presence of HBV integration. Serum ORC1 expression, dichotomized at the median, served as the dependent variable in logistic regression (Table 2). Univariate analysis showed no significant associations with gender, age, antiviral therapy, HBV DNA load, hepatitis B e antigen, HBsAg, alanine aminotransferase, aspartate aminotransferase, or AFP. However, HBV integration was strongly associated with increased ORC1 expression. Multivariate logistic regression confirmed HBV integration as the only independent predictor (Table 3). These results indicate that HBV integration enhances ORC1 transcription, driving its upregulation.

Table 2 Variable descriptions and assignments for logistic regression analysis.

Variable	Description	Assignment and notes
X1	Gender	Female = 0, male = 1
X2	Age	≤ 45 years = 0, > 45 years = 1
X3	History of anti-HBV therapy	No = 0, unknown = 1, yes = 2
X4	HBV DNA load	Log10-transformed
X5	HBeAg status	Negative = 0, positive = 1
X6	HBsAg level	Log10-transformed
X7	ALT level	Measured value (U/L)
X8	AST level	Measured value (U/L)
X9	AFP level	Measured value (ng/mL)
X10	HBV integration	No = 0, yes = 1
Y	Serum ORC1 level	≤ median = 0, > median = 1

HBV: Hepatitis B virus; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; AFP: Alpha-fetoprotein; ORC1: Origin recognition complex subunit 1.

Table 3 Univariate and multivariate logistic regression analysis of factors associated with elevated serum origin recognition complex subunit 1 levels.

Factor	Univariate analysis			Multivariate analysis
	B	OR (95%CI)	P value	B	Adjusted OR (95%CI)	P value
Gender	-0.198	0.821	0.725	-0.751	0.472	0.358
Age	-1.168	0.311	0.034	-0.371	0.690	0.598
History of anti-HBV therapy	0.134	1.143	0.820	0.028	1.029	0.972
HBV DNA load	0.453	1.573	0.124	0.789	2.201	0.066
HBeAg status	-0.693	0.500	0.579	-2.140	0.118	0.297
HBsAg level	0.166	1.181	0.563	0.169	1.184	0.622
ALT level	0.002	1.002	0.904	-0.021	0.979	0.523
AST level	0.013	1.013	0.636	0.001	1.001	0.982
AFP level	0.000	1.000	0.533	0.023	1.023	0.690
HBV integration	2.955	19.200	0.000	2.919	18.518	0.002

OR: Odds ratio; CI: Confidence interval; HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; HBeAg: Hepatitis B e antigen; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; AFP: Alpha-fetoprotein.

Correlation between ORC1 and ESPL1

In 400 HBV-infected patients with matched data, serum ORC1 levels were positively correlated with ESPL1 levels (Spearman’s r = 0.231, P < 0.001; Figure 3), suggesting complementary biomarker roles in HBV-related disease progression.

Figure 3 Correlation between serum origin recognition complex subunit 1 and extra spindle pole bodies-like 1 levels in hepatitis B virus-infected patients (n = 400). A significant positive correlation was observed between serum origin recognition complex subunit 1 and extra spindle pole bodies-like 1 levels, suggesting functional linkage in hepatitis B virus-driven hepatocarcinogenesis. ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; CI: Confidence interval.

Diagnostic performance in AFP-negative HBV-HCC

Among 400 participants with complete biomarker data, 90 were histologically confirmed to have HBV-HCC. Of these, only 21.1% (19/90) were AFP-positive, while 78.9% (71/90) were AFP-negative (Figure 4A). In AFP-negative patients, ESPL1 and ORC1 detected 73.2% (52/71) and 64.8% (46/71) of cases, respectively (Figure 4B), demonstrating superior sensitivity compared with AFP. Compared with healthy controls, both AFP-negative and AFP-positive HBV-HCC patients exhibited significantly higher ORC1 levels (1051.38 ng/L vs 731.51 ng/L; 1093.14 ng/L vs 731.51 ng/L, P < 0.05; Figure 4C) and ESPL1 levels (417.27 ng/L vs 128.89 ng/L; 1093.14 ng/L vs 128.89 ng/L, P < 0.05; Figure 4D). No significant differences were observed between AFP-negative and AFP-positive patients, indicating that ORC1 and ESPL1 expression is independent of AFP status. Both biomarkers were consistently elevated in HBV-HCC patients regardless of AFP status, underscoring their utility in detecting AFP-negative disease.

Figure 4 Detection of hepatitis B-related hepatocellular carcinoma cases by alpha-fetoprotein, origin recognition complex subunit 1, and extra spindle pole bodies-like 1 among alpha-fetoprotein-negative patients. A: Among 90 hepatitis B-related hepatocellular carcinomas (HBV-HCC) cases, 71 (78.9%) were alpha-fetoprotein (AFP)-negative; B: Among these AFP-negative cases, serum extra spindle pole bodies-like 1 and origin recognition complex subunit 1 identified 52 (73.2%) and 46 (64.8%) cases, respectively; C: Serum origin recognition complex subunit 1 levels were significantly elevated in both AFP-negative and AFP-positive HBV-HCC patients compared to healthy controls (P < 0.05); D: Serum extra spindle pole bodies-like 1 levels were significantly higher in AFP-negative and AFP-positive HBV-HCC patients than in healthy controls (P < 0.001). ESPL1: Extra spindle pole bodies-like 1; HBV-HCC: Hepatitis B virus-related hepatocellular carcinoma; ORC1: Origin recognition complex subunit 1; AFP: Alpha-fetoprotein; NS: Not significant.

ROC curve and AUC analysis

ROC curve analysis demonstrated that the combined ORC1 + ESPL1 + AFP panel achieved an AUC of 0.887 (95%CI: 0.784-0.984), significantly higher than ESPL1 alone (0.887 vs 0.776, P < 0.05) and ORC1 alone (0.887 vs 0.587, P < 0.05), but not significantly different from AFP alone (0.887 vs 0.844, P > 0.05; Figure 5, Table 4). Individually, AFP (AUC = 0.844) and ESPL1 (AUC = 0.776) outperformed ORC1 (AUC = 0.587, P < 0.05 for both). The triple-marker panel significantly improved specificity (84.19% vs 54.84%, P < 0.05) and diagnostic accuracy (84.25% vs 57.25%, P < 0.05) compared with ORC1 alone. Importantly, it maintained high sensitivity, exceeding that of ESPL1 (84.44% vs 71.11%, P < 0.05) and AFP (84.44% vs 21.17%, P < 0.05). The panel also achieved an exceptional negative predictive value (negative predictive value = 94.91%, 261/275), which is particularly relevant for screening applications. Key diagnostic indices - including sensitivity, specificity, and accuracy - are summarized in Table 5. Collectively, these findings support the clinical utility of integrating ORC1 and ESPL1 with AFP for improved detection of HBV-HCC, especially in AFP-negative populations.

Figure 5 Receiver operating characteristic curve analysis for serum biomarkers in hepatitis B-related hepatocellular diagnosis. ^aP < 0.05 vs ESPL1; ^bP < 0.05 vs ORC1. ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; AFP: Alpha-fetoprotein; AUC: Area under the receiver operating characteristic curve; CI: Confidence interval.

Table 4 Receiver operating characteristic curve analysis of serum biomarkers in the diagnosis of hepatitis B virus-related hepatocellular carcinoma.

Group	AUC	Youden’s index	Cut-off value	95%CI	Z value	P value
ORC1	0.587	0.204	967.814	0.520-0.653	2.599	0.012
ESPL1	0.776b	0.579	345.654	0.713-0.893	8.547	< 0.001
AFP	0.844b	0.566	400.000	0.794-0.984	13.498	< 0.001
ORC1 + ESPL1 + AFP	0.887a,b	0.686	0.152	0.784-0.891	17.541	< 0.001

^aP < 0.05 vs ESPL1.

^bP < 0.05 vs ORC1.

AUC: Area under the curve; CI: Confidence interval; ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; AFP: Alpha-fetoprotein.

Table 5 Diagnostic reliability analysis of serum biomarkers for hepatitis B virus-related hepatocellular carcinoma, (n/N, %).

Group	Sensitivity	Specificity	Accuracy	PPV	NPV
ORC1	65.56 (59/90, 0.551-0.750)	54.84 (170/310, 0.493-0.603)	57.25 (229/400, 0.523-0.621)	29.65 (59/199, 0.235-0.365)	84.58 (170/201, 0.792-0.889)
ESPL1	71.11 (64/90, 0.612-0.798)	86.77 (269/310, 0.827-0.901)	83.25 (333/400, 0.792-0.868)	60.95 (64/105, 0.514-0.697)	91.19 (269/295, 0.876-0.939)
AFP	21.11 (19/90, 0.138-0.309)	100.00 (310/310, 0.988-1.000)	82.25 (329/400, 0.782-0.858)	100.00 (19/19, 0.824-1.000)	81.36 (310/381, 0.773-0.849)
ORC1 + ESPL1 + AFP	84.44a,b (76/90, 0.756-0.909)	84.19c (261/310, 0.798-0.879)	84.25c (337/400, 0.803-0.876)	60.80 (76/125, 0.521-0.690)	94.91 (261/275, 0.917-0.971)

^aP < 0.05 vs ESPL1.

^bP < 0.05 vs AFP.

^cP < 0.05 vs ORC1.

PPV: Positive predictive value; NPV: Negative predictive value; ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; AFP: Alpha-fetoprotein.

DISCUSSION

The ORC1 gene encodes the largest subunit of the origin recognition complex, an essential adenosinetriphosphatase that regulates DNA replication licensing during the G1/S transition of the cell cycle[20,21]. ORC1 overexpression has been reported in several malignancies, including lung adenocarcinoma, glioblastoma, and HCC, and has been linked to poor prognosis[27-29]. Our study extends these findings to the serological level, showing that serum ORC1 concentrations are elevated in HBV-infected individuals, particularly those with HBV-HCC, thereby supporting its potential as a minimally invasive biomarker for early detection.

Genomic integration analysis revealed that 71.4% (10/14) of HBV-HCC cases with detectable integration exhibited viral insertions within a 6 Mb region surrounding the ORC1 locus on chromosome 1p32.3. This localized enrichment, accounting for only 2.4% of the chromosome, suggests a potential hotspot for HBV integration. Notably, one case demonstrated direct HBV S gene integration at the ORC1 promoter. Given the oncogenic consequences of HBV DNA integration[30] and prior reports of recurrent hotspots such as ESPL1 and TERT[16,31], our findings suggest that ORC1 may represent a novel integration target. Such events could disrupt DNA replication licensing and contribute to hepatocarcinogenesis.

Elevated serum ORC1 levels in HBV-infected patients - particularly those in the HBV-S-Int and HBV-HCC subgroups - suggest that viral integration near ORC1 may drive its overexpression and systemic release. This HBV-specific increase was not observed in non-HBV-HCC patients or healthy controls, underscoring its pathogenetic relevance. We hypothesize that HBV DNA integration into or adjacent to the ORC1 locus enhances transcriptional activity, resulting in elevated serum ORC1 levels. This aberrant overexpression may disrupt regular cell cycle checkpoints, activate oncogenic signaling pathways, and ultimately promote HBV-HCC development. Although causality cannot be established in this observational study, these findings highlight a plausible mechanistic link between viral integration and ORC1-mediated hepatocarcinogenesis.

ESPL1, a cysteine endopeptidase essential for chromatid separation during mitosis[32], has also been implicated in HBV-driven hepatocarcinogenesis through viral integration. Previous studies demonstrated that serum ESPL1 is upregulated in HBV-HCC patients[33]. Our findings confirm its diagnostic utility and complementarity with ORC1. The moderate positive correlation between ORC1 and ESPL1 levels (r = 0.231, P < 0.001) suggests potential co-regulation in HBV-infected hepatocytes, although this likely reflects parallel dysregulation rather than direct molecular interaction. Given that ORC1 regulates the S phase and ESPL1 functions during the M phase of the cell cycle[34-37], their concurrent dysregulation may drive abnormal cell cycle progression, contributing to aneuploidy and malignant transformation (Figure 6).

Figure 6 Schematic model illustrating the molecular mechanism and diagnostic workflow of hepatitis B virus integration near origin recognition complex subunit 1 and extra spindle pole bodies-like 1. The hepatitis B virus S gene integrates near the origin recognition complex subunit 1 and extra spindle pole bodies-like 1 loci, promoting their overexpression. These genes may be co-regulated in hepatitis B virus-related hepatocellular carcinoma. Since origin recognition complex subunit 1 controls the S phase while extra spindle pole bodies-like 1 regulates the M phase, their concurrent dysregulation could drive aberrant cell cycle progression, leading to hepatocyte aneuploidy and malignant transformation. ORC1: Origin recognition complex subunit 1; ESPL1: Extra spindle pole bodies-like 1; HBV: Hepatitis B virus.

Consistent with previous reports[38], AFP alone detected only 21.1% of HBV-HCC cases, underscoring its limited sensitivity. In contrast, ESPL1 and ORC1 were elevated in HBV-HCC regardless of AFP status, and detected 73.2% and 64.8% of AFP-negative cases, respectively. The combined biomarker panel (ORC1 + ESPL1 + AFP) demonstrated significantly improved diagnostic accuracy, with an AUC of 0.887, sensitivity of 84.44%, and specificity of 84.19%, surpassing the performance of any single marker. These results emphasize the potential of ORC1 and ESPL1 to complement AFP, thereby addressing diagnostic gaps, particularly in AFP-negative patients where early detection is most challenging.

Several limitations should be acknowledged. First, the retrospective design may have introduced selection bias and may limit the generalizability of our findings. Second, the relatively small sample size, particularly for the HBV integration analysis, and the absence of external validation restrict the robustness of our conclusions. The ROC analysis was performed on the full dataset without a training/validation split, and no external cohorts were included. Third, functional validation of the biological effects of ORC1 promoter integration was beyond the scope of this study. Finally, our conclusions are specific to HBV-related HCC, and the expression and diagnostic value of ORC1 in HCCs of other etiologies remain to be clarified. Future multicenter prospective studies with larger cohorts and mechanistic experiments are warranted to validate the clinical utility and biological significance of serum ORC1 and ESPL1 in HBV-HCC.

CONCLUSION

In summary, our study demonstrates that HBV integration near the ORC1 locus may drive its overexpression and contribute to elevated serum ORC1 levels in HBV-HCC patients. ORC1 and ESPL1 emerge as complementary serum biomarkers with significant value for early diagnosis, particularly in AFP-negative cases. The combined use of ORC1, ESPL1, and AFP markedly enhances diagnostic sensitivity and specificity. Prospective, multicenter studies are needed to validate these findings and establish their clinical applicability.

ACKNOWLEDGEMENTS

We express our profound gratitude to the patients and their families for their indispensable participation in this study.