Hepatocellular carcinoma (HCC) is the sixth most common cancer and ranks the fourth in cancer mortality worldwide, and patients with liver cirrhosis (LC) are at high risk of HCC [1, 2]. Constantly elevated levels of alpha-fetoprotein (AFP) and other serum biomarkers including AFP-L3 and PIVKA-II generally indicate development of HCC; however, the performance of these biomarkers as diagnostic models for early HCC remains unpromising [3].

The utility of cancer-associated aberrations including genic mutations in cell-free DNA (cfDNA) for cancer detection is a global research hot spot [4, 5]. Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) is a detection platform that can simultaneously detect and quantitate multiple plasma DNA variants based on next-generation sequencing [6, 7]. A total of 1702 individuals (healthy cohort, LC cohort, and HCC cohort) from nine clinical sites across China were enrolled from June 2018 through January 2019 in this study. In HCC cohort, 27 were excluded according to pathology diagnosis. Finally, 1675 participants (490 healthy controls, 577 LC patients, and 608 HCC patients) were randomly assigned to training/validation/test cohorts (Additional file 1: Fig S1). Detail information of these participants is shown in Additional files 1, 2: Tables S1–S8. 10 mL peripheral blood was provided from each individual for cSMART test at enrollment time.

We first constructed negative background pool using cfDNA samples from 490 healthy individuals. To explore the feature of cfDNA mutations in HCC and LC, 931 regions among 21 genes of 608 HCCs and 577 LCs were detected by cSMART. Top 20 gene mutation sites with high mutation frequency are detailed in Additional files 3, 4: Tables S9–S12. The overall mutation ratio of cfDNA in HCC was significantly higher than that in LC (Fig. 1a and Additional file 1: Fig. S2). Then, detected mutations were minimized and finally three mutation sites located in different regions of gene TERT, TP53, and CTNNB1 were screened out to be further analysis. The performance of the single mutation gene site in the diagnosis of HCC is shown in Additional file 1: Table S13. A gradual increasing trend in variant allele frequency (VAF) at HCC-specific mutation sites from early HCC (BALC 0/A) to advanced HCC (BCLC C) was identified (Additional file 1: Fig. S3), proving that cSMART was sensitive for quantification of low-copy number DNA in plasma and could accurately reflect the tumor mutational burden.

Combined method holds a strong value in diagnosis of HCC. a Basic information (age, gender), cirrhosis background, tumor serological biomarkers (CA199, PIVKA-II, AFP-L3, AFP), and HCC related parameters (MVI and BCLC stages) of all HCC samples with positive mutations at the top 20 high-frequency mutation sites. CA199: carbohydrate antigen199; MVI: microvascular invasion; and BCLC: Barcelona clinic liver cancer. b ROC curves of Combined method and AFP for HCC patients versus LC patients in the training, test, and validation cohorts. c Proportions of positive and negative calling by Combined method, GALAD, and AFP in all participants with different AFP, AFP-L3, and PIVKA-II levels in test cohort. d Proportions of positive and negative calling by Combined method, GALAD, and AFP in all participants with different age, gender, Child–Pugh stages, HBV infection status, and cirrhosis history in test cohort. e Proportions of positive and negative calling by Combined method, GALAD, and AFP in HCC patients with different tumor sizes and BCLC stages in test cohort

By integrating three mutations of cfDNA and three serum biomarkers (AFP, AFP-L3, and PIVKA-II), Combined method was developed for diagnosis of HCC. AFP, the most commonly used biomarker, could detect 43 of 151 HCCs in test cohort, and 26 of 112 HCCs in validation cohort at the cutoff value of 400 ng/mL, and achieved diagnostic sensitivity of 56.29%/48.21% at specificity of 91.03%/93.18% in test cohort or validation cohort at 20 ng/mL cutoff value. Combined method showed better performance compared with AFP, detecting 135 of 151 HCCs with a sensitivity of 89.40% at 80.69% specificity in test cohort. More, the sensitivities of this model to detect HCC at BCLC 0 and A were 60.00% and 83.87%, respectively (Table 1). The same conclusion could also be drawn from the data of the independent validation cohort (Table 1). Receiver operating characteristic (ROC) curve further corroborated that this cfDNA-based integrated diagnostic model was significantly superior to AFP in the diagnosis of HCC (Fig. 1b).

Next, the accuracy of Combined method to differentiate HCC from LC was evaluated in different subgroups and compared with GALAD and AFP. In test cohort, this model could not only distinguish AFP-positive HCC from LC (accuracy: 95.56%), but also detect AFP-negative HCC who might be missed by conventional diagnostic approaches (accuracy: 83.27%). Furthermore, Combined method exhibited high accuracy for HCC diagnosis in both AFP-L3/PIVKA-II-positive and AFP-L3/PIVKA-II-negative subgroups, outperforming current commonly used biomarkers without over diagnosis (Fig. 1c). In addition, Combined method held high accuracy in diagnosis of liver tumors with any size irrespective of age, gender, Child–Pugh stage, HBV infection status, and cirrhosis history and showed much better performance in detecting early and very early HCC (accuracy: BCLC 0: 60.00%; BCLC A: 83.33%) than GALAD and AFP (Fig. 1d, e). Subsequently, the above conclusions were further confirmed in validation cohort (Additional file 1: Fig. S4).

In conclusion, we developed a retrospective phase 3 study according to the criteria for biomarker development delineated by Pepe et al., identified the unique cfDNA hotspot mutation signature of HCC, and constructed Combined method based on three mutation sites and three serum biomarkers [8]. Combined method has fixed indicators and simple detection process, outperforming conventional approaches in the diagnosis of HCC, especially early HCC, in a noninvasive way. Our model holds great potentials to be incorporated into current clinical care considering its cost-effectiveness and practicality, which is expected to improve the outcomes for HCC patients missed by traditional methods in the future.