AFP-positive Hepatocellular Carcinoma Research Articles

Development of a machine learning-based model to predict prognosis of alpha-fetoprotein-positive hepatocellular carcinoma

BackgroundPatients with alpha-fetoprotein (AFP)-positive hepatocellular carcinoma (HCC) have aggressive biological behavior and poor prognosis. Therefore, survival time is one of the greatest concerns for patients with AFP-positive HCC. This study aimed to demonstrate the utilization of six machine learning (ML)-based prognostic models to predict overall survival of patients with AFP-positive HCC.MethodsData on patients with AFP-positive HCC were extracted from the Surveillance, Epidemiology, and End Results database. Six ML algorithms (extreme gradient boosting [XGBoost], logistic regression [LR], support vector machine [SVM], random forest [RF], K-nearest neighbor [KNN], and decision tree [ID3]) were used to develop the prognostic models of patients with AFP-positive HCC at one year, three years, and five years. Area under the receiver operating characteristic curve (AUC), confusion matrix, calibration curves, and decision curve analysis (DCA) were used to evaluate the model.ResultsA total of 2,038 patients with AFP-positive HCC were included for analysis. The 1-, 3-, and 5-year overall survival rates were 60.7%, 28.9%, and 14.3%, respectively. Seventeen features regarding demographics and clinicopathology were included in six ML algorithms to generate a prognostic model. The XGBoost model showed the best performance in predicting survival at 1-year (train set: AUC = 0.771; test set: AUC = 0.782), 3-year (train set: AUC = 0.763; test set: AUC = 0.749) and 5-year (train set: AUC = 0.807; test set: AUC = 0.740). Furthermore, for 1-, 3-, and 5-year survival prediction, the accuracy in the training and test sets was 0.709 and 0.726, 0.721 and 0.726, and 0.778 and 0.784 for the XGBoost model, respectively. Calibration curves and DCA exhibited good predictive performance as well.ConclusionsThe XGBoost model exhibited good predictive performance, which may provide physicians with an effective tool for early medical intervention and improve the survival of patients.

Multi-dimensional single-cell characterization revealed suppressive immune microenvironment in AFP-positive hepatocellular carcinoma

Alpha-fetoprotein (AFP)-secreting hepatocellular carcinoma (HCC), which accounts for ~75% of HCCs, is more aggressive with a worse prognosis than those without AFP production. The mechanism through which the interaction between tumors and the microenvironment leads to distinct phenotypes is not yet clear. Therefore, our study aims to identify the characteristic features and potential treatment targets of AFP-negative HCC (ANHC) and AFP-positive HCC (APHC). We utilized single-cell RNA sequencing to analyze 6 ANHC, 6 APHC, and 4 adjacent normal tissues. Integrated multi-omics analysis together with survival analysis were also performed. Further validation was conducted via cytometry time-of-flight on 30 HCCs and multiplex immunohistochemistry on additional 59 HCCs. Our data showed that the genes related to antigen processing and interferon-γ response were abundant in tumor cells of APHC. Meanwhile, APHC was associated with multifaceted immune distortion, including exhaustion of diverse T cell subpopulations, and the accumulation of tumor-associated macrophages (TAMs). Notably, TAM-SPP1+ was highly enriched in APHC, as was its receptor CD44 on T cells and tumor cells. Targeting the Spp1-Cd44 axis restored T cell function in vitro and significantly reduced tumor burden when treated with either anti-Spp1 or anti-Cd44 antibody alone or in combination with anti-Pd-1 antibody in the mouse model. Furthermore, elevated IL6 and TGF-β1 signaling contributed to the enrichment of TAM-SPP1+ in APHC. In conclusion, this study uncovered a highly suppressive microenvironment in APHC and highlighted the role of TAM-SPP1+ in regulating the immune microenvironment, thereby revealing the SPP1-CD44 axis as a promising target for achieving a more favorable immune response in APHC treatment.

Circulating the HLA-DR+ T Cell Ratio Is a Prognostic Factor for Recurrence of Patients with Hepatocellular Carcinoma after Curative Surgery.

HLA-DR+ T cell, accounting for 1.2%-5.8% of peripheral lymphocyte, is a type of activated T lymphocyte. This retrospective study aimed to evaluate the prognostic value of HLA-DR+ T cell for progression-free survival (PFS) and overall survival (OS) in hepatocellular carcinoma (HCC) patients after curative surgery. Patients and Methods. Clinicopathological data of 192 patients who underwent curative resection for hepatocellular carcinoma in the affiliated hospital of Qingdao University between January 2013 and December 2021 were collected and analyzed. Statistical tests used in this study were the chi-square test and Fisher's exact test. The prognostic value of the HLA-DR+ T cell ratio was analyzed using univariate and multivariate Cox regression analyses. The Kaplan-Meier curves were drawn by the R programming language. HCC patients were divided into high (≥5.8%) and low (<5.8%) HLADR+ T cell ratio groups. Cox regression analysis indicated that a high HLA-DR+ T cell ratio was positively related to the PFS in HCC patients (P=0.003) and AFP-positive (≥20 ng/ml) HCC patients (P=0.020). HCC patients and AFP-positive HCC patients in the high HLA-DR+ T cell ratio group were prone to have a higher T cell ratio, a higher CD8+T cell ratio, and a lower B cell ratio than the low HLA-DR+ T cell ratio group. However, the HLA-DR+ T cell ratio was not a statistically significant predictor for OS in HCC patients (P=0.57) as well as PFS (P=0.088) and OS (P=0.63) in AFP-negative HCC patients. This study confirmed that the HLA-DR+ T cell ratio was a significant predictor of PFS in HCC patients and AFP-positive HCC patients after curative surgery. This association may have guiding significance for the follow-up work of HCC patients after surgery.

FOXM1 Is a Novel Molecular Target of AFP-Positive Hepatocellular Carcinoma Abrogated by Proteasome Inhibition.

Alpha-fetoprotein (AFP) is an oncofetal protein that is elevated in a subset of hepatocellular carcinoma (HCC) with poor prognosis, but the molecular target activated in AFP-positive HCC remains elusive. Here, we demonstrated that the transcription factor forkhead box M1 (FOXM1) is upregulated in AFP-positive HCC. We found that FOXM1 expression was highly elevated in approximately 40% of HCC cases, and FOXM1-high HCC was associated with high serum AFP levels, a high frequency of microscopic portal vein invasion, and poor prognosis. A transcriptome and pathway analysis revealed the activation of the mitotic cell cycle and the inactivation of mature hepatocyte metabolism function in FOXM1-high HCC. The knockdown of FOXM1 reduced AFP expression and induced G2/M cell cycle arrest. We further identified that the proteasome inhibitor carfilzomib attenuated FOXM1 protein expression and suppressed cell proliferation in AFP-positive HCC cells. Carfilzomib in combination with vascular endothelial growth factor receptor 2 (VEGFR2) blockade significantly prolonged survival by suppressing AFP-positive HCC growth in a subcutaneous tumor xenotransplantation model. These data indicated that FOXM1 plays a pivotal role in the proliferation of AFP-positive liver cancer cells. Carfilzomib can effectively inhibit FOXM1 expression to inhibit tumor growth and could be a novel therapeutic option in patients with AFP-positive HCC who receive anti-VEGFR2 antibodies.

Serum alpha-fetoprotein as a predictive biomarker for tissue alpha-fetoprotein status and prognosis in patients with hepatocellular carcinoma.

BackgroundAlpha-fetoprotein (AFP) expression is closely related to hepatocarcinogenesis, and it is an important prognostic factor for hepatocellular carcinoma (HCC). We aimed to investigate the relationship between serum AFP concentration and tissue AFP status and identify the prognostic value of serum and tissue AFP for HCC.MethodsThis is a retrospective review of 248 patients with HCC from January 2012 to December 2018. Receiver operating characteristic (ROC) curves were plotted to investigate the predictive value of serum AFP for tissue AFP status. Overall survival (OS) was analyzed using the Kaplan-Meier method and log-rank tests were used for comparison between two groups. Multivariate Cox proportional hazards regression analysis was performed for various risk factors.ResultsThe serum AFP level in patients with tissue AFP-positive HCC was higher than those with tissue AFP-negative HCC (506.7 vs. 7.7 ng/mL, P<0.0001). Youden’s index yielded an optimal cut-off value of serum AFP for tissue AFP status of 92.33 ng/mL with a sensitivity and specificity of 0.84 (95% CI: 0.74–0.90) and 0.88 (95% CI: 0.82–0.92), respectively. Moreover, high serum AFP concentrations (≥92.33 ng/mL) were significantly correlated with positive hepatitis B virus (HBV, P=0.012), tumor size (P=0.025) and histological grade (P=0.001); tissue AFP-positive status was associated with positive HBV (P=0.006), tumor number (P=0.033) and histological grade (P<0.001). Further, serum AFP level ≥92.33 ng/mL and tissue AFP-positive status were associated with poorer OS, and positive HBV (Positive: HR 3.496; 95% CI: 1.349–9.064; P=0.010) and larger tumor size (≥5; HR 2.617; 95% CI: 1.372–4.992; P=0.003) were independent factors of OS.ConclusionsThis study showed that serum AFP level could be a highly predictive biomarker for tissue AFP status in patients with HCC. Furthermore, serum AFP levels ≥92.33 ng/mL and tissue AFP-positive status were associated with poorer OS but were not independent factors of OS.

Alpha-Fetoprotein Ratio Predicts Alpha-Fetoprotein Positive Hepatocellular Cancer Patient Prognosis after Hepatectomy.

Background This study was conducted to investigate the effect of alpha-fetoprotein (AFP) ratio on the prognosis of AFP-positive hepatocellular carcinoma (HCC) patients after hepatectomy. Methods We retrospectively included 879 HCC patients with AFP-positive who underwent hepatectomy from February 2012 to October 2017 and randomly divided into training cohort and validation cohort. AFP ratio was equal to the AFP level within one week before hepatectomy to AFP level within 20-40 days after surgery. The end point of follow-up was disease-free survival (DFS) and overall survival (OS). Results AFP ratio was not associated with clinical characteristics in training cohort and validation cohort. According to the X-tile software, the optimum cut-off point was 17.8 for AFP ratio. Significant differences between AFP ratio high and AFP ratio low were observed in DFS and OS in both cohort (p < 0.05). Kaplan-Meier curves and receiver-operating curves were showed that AFP ratio was better than AFP level preoperation in predicting the prognosis of AFP-positive HCC patients after hepatectomy. The multivariate analysis demonstrated that AFP ratio was a significant independent risk factor for both OS and DFS in HCC patients with AFP-positive. Conclusions AFP ratio might be a prognosis predictor for HCC patients with AFP-positive after hepatectomy.

Investigation of Potential Molecular Biomarkers for Diagnosis and Prognosis of AFP-Negative HCC.

BackgroundAlpha-fetoprotein (AFP) is the most important diagnostic and prognostic index of hepatocellular carcinoma (HCC). AFP-positive HCC can be easily diagnosed based on the serum AFP level and typical imaging features, but a number of HCC patients are negative (AFP < 20 ng/mL) for AFP. Therefore, it is necessary to develop novel diagnostic and prognostic biomarkers for AFP-negative HCC.MethodsRNA data from TCGA and differential expression of lncRNAs, miRNAs, and mRNAs were downloaded to analyze the differential RNA expression patterns between AFP-negative HCC tissues and normal tissues. A lncRNA-miRNA-mRNA ceRNA regulatory network was constructed to elucidate the interaction mechanism of RNAs. Functional enrichment analysis of these DEmRNAs was performed to indirectly reveal the mechanism of action of lncRNAs. A PPI network was built using STRING, and the hub genes were identified with Cytoscape. The diagnostic value of hub genes was assessed with receiver operating characteristic (ROC) analysis. And the prognostic value of RNAs in the ceRNA was estimated with Kaplan–Meier curve analysis.ResultsA total of 131 lncRNAs, 185 miRNA, and 1309 mRNAs were found to be differentially expressed in AFP-negative HCC. A ceRNA network consisting of 12 lncRNA, 23 miRNA, and 74 mRNA was constructed. The top ten hub genes including EZH2, CCNB1, E2F1, PBK, CHAF1A, ESR1, RRM2, CCNE1, MCM4, and ATAD2 showed good diagnostic power under the ROC curve; and 2 lncRNAs (LINC00261, LINC00482), 3 miRNAs (hsa-miR-93, hsa-miR-221, hsa-miR-222), and 2 mRNAs (EGR2, LPCAT1) were found to be associated with the overall survival of AFP-negative patients.ConclusionThis study could provide a novel insight into the molecular pathogenesis of AFP-negative HCC and reveal some candidate diagnostic and prognostic biomarkers for AFP-negative HCC.

Alpha-Fetoprotein- and CD40Ligand-Expressing Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma.

Simple SummaryIn first clinical trials, vaccinations against tumor-associated antigens (TAA), such as Alpha-Fetoprotein (AFP) using antigen presenting cells, such as dendritic cells (DC), failed to achieve effective immune responses towards hepatocellular carcinoma (HCC). CD40Ligand is a potent immune checkpoint, which can increase the antitumoral immune response of DC. In this study, a subcutaneous vaccination with DCs, which were transduced with AFP-coding adenoviruses and an intratumoral treatment with DCs, which were transduced with CD40L-coding adenoviruses, induced an antitumoral immune response and led to complete remissions and long-term survival in 62% of mice with established HCC. Combined strategy causes rapid and profound changes in the tumor environment with enhanced Th1-cytokine expression, strong tumor infiltration of cytotoxic T lymphocytes and DC, and higher tumor apoptosis, leading to effective tumor regression of HCC. Thus, intratumoral CD40L co-stimulation represents a promising tool for improving tumor-antigen DC-based immunotherapy of HCC.Dendritic cells (DC) as professional antigen presenting cells are able to prime T-cells against the tumor-associated antigen α-fetoprotein (AFP) for immunotherapy of hepatocellular carcinoma (HCC). However, a strong immunosuppressive tumor environment limits their efficacy in patients. The co-stimulation with CD40Ligand (CD40L) is critical in the maturation of DC and T-cell priming. In this study, the impact of intratumoral (i.t.) CD40L-expressing DC to improve vaccination with murine (m)AFP-transduced DC (Ad-mAFP-DC) was analyzed in subcutaneous (s.c.) and orthotopic murine HCC. Murine DC were adenovirally transduced with Ad-mAFP or Ad-CD40L. Hepa129-mAFP-cells were injected into the right flank or the liver of C3H-mice to induce subcutaneous (s.c.) and orthotopic HCC. For treatments, 106 Ad-mAFP-transduced DC were inoculated s.c. followed by 106 CD40L-expressing DC injected intratumorally (i.t.). S.c. inoculation with Ad-mAFP-transduced DC, as vaccine, induced a delay of tumor-growth of AFP-positive HCC compared to controls. When s.c.-inoculation of Ad-mAFP-DC was combined with i.t.-application of Ad-CD40L-DC synergistic antitumoral effects were observed and complete remissions and long-term survival in 62% of tumor-bearing animals were achieved. Analysis of the tumor environment at different time points revealed that s.c.-vaccination with Ad-mAFP-DC seems to stimulate tumor-specific effector cells, allowing an earlier recruitment of effector T-cells and a Th1 shift within the tumors. After i.t. co-stimulation with Ad-CD40L-DC, production of Th1-cytokines was strongly increased and accompanied by a robust tumor infiltration of mature DC, activated CD4+-, CD8+-T-cells as well as reduction of regulatory T-cells. Moreover, Ad-CD40L-DC induced tumor cell apoptosis. Intratumoral co-stimulation with CD40L-expressing DC significantly improves vaccination with Ad-mAFP-DC in pre-established HCC in vivo. Combined therapy caused an early and strong Th1-shift in the tumor environment as well as higher tumor apoptosis, leading to synergistic tumor regression of HCC. Thus, CD40L co-stimulation represents a promising tool for improving DC-based immunotherapy of HCC.

A Study to Access the Correlation between Serum Alpha Fetoprotein and Radiological Image Finding in Patients with Malignant Liver Mass

Malignant liver mass remains a major health problem worldwide. Patients with chronic liver disease, the accuracy of ultrasound scan (US), spiral computed tomography (CT), magnetic resonance imaging (MRI), and alpha-fetoprotein (AFP) in diagnosing hepatocellular carcinoma (HCC) and malignant liver disease is assessed in this study. This study helps to find out the significance of serum alpha protein in patients with malignant liver mass. Retrospective cross-sectional study was done on patients with malignant liver mass. A total of 62 patients were diagnosed with malignant liver mass, out of which 44 are male, and 18 were female. They were grouped into three age groups 30-50 years, 51-70 years and above 70 years. In this study, out of the radiologically diagnosed malignant liver mass, HPE has proven hepatocellular carcinoma where 67.7% and liver secondaries where 29.03%. 55% of malignant liver mass has raised alpha feto proteins. 74% of HCC diagnosed and confirmed on biopsy have elevated alpha feto proteins. Only 11% of multiple malignant liver lesions have elevated alpha feto proteins.AFP receptors are expressed only in the AFP-positive HCC tissues. In the AFP-negative HCC, the rate of tumour growth would probably be expected to be relatively slow, and tumour staging might be lower than in AFP-positive. Based on this study, radiological imaging has been very useful in the diagnosis of various malignant liver masses in both elevated and normal AFP.

AFP promotes HCC progression by suppressing the HuR-mediated Fas/FADD apoptotic pathway

Hepatocellular carcinoma (HCC) is a major leading cause of cancer-related death worldwide. Alpha fetoprotein (AFP) is reactivated in a majority of hepatocellular carcinoma (HCC) and associated with poor patient outcomes. Although increasing evidence has shown that AFP can regulate HCC cell growth, the precise functions of AFP in hepatocarcinogenesis and the associated underlying mechanism remain incompletely understood. In this study, we demostrated that depleting AFP significantly suppressed diethylnitrosamine (DEN)-induced liver tumor progression in an AFP gene-deficient mouse model. Similarly, knocking down AFP expression inhibited human HCC cell proliferation and tumor growth by inducing apoptosis. AFP expression level was inversely associated with the apoptotic rate in mouse and human HCC specimens. Investigation of potential cross-talk between AFP and apoptotic signaling revealed that AFP exerted its growth-promoting effect by suppressing the Fas/FADD-mediated extrinsic apoptotic pathway. Mechanistically, AFP bound to the RNA-binding protein HuR, increasing the accumulation of HuR in the cytoplasm and subsequent inhibition of Fas mRNA translation. In addition, we found that inhibiting AFP enhanced the cytotoxicity of therapeutics to AFP-positive HCC cells by activating HuR-mediated Fas/FADD apoptotic signaling. Conclusion: Our study defined the pro-oncogenic role of AFP in HCC progression and uncovered a novel antiapoptotic mechanism connecting AFP to HuR-mediated Fas translation. Our findings suggest that AFP is involved in the pathogenesis and chemosensitivity of HCC and that blockade of AFP may be a promising strategy to treat advanced HCC.

Upregulation of Serum Sphingosine (d18:1)-1-P Potentially Contributes to Distinguish HCC Including AFP-Negative HCC From Cirrhosis.

BackgroundSerum sphingolipids are widely involved in the development of hepatocellular carcinoma (HCC). We investigated the serum sphingolipid profile in patients with HCC or cirrhosis and explored the potential diagnostic efficiency of serum sphingolipid metabolites which may be helpful in differentiating HCC including α-fetoprotein (AFP)-negative HCC from cirrhosis.MethodsSeventy-two HCC patients (including 24 AFP-negative HCC) and 104 cirrhotic patients were consecutively enrolled in this study. High-performance liquid chromatography–tandem mass spectrometry was used to detect a panel of 57 serum sphingolipid metabolites.ResultsTwenty-four sphingolipid metabolites showed significant differences between HCC and cirrhotic patients (all P < 0.05). Sphingosine (d18:1)-1-P was found to have the potential to differentiate HCC from cirrhosis by orthogonal partial least squares discriminant analysis (OPLS-DA). There was no significant difference in the efficacy of Sphingosine (d18:1)-1-P and AFP to distinguish HCC from cirrhosis, and the area under the receiver operating curve (AUC) were 0.85 and 0.83 (P > 0.05), respectively. When the cut-off value of Sphingosine (d18:1)-1-P was set at 56.29 pmol/0.1 ml, the sensitivity and specificity were 79.20% and 78.70%, respectively. Notably, the upregulation of Sphingosine (d18:1)-1-P could also distinguish AFP-negative HCC from cirrhosis with an AUC of 0.79. The sensitivity and specificity were 62.50% and 77.90% at a cut-off value of 56.29 pmol/0.1 ml. Spearman rank correlation analysis revealed that serum Sphingosine (d18:1)-1-P was not correlated with AFP in patients with cirrhosis, AFP-positive HCC, and AFP-negative HCC. Moreover, the difference in the diagnostic efficiency of serum Sphingosine (d18:1)-1-P was not statistically significant between tumor size (≤2 cm vs. >2 cm, P = 0.476). Also, there was no difference among patients with different TNM stages and BCLC stages.ConclusionThe upregulation of serum Sphingosine (d18:1)-1-P exhibits good diagnostic performance for HCC. Particularly, Sphingosine (d18:1)-1-P could also serve as a biomarker for the diagnosis of AFP-negative HCC. These findings may contribute to the non-invasive diagnosis of HCC including AFP-negative HCC.

New Blood Biomarkers for the Diagnosis of AFP-Negative Hepatocellular Carcinoma.

An early diagnosis of hepatocellular carcinoma (HCC) followed by effective treatment is currently critical for improving the prognosis and reducing the associated economic burden. Alpha-fetoprotein (AFP) is the most widely used biomarker for HCC diagnosis. Based on elevated serum AFP levels as well as typical imaging features, AFP-positive HCC (APHC) can be easily diagnosed, but AFP-negative HCC (ANHC) is not easily detected due to lack of ideal biomarkers and thus mainly reliance on imaging. Imaging for the diagnosis of ANHC is probably insufficient in sensitivity and/or specificity because most ANHC tumors are small and early-stage HCC, and it is involved in sophisticated techniques and high costs. Moreover, ANHC accounts for nearly half of HCC and exhibits a better prognosis compared with APHC. Therefore, the diagnosis of ANHC in clinical practice has been a critical issue for the early treatment and prognosis improvement of HCC. In recent years, tremendous efforts have been made to discover new biomarkers complementary to AFP for HCC diagnosis. In this review, we systematically review and discuss the recent advances of blood biomarkers for HCC diagnosis, including DNA biomarkers, RNA biomarkers, protein biomarkers, and conventional laboratory metrics, focusing on their diagnostic evaluation alone and in combination, in particular on their diagnostic performance for ANHC.

Abstract 3715: Long-chain non-coding RNA H19-miR-152-3p axis regulates AFP expression in hepatocellular carcinoma

Abstract Background: Serum alpha-fetoprotein (AFP) is the most widely used serological marker used to diagnose hepatocellular carcinoma (HCC). High expression of AFP is significantly associated with early postoperative recurrence and poor prognosis. However, the upstream regulation mechanism of AFP expression is not fully understood, and the role of long-chain non-coding RNA (lncRNA) in AFP expression regulation has not been reported. Methods: HCC cases from The Cancer Genome Atlas (TCGA) database were divided into 3 groups: (1) AFP&amp;lt;10ng/ml, (2) 10ng/ml≤AFP&amp;lt;1000ng/ml, and (3) AFP≥1000ng/ml. The differential expression and co-expression of lncRNAs and miRNAs with AFP among the 3 groups was analyzed. lncRNA-ceRNA for AFP expression regulation was constructed by using LncATLAS to predict lncRNAs localization, miRWalk3.0 to predict miRNA-AFP targets, and LncBase Predicted v.2 to predict miRNA-lncRNA targets. Results: In the 3 groups, 32 lncRNAs were differentially expressed (P&amp;lt;0.05). SNHG7, SNHG3, SNHG8, DSCR9, KCNQ1OT1, SNHG6, GAS5, SNHG11, H19, TMEM191A, and MEG3 were significantly positively correlated with AFP expression (r&amp;gt;0, P&amp;lt;0.05). SEC22B and SRD5A2 were significantly negatively correlated with AFP expression (r&amp;lt;0, P&amp;lt;0.05). Among the positively correlated lncRNAs, SNHG7, SNHG3, SNHG8, SNHG6, GAS5, and H19 were mainly located in the HepG2 cytoplasm, DSCR9, KCNQ1OT1, SNHG11, and MEG3 were mainly located in the HepG2 nucleus, and TMEM191A had no location information in HepG2 cells. We found 134 differentially expressed miRNAs (P&amp;lt;0.05), and hsa-miR-196a-5p, hsa-miR-18a-5p, hsa-miR-148b-3p, hsa-miR-483-5p, hsa-miR-1307-3p, hsa-miR-1301-3p, hsa-miR-222-5p, hsa-miR-18a-3p, hsa-miR-501-5p, hsa-miR-483-3p, hsa-miR-222-3p, hsa-miR-19a-3p, and hsa-miR-221-3p were significantly positively correlated with AFP expression (r&amp;gt;0, P&amp;lt;0.05). hsa-miR-99a-5p, hsa-miR-152-3p, hsa-miR-125b-2-3p, hsa-let-7c-5p, and hsa-miR-1295b-5p were significantly negatively correlated with AFP expression (r&amp;lt;0, P&amp;lt;0.05). Among the negatively correlated miRNAs, hsa-miR-152-3p, hsa-miR-125b-2-3p, and hsa-let-7c-5p were predicted to be able to target the AFP 3' UTR. LncRNAs were predicted for the 3 miRNAs mentioned above. At the LncBase score&amp;gt;0.60, 769 pairs of miRNA-lncRNA interaction were recognized. These predicted lncRNAs were then intersected with the 6 cytoplasm-located lncRNAs. H19 satisfied the constraints, and therefore, a lncRNA H19-miR-152-3p-AFP ceRNA was proposed. Conclusions: Our study revealed that the proposed H19-miR-152-3p-AFP pathway affects the expression of AFP in HCC, suggesting that the H19-miR-152-3p axis may play an important role in the development of typical AFP-positive HCC. Citation Format: Yang Ke, Xiang Kui, Hang Jiang, Tiangen Wu, Cheng Zhang, Jian Zhou, Jinze Li, Heng Zhang, Yueqiang Song, Juan Zhao, Huafeng Duan, Lin Wang, Yan Wang. Long-chain non-coding RNA H19-miR-152-3p axis regulates AFP expression in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3715.

Preoperative color Doppler ultrasonography predicts early recurrence in AFP-positive hepatocellular carcinoma.

The present study aimed to evaluate the hepatic hemodynamics of patients with α-fetoprotein (AFP)-positive hepatocellular carcinoma (HCC) and early recurrence (ER), as determined by color Doppler ultrasound. Screening with color Doppler ultrasound was performed in 150 patients with AFP-positive HCC, yielding 43 cases with postoperative ER. In addition, 35 healthy controls were enrolled in the study for comparison of the blood flow parameters between the healthy control and the HCC groups. Receiver operating characteristic curve analysis was performed to evaluate the predicted effect of Doppler for the ER of HCC. The average mean flow velocity of portal vein in patients with HCC (14.686±5.873 cm/s) was significantly lower in the HCC group compared with the healthy control group (17.631±3.569 cm/s; P=0.005). Additionally, the preoperative portal vein diameter in the HCC group was significantly higher compared with that in the healthy control group (P=0.001). Regarding the effect of surgery, the portal vein diameter was significantly increased in the patients postoperatively compared with preoperatively (P=0.003), while the pulsation and resistance indexes exhibited a decreasing trend in the postoperative group (P=0.001 and P=0.003, respectively). Notably, patients with HCC and ER presented with a higher resistance index (1.163±0.342) compared with the ER-free group (1.023±0.176; P=0.004). Furthermore, a significant difference in hepatic artery pulsation index was also observed between the ER group (0.673±0.075) and the ER-free group (0.624±0.056; P=0.018), indicating that an increased hepatic artery pulsation index may significantly predict the ER of HCC. In addition, areas under the curve of 0.683 and 0.700, respectively, suggested that the hepatic artery resistance and pulsation indexes may be used to diagnose ER in HCC (P=0.009 and P=0.004, respectively). Taken together, Doppler ultrasound provides a reliable and accurate quantification of hepatic hemodynamics for detecting ER in HCC with a good diagnostic accuracy.

Molecular differences between AFP-negative and AFP-positive hepatocellular carcinoma.

e15681 Background: Alpha-fetoprotein (AFP) is a major biomarker for diagnosis of hepatocellular carcinoma (HCC). Screening of molecular differences between AFP-negative and AFP-positive HCC can be helpful for its diagnosis. Methods: Genomic mutations, gene expressions and clinicopathological data of 270 HCC patients were collected from TCGA database. According to preoperative AFP level, patients were categorized into AFP- (0-20ng/mL, n = 112) and AFP+ ( &gt; 20 ng/mL, n = 158) groups. We identified significantly mutated genes and differentially expressed genes (DEGs) between the two groups and analyzed their correlation with prognosis. Results: The 1-, 3-, and 5-year overall survival (OS) rates were 91.5%, 76.3%, and 67.9% in AFP- and 84.3%, 62.5%, and 34.2% in AFP+. The difference was statistically significant (p = 0.0053). To identify AFP-biased mutation genes, we focused on highly mutated genes in each group (≥5% mutation frequency). At FDR = 0.05, we identified 11 AFP-biased genes: CTNNB1, VWA5B2, HERC2, ZFYVE26, SPEN, MUC6, DST, WDR87, DNAH1, PTPN13, and FANCM. The most striking gene identified was CTNNB1, which is a WNT pathway oncogene. CTNNB1 was more frequently mutated in AFP- than in AFP+ (35.8% vs. 17.0%, p = 0.00059). The OS for HCC was calculated on the basis of the mutation of each gene, which showed that MUC6 mutation was negatively associated with OS (p = 0.018), while other ten genes showed no obvious difference. With the mRNA expression data, a total of 949 DEGs were identified, consisting of 718 upregulated in AFP+, and 231 upregulated in AFP-. The enriched pathways of the DEGs included cell cycle, metabolism of xenobiotics by cytochrome P450 and chemical carcinogenesis. AVP, CCL20 and MUC6 genes were identified as hub genes in the PPI network. Conclusions: According to the results of this study, the AFP level is associated with patients’ OS rates, and AFP- patients tend to have higher CCTNB1 mutation rate. Our ongoing study will verify this result by enrolling new HCC patients.

Long Non-Coding RNA IGF2AS in Serum may be a Biomarker for Diagnosis of Hepatitis B Virus-Related Hepatocellular Carcinoma.

The current study aims to evaluate whether serum IGF2AS can be used as a marker for early diagnosis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). First, the expression of IGF2AS in serum and cancer tissues of HBV-related HCC patients was detected using RT-PCR. Then, according to the Barcelona Staging Method (BCLC), HBV-related HCC patients were further divided into group A, B, C, and D. The serum IGF2AS levels of different groups were further detected. Additionally, HBV-related HCC patients were divided into alpha-fetoprotein (AFP) negative cases and AFP positive cases. The serum level of IGF2AS was also evaluated. Our data showed that the level of IGF2AS in liver cancer tissues of HBV-related HCC was significantly higher than that in the adjacent non-cancerous tissues. Meanwhile, the serum level of IGF2AS in the HBV-related HCC group was higher than that in healthy control group, chronic HBV group, and HBV-related cirrhosis. Moreover, serum IGF2AS in patients with HBV-related HCC gradually increased in groups A, B, C, and D of HBV-re-lated HCC patients according to BCLC classification. Further analysis showed that serum IGF2AS levels were increased in both AFP-negative HCC patients and AFP-positive HCC patients compared to that in HBV-related cirrhosis patients, suggesting that serum IGF2AS can be used in early identification and diagnosis of HBV-related HCC irrespective of AFP levels. In summary, for the first time, the current study demonstrated that serum IGF2AS may be a potential biomarker for the diagnosis of HBV-related HCC.

Metastatic Hepatocellular Carcinoma Secreting Intact PTH: A Case Report

Hypercalcemia is a known paraneoplastic complication of hepatocellular carcinoma (HCC), however it is more commonly described in advanced-stage cancer associated with high alpha fetoprotein (AFP) levels. Furthermore, few cases report intact-PTH (iPTH) secretion as the driving mechanism. We describe a case of a patient with prior HCC and previously elevated AFP who presented with profound hypercalcemia secondary to recurrent, metastatic HCC and biopsy-proven ectopic iPTH secretion despite low AFP. A 68-year-old man with cirrhosis attributed to chronic hepatitis C infection (successfully treated with antiviral therapy) complicated by BCLC stage B HCC previously treated with radiation therapy presented with duodenal perforation in the setting of severe hypercalcemia. During exploratory laparotomy and repair, a vascular, peri-splenic mass was identified incidentally. It was not biopsied given concern for bleeding. Workup revealed a corrected calcium level of 13.4 mg/dL, elevated iPTH (397 pg/mL), normal parathyroid hormone related-peptide (PTHrP), 1,25 vitamin D, and SPEP. AFP was 2.3 ng/mL (though peaked at 1,107 ng/mL prior to radiation treatment). A parathyroid ultrasound and Sestamibi scan were non-revealing. Abdominal MRI demonstrated an enhancing mass correlating with that seen intraoperatively and multiple liver lesions unchanged from prior. He was managed with intravenous fluids, calcitonin, cinacalcet, and intermittent diuretics, but sustained normocalcemia was never achieved. Repeat PET scan weeks later revealed multiple new FDG-avid liver lesions and an enlarging FDG-avid peri-splenic mass. Eventual biopsy of the peri-splenic mass revealed morphology consistent with an HCC metastasis. PTH staining was positive. With largely refractory hypercalcemia, bisphosphonates were initiated and liver embolization was pursued. Unfortunately the patient died of hypercalcemia-related complications. This is a very rare case of a patient with prior AFP-positive HCC who presented with hypercalcemia and a new peri-splenic mass found to be an HCC metastasis secreting iPTH. What perhaps delayed diagnosis was that repeat AFP levels were low, suggesting that recurrence of HCC was unlikely, thus prompting workup for primary hyperparathyroidism and neuroendocrine malignancy instead. This case is an excellent reminder that while rare, HCC recurrences can transition from high to low AFP types and metastatic HCC should be considered a potential source of ectopic iPTH.

Direct comparison of five serum biomarkers in early diagnosis of hepatocellular carcinoma.

BackgroundAlthough a number of serum biomarkers for detection of hepatocellular carcinoma (HCC) have been explored, their exact diagnostic value remains unclear. We aimed to conduct a direct comparison of five representative serum biomarkers for detecting HCC and to derive multi-marker prediction algorithms.Patients and methodsIn total, 846 patients were recruited from three hospitals in China, including 202 HCC patients, 226 liver cirrhosis patients, 215 chronic hepatitis B virus-infected patients, and 203 healthy volunteers. Serum levels of alpha-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3), des-gamma-carboxyprothrombin (DCP), squamous cell carcinoma antigen, and centromere protein F autoantibody were measured by ELISA. The diagnostic performances of individual biomarkers and multi-marker combinations were evaluated by receiver operating characteristics analysis. The bootstrapping method was adopted to adjust for potential overfitting of all diagnostic indicators.ResultsDCP exhibited the best diagnostic performance, with areas under the curve (AUC) for detecting HCC of 0.82 (95% CI 0.64–0.80) and sensitivity of 65.2% (95% CI 63.3–82.1%) at 90% specificity. Of note, DCP showed similar diagnostic efficacy for detecting AFP-positive and AFP-negative HCC. After a comprehensive search for multi-marker combinations, a two-marker prediction algorithm including AFP and DCP was constructed and yielded an AUC of 0.87 (95% CI 0.68–0.84) for detecting HCC. In addition, the combination showed good ability in discriminating early-stage HCC and decompensated liver cirrhosis, with an AUC of 0.81 (95% CI 0.75–0.86).ConclusionDCP could be a complementary biomarker in the early diagnosis of HCC. The constructed multi-marker prediction algorithms could contribute toward distinguishing HCC from non-malignant chronic liver diseases.

Hepatocellular carcinoma-targeting oncolytic adenovirus overcomes hypoxic tumor microenvironment and effectively disperses through both central and peripheral tumor regions

Cancer-specific promoter driven replication of oncolytic adenovirus (Ad) is cancer-specific, but shows low transcriptional activity. Thus, we generated several chimeric α-fetoprotein (AFP) promoter variants, containing reconstituted enhancer and silencer regions, to preferentially drive Ad replication in hepatocellular carcinoma (HCC). Modified AFP promoter, containing 2 enhancer A regions and a single enhancer B region (a2bm), showed strong and HCC-specific transcription. In AFP-positive HCCs, gene expression was 43- to 456-fold higher than those of control AFP promoter lacking enhancers. a2bm promoter was further modified by inserting multiple hypoxia-responsive elements (HRE) to generate Ha2bm promoter, which showed stronger transcriptional activity than a2bm promoter under hypoxic conditions. Ha2bm promoter-regulated oncolytic Ad (Ha2bm-d19) showed a stronger antitumor and proapoptotic effect than did a2bm promoter-regulated oncolytic Ad (a2bm-d19) in HCC xenograft tumors. Systemically administered Ha2bm-d19 caused no observable hepatotoxicity, whereas control replication-competent Ad, lacking cancer specificity (d19), induced significant hepatic damage. Ha2bm-d19 caused significantly lower expression of interleukin-6 than d19, showing that HCC-targeted delivery of Ad attenuates induction of the innate immune response against Ad. This chimeric AFP promoter enabled Ad to overcome the hypoxic tumor microenvironment and target HCC with high specificity, rendering it a promising candidate for the treatment of aggressive HCCs.

GTSF1 gene may serve as a novel potential diagnostic biomarker for liver cancer.

The gametocyte-specific factor 1 (GTSF1) gene participates in DNA methylation and retrotransposon activation in germ cells, particularly during cell proliferation. The present study aimed to assess the level of GTSF1 gene expression in liver cancer tumor tissues, and its role in human hepatoma cell lines in vitro and in a nude mouse model in vivo. GTSF1 gene expression was detected in liver cancer tumor tissues, compared with in healthy controls, via reverse transcription quantitative polymerase chain reaction. An adeno-associated virus vector was used to study tumor stem cell proliferation in vivo. A plasmid expressing GTSF1 was constructed and transfected into various human hepatoma cell lines, in order to analyze the cellular proliferation and apoptosis of liver cancer cells using small interfering (si)RNAs in vitro. In the present study, GTSF1 gene expression was detected in 18/24 (75.0%) liver cancer tumor tissues from patients with hepatocellular carcinoma (HCC), and elevated GTSF1 expression was identified in the tissue of one of 32 healthy control samples (3.13%; P<0.05). Notably, the GTSF1 gene was expressed at a higher frequency in AFP-positive HCC samples (14/16, 87.50%) compared with in AFP-negative HCC samples (4/8, 50.0%; P=0.129). In addition, there was no statistical significance between GTSF1 expression in non-HBV-infected (71.42%) and HBV-infected HCC specimens (76.47%), as determined by a χ2 test (P=0.921). It was demonstrated that GTSF1 significantly increased the tumorigenicity of Ad-shNC-transfected (GTSF1-positive) HepG2 cells in the nude mouse xenograft model, whereas the sizes and weights of the tumors in the GTSF1-negative group were dercreased in comparison with the GTSF1-positive group (P<0.05). Reduced levels of GTSF1 mRNA, along with fewer and smaller colonies, were identified in two groups of human liver cancer cells treated with with GTSF1-targeting siRNA, when compared with cells without GTSF1 mRNA interference (P<0.05). In summary, the present study elucidated the GTSF1 mRNA expression pattern in liver cancer, and investigated the potential role of GTSF1 in tumorigenesis. The data suggest an important role for the GTSF1 gene in the molecular etiology of hepatocarcinogenesis, and indicate a potential application of GTSF1 mRNA expression in liver cancer diagnosis and therapy.