PIVKA-II Research Articles

Comprehensive Liquid Profiling of Circulating Tumor DNA and Protein Biomarkers in Long-Term Follow-Up Patients with Hepatocellular Carcinoma

Circulating tumor DNA (ctDNA) provides a novel approach for detecting tumor burden and predicting clinical outcomes of hepatocellular carcinoma (HCC). Here, we performed a thorough evaluation of HCC circulating genetic features and further fully integrated them to build a robust strategy for HCC monitoring and prognostic outcome assessment. We performed target sequencing and low-coverage whole-genome sequencing on plasma samples collected from 34 long-term follow-up patients with HCC to capture tumor somatic SNVs and CNVs, respectively. Clinical information was also obtained to evaluate the prognostic performance of ctDNA comparing with clinically applied protein biomarkers. All plasma samples before surgery showed somatic genetic variations resembling corresponding tumor tissues. During follow-up, SNVs and CNVs dynamically changed correlating to patients' tumor burden. We integrated the comprehensive ctDNA mutation profiles to provide a robust strategy to accurately assess patients' tumor burden with high consistence comparing with imaging results. This strategy could discover tumor occurrence in advance of imaging for an average of 4.6 months, and showed superior performance than serum biomarkers AFP, AFP-L3%, and Des-Gamma-Carboxy Prothrombin (DCP). Furthermore, our strategy could precisely detect minimal residual disease (MRD) in advance and predict patients' prognostic outcomes for both relapse-free survival (P = 0.001) and overall survival (P = 0.001); further combining ctDNA with DCP could increase the sensitivity for MRD detection. We demonstrated that plasma CNV and SNV levels dynamically correlated with patients' tumor burden in HCC. Our strategy of comprehensive mutation profile integration could accurately and better evaluate patients' prognostic risk and detect tumor occurrence in advance than traditional strategies.

Ramucirumab and GSK1838705A Enhance the Inhibitory Effects of Low Concentration Sorafenib and Regorafenib Combination on HCC Cell Growth and Motility.

Several new multikinase inhibitors have recently been introduced into clinical practice for hepatocellular carcinoma (HCC) therapy. Small increases in survival were reported as well as considerable toxicity. There is thus a need for effective therapies with lower toxicities. We examined whether a combination of sorafenib and regorafenib might also be effective at very low concentrations, with resulting potential for lessened clinical toxicity. MTT test, clonogenic assay, Ki67 staining and cell cycle analysis were assessed for cell proliferation and Annexin V and western blotting analysis relative to the expression of cleaved Caspase-3 and BID for cell apoptosis. In these experimental conditions cell growth and migration were potently inhibited and apoptosis induced even in HCC cells producing high alpha fetoprotein (AFP) levels (clinically worse prognosis). The combination also inhibited levels of the two HCC biomarkers, AFP and des gamma carboxy prothrombin (DCP). Additional inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR) or Insulin-like Growth Factor 1 Receptor (IGF1R) enhanced effects on AFP and DCP levels, cell growth inhibition and MAPK and PI3K/Akt signaling inhibition due to sorafenib/regorafenib combination. These combinations have the potential for decreased toxicity while simultaneously enhancing therapeutic effects. This potential decrease in toxicity is being explored in ongoing studies.

Refractoriness to transarterial chemoembolization in patients with recurrent hepatocellular carcinoma after curative resection.

Background/AimsIt is important to identify patients who are refractory to transarterial chemoembolization (TACE), which is performed for the treatment of hepatocellular carcinoma (HCC). We investigated the predictors of poor treatment outcomes in patients with recurrent HCC treated who were treated with TACE after curative resection.Methods428 patients with recurrent HCC after curative resection who were treated with TACE were enrolled.ResultsThe median age of the study population was 59.2 years. On multivariate analysis, ≥2 TACE procedures within 6 months (hazard ratio [HR] = 1.898), and the des-gamma carboxyprothrombin level (HR = 1.000) independently predicted the progression to Barcelona Clinic Liver Cancer (BCLC) stage C in patients with BCLC stage 0-B HCC (both P<0.05). In addition, ≥2 and ≥3 TACE procedures within 6 months independently predicted mortality in the entire study population (HR = 1.863 and 1.620, respectively). The probability of progression to BCLC stage C in patients with BCLC stage 0-B HCC and the mortality rate in the entire study population were significantly higher in patients treated with ≥2 TACE within 6 months than in those who underwent fewer procedures (P = 0.002 and P<0.001, respectively).ConclusionsMore than 2 TACE procedures within 6 months might be associated with the refractoriness to TACE in patients with recurrent HCC after curative resection.

Longitudinal Assessment of Three Serum Biomarkers to Detect Very Early-Stage Hepatocellular Carcinoma.

We aimed to determine the surveillance performance of alpha-fetoprotein (AFP), lectin-reactive AFP (AFP-L3), des-gamma-carboxy prothrombin (DCP), and their combinations for the early detection of hepatocellular carcinoma (HCC) by using prospectively collected longitudinal samples in patients at risk. Among 689 patients with cirrhosis and/or chronic hepatitis B who participated in four prospective studies, 42 HCC cases were diagnosed, selected, and matched with 168 controls for age, sex, etiology, cirrhosis, and duration of follow-up in a 1:4 ratio. Levels of AFP, AFP-L3, and DCP at the time of HCC diagnosis, month -6, and month -12 were compared between cases and controls. Of 42 HCC cases, 39 (93%) had cirrhosis, 36 (85.7%) had normal alanine aminotransferase levels, and 31 (73.8%) had very early-stage HCC (single <2 cm). AFP and AFP-L3 began to increase from 6 months before diagnosis of HCC in cases (P < 0.05), while they remained unchanged in controls. At HCC diagnosis, the area under the receiver operator characteristic curves (AUROCs) for AFP, AFP-L3, and DCP were 0.77, 0.73, and 0.71, respectively. Combining AFP and AFP-L3 showed a higher AUROC (0.83), while adding DCP did not further improve the AUROC (0.86). With the optimal cutoff values (AFP, 5 ng/mL; AFP-L3, 4%), the sensitivity and specificity of AFP and AFP-L3 combination were 79% and 87%, respectively. The sensitivity of ultrasonography was 48.6%, which was increased to 88.6% and 94.3% by adding AFP and AFP + AFP-L3, respectively. Conclusion: Among three biomarkers, AFP showed the best performance in discriminating HCC cases from controls; the AFP and AFP-L3 combination, adopting cutoff values (5 ng/mL and 4%, respectively), significantly improved the sensitivity for detecting HCC at a very early stage.

Clinical Significance of Preoperative Hepatocellular Carcinoma With High Lens culinaris Agglutinin-reactive Fraction of Alpha-Fetoprotein, But Low Alpha-Fetoprotein.

The aim of this study was to verify the significance of high Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) in patients with hepatocellular carcinoma (HCC) with low AFP. There were 283 patients with low AFP who underwent initial hepatic resection with or without radiofrequency ablation for HCC. Patients were divided into two groups based on AFP-L3 values: >10%: high AFP-L3 (n=24); and ≤10%: low AFP-L3 (n=259). Overall survival (OS) and 2-year recurrence rates were compared, and independent prognostic factors were identified. The OS and 2-year recurrence rates of the high AFP-L3 group were significantly worse than those of the low AFP-L3 group. The independent prognostic factors for poor OS were des-gamma-carboxy prothrombin (DCP) of >40 mAU/ml, microvascular invasion, and invasive growth, and those for 2-year recurrence were 99mTc-galactosyl human serum albumin uptake ratio of <0.90, DCP of >40 mAU/ml, multiple tumors, microvascular invasion, and poor differentiation. DCP levels increased with AFP-L3, and cases with high DCP and AFP-L3 had worse prognoses and higher 2-year recurrence rates compared to those with elevation of only one of these. Patients with high AFP-L3 but low AFP have poor prognosis and high 2-year recurrence rates. DCP strongly reflects HCC malignancy in patients with low AFP.

Current Status Regarding Tumour Progression, Surveillance, Diagnosis, Staging, and Treatment Of HCC: A Literature Review

Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver, and is globally considered to be a major causes of cancer-associated mortality. The early diagnosis of HCC improves overall survival through the application of suitable treatment options. This article presents some of the techniques for the surveillance of HCC like ultrasonography and the use of tumour biomarkers such as α-fetoprotein (AFP), DesGamma-Carboxy Prothrombin (DCP) and others. Included in the discussion will be diagnostic methods like computed tomography (CT), magnetic resonance imaging (MRI), contrast enhancement ultrasound (CEUS), and fluorodeoxyglucose positron emission tomography hybrid with computed tomography (FDG PET/CT). Current molecular pathogenesis related to HCC and the molecular steps that determine the transition from benign to malignancy are also analysed. The HCC stages which depends on the Barcelona Clinic Liver Cancer (BCLC) algorithm are also discussed. Finally, this review article discusses the present therapeutic and treatment options for HCC such as resection, transplantation, or ablation used to treat early stage cancer. Also included will be trans-arterial chemoembolization (TACE) and Sorafenib for patients with intermediate and advanced-stage cancer, respectively.

Clinical application of alpha fetoprotein combined with abnormal prothrombin in detecting primary liver cancer

Objective To investigate the clinical value of serum alpha fetoprotein(AFP) combined with abnormal prothrombin(PIVKA-Ⅱ) in the detection of primary liver cancer. Methods From May 2017 to October 2017, 80 cases of primary liver cancer in Department of Hepatobiliary Surgery of Deyang People's Hospital, 100 cases of non HCC patients(including chronic liver disease, cirrhosis, metastatic liver cancer) were selected, and another 100 healthy people were selected as control group.The levels of serum AFP and PIVKA-Ⅱ were detected by SIEMENS XP automatic immunoassay analyzer and lumipulseg1200 automatic immune analyzer respectively.All data were analyzed by SPSS17.0 analysis software. Results Comparison between primary liver cancer and non-primary liver cancer: the levels of serum AFP and PIVKA-Ⅱ(F=1.763; F=1.788, sig=0.000, P<0.05). Comparison between non-primary liver cancer and normal group: the levels of serum AFP and PIVKA-Ⅱ(F=0.896; F=0.346, sig=0.000, P<0.05). Serum AFP had a sensitivity of 75.0% and a specificity of 71.5% in the diagnosis of primary liver cancer, while PIVKA-Ⅱ had a sensitivity of 82.0% and a specificity of 78.7%.The diagnosis sensitivity and specificity of AFP combined with PIVKA-Ⅱ reached to 92.0% and 95.0%.The sensitivity of AFP combined with PIVKA-Ⅱ in the diagnosis of primary liver cancer had statistically significant differences compared with AFP and PIVKA-Ⅱ alone(χ2=4.112, P=0.008; χ2=2.991, P=0.010). Conclusion Combined detection of serum AFP and PIVKA-II has important clinical significance in the diagnosis of primary liver cancer. Key words: Liver Neoplasms; Alpha-fetoproteins; Prothrombin

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.

Role of des-gamma-carboxy prothrombin in assessment of liver function and prognosis of patients with liver cirrhosis

Objective To investigate the role of des-gamma-carboxy prothrombin (DCP) in assessment of liver function and prognosis of patients with liver cirrhosis. Methods From January 2013 to August 2016, a total of 137 patients with liver cirrhosis in Shanghai Changzheng Hospital were enrolled. The serum DCP level was measured, the clinical data was collected and the complication and survival situation was followed up. The 137 patients were divided into DCP negative group (DCP≤40 mAU/mL, 118 cases) and DCP positive group (DCP>40 mAU/mL, 19 cases). Forty-five patients with compensated liver cirrhosis were divided into high-level DCP group (DCP>16.5 mAU/mL, 32 cases) and low-level DCP group (DCP≤16.5 mAU/mL, 13 cases). Chi square test was used to analyze the difference in the positive rate of DCP in patients with different Child-Pugh classification. Spearman correlation test was performed to analyze the correlation between DCP and model for end-stage liver disease (MELD) scores. Kaplan-Meier survival curve was used to analyze the correlation between DCP and liver disease related mortality. Results Compared to that of DCP negative group, albumin level of patients in DCP positive group decreased (35 g/L, 20 to 57 g/L vs. 29 g/L, 17 to 42 g/L), however, total bilirubin (TBil), prothrombin time (PT), and international normalized ratio all increased (12.9 mg/L, 1.80 to 83.0 mg/L vs.22.2 mg/L, 6.4 to 169.0 mg/L; 15.5 s, 11.7 to 35.7 s vs.17.5 s, 13.9 to 33.4 s; 1.24, 0.96 to 3.72 vs.1.44, 1.09 to 3.22), and the differences were statistically significant (Z=-2.785, -2.891, -2.945 and -2.879, all P 40 mAU/mL) rates of Child-Pugh A, B and C patients were 1.8% (1/55), 21.2% (11/52) and 23.3% (7/30), respectively, and the difference was statistically significant (χ2=11.246, P=0.003). The DCP levels of patients with Child-Pugh class B and C cirrhosis were significantly correlated with MELD scores (r=0.259, P=0.021). There were 16 and three patients with ascites and spontaneous bacterial peritonitis (SBP) of DCP positive group, and the incidence was higher than that of DCP negative group (55.1%, 65/118 and 1.7%, 2/118), and the differences were statistically significant (χ2=5.744 and 97.636, both P<0.05). Patients in high-level DCP group had a higher proportion of clinical decompensation than low-level DCP group (53.1% (17/32) and two cases), the difference was statistically significant (χ2=5.397, P=0.024). The overall survival rate of DCP positive group (nine survival cases) were lower than that of DCP negative group (87.9%, 87/99), and the difference was statistically significant (χ2=5.442, P=0.020). Conclusions Serum DCP level is closely related to liver function, ascites and SBP in patients with liver cirrhosis. Furthermore, it is associated with occurrence of decompensation in compensated patients and liver-related mortality in patients with liver cirrhosis. DCP may be a useful serum marker for evaluation of disease severity and prognosis in patients with liver cirrhosis in clinical practice. Key words: Liver cirrhosis; Des-gamma-carboxy prothrombin; Liver fuction; Prognosis

Microvascular invasion of single small hepatocellular carcinoma ≤3 cm: Predictors and optimal treatments.

BackgroundSmall hepatocellular carcinomas (HCC ≤3 cm) are generally considered to have low malignant potential; however, some of them display pathological microvascular invasion (MVI).MethodsBetween 1991 and 2013, 414 patients with a single HCC ≤3 cm underwent curative hepatic resection (HR). Predictors for MVI were identified. Using another cohort (149 patients during 2000‐2014), our predictors for MVI in HCC ≤3 cm were validated. In 428 patients with a single HCC ≤3 cm who had predictors for MVI, survival was compared among anatomical HR (n = 149), partial HR (n = 227), and radiofrequency ablation (RFA) (n = 52).ResultsThe positive rate of MVI reached 40.6% (168/414 patients). Independent predictors for MVI were as follows: tumor diameter ≥2 cm (odds ratio 1.84, P = .0052), alpha‐fetoprotein (AFP) ≥200 ng/mL (odds ratio 1.82, P = .0466), and des‐gamma‐carboxy prothrombin (DCP) ≥40 mAU/mL (odds ratio 1.79, P = .0126). Matching at least one predictor among these three could predict MVI in HCC ≤3 cm well (sensitivity 82.8%, positive predictive value [PPV] 48.7%). This criterion could also predict MVI in HCC ≤3 cm well in another cohort (sensitivity 82.8%, PPV 30.3%). In patients with single HCC ≤3 cm matching our criterion for predicting MVI, anatomical HR led to significantly better survival in both disease‐free (hazard ratio 0.689, P = .0231) and overall (hazard ratio 0.589, P = .0316) survivals.ConclusionMatching at least one factor among three (tumor diameter ≥2 cm, AFP ≥200 ng/mL, or DCP ≥40 mAU/mL) can predict MVI in HCC ≤3 cm. In such patients, anatomical HR would be recommended to improve survival.

Differences in the impact of prognostic factors for hepatocellular carcinoma over time.

The aim of the present study was to evaluate the prognostic significance of serum markers that reflect tumor progression, liver function, or liver fibrosis in patients with hepatocellular carcinoma (HCC), focusing on how their impact changes over time after diagnosis. Alpha‐fetoprotein (AFP), des‐gamma‐carboxy prothrombin (DCP), albumin‐bilirubin (ALBI) score, aspartate aminotransferase to platelet ratio index (APRI), and FIB‐4 index were measured at the time of initial non‐recurrent HCC diagnosis in 1669 patients between 1997 and 2016. Survival rates after diagnosis were compared after stratifying patients by these markers. Time‐dependent receiver‐operating characteristics (ROC) analysis was carried out to assess how these markers predict patient survival or death. Serum AFP and DCP levels, ALBI score, and APRI and FIB‐4 index were strongly correlated with HCC progression, liver function, and degree of liver fibrosis, respectively. Survival rates after diagnosis were significantly different when patients were stratified by these markers. In the time‐dependent ROC analysis, AFP and DCP had a high prognostic impact within 3 years of diagnosis but the impact decreased thereafter. In contrast, APRI and FIB‐4 index had higher prognostic impact 10 years after diagnosis. ALBI score had a high prognostic impact throughout the study period. Time‐dependent ROC analysis clearly showed changes in the prognostic importance of serum markers based on the duration after diagnosis. Whereas the prognostic impact of tumor progression markers was strong in the short term, liver fibrosis markers had higher prognostic impact long after diagnosis. Liver function had constant prognostic impact on patient survival after diagnosis.

Des-gamma-carboxy prothrombin in hepatocellular cancer patients waiting for liver transplant: a systematic review and meta-analysis.

The use of des-gamma-carboxy prothrombin (DCP) as a predictor of the risk of recurrence of hepatocellular cancer (HCC) after liver transplant (LT) has recently gained interest, especially in view of the recent extension of the eligibility criteria of these patients for LT. The aim of the present study is to look into this important matter based on a systematic review and meta-analysis. A systematic literature review about the role of DCP in the specific setting of LT for HCC has been conducted. Three selected studies, which showed a high rate of homogeneity (I2 = 0.0%), confirmed that the tumor marker DCP is a useful predictive factor, indicating a 5-fold increased risk for HCC recurrence after LT (p<0.001). The meta-analysis enabled us to underline the importance of DCP in the refinement of the eligibility criteria of HCC patients for LT. This information, based on Japanese studies performed in the setting of living-donor LT only, needs further validation in the Western world both in the setting of post-mortem and living-donor LT.

Combining des-gamma-carboxyprothrombin and alpha-fetoprotein for hepatocellular carcinoma diagnosing: an update meta-analysis and validation study.

Controversies about the combination of des-gamma-carboxyprothrombin (DCP) and alpha-fetoprotein (AFP) for hepatocellular carcinoma diagnosing still exist. Hence, we performed this updated meta-analysis to estimate the diagnostic value of DCP , AFP and DCP + AFP in HCC. In addition, we conducted a validation study to analyze the performance of the candidate makers. After a systematic literature review, 27 studies from 20 articles were identified from four major databases. The pooled sensitivity and specificity were 69% and 89%, respectively, for DCP; for AFP, they were 65% and 88%, respectively; and they were 82% and 85%, respectively, for DCP + AFP. The values of the area under the curve (AUC) for DCP, AFP, DCP + AFP, respectively, were 0.88, 0.75, and 0.90. The validation study confirmed that the performance of DCP + AFP (sensitivity = 84%, specificity = 86%; AUC = 0.887) was higher than that of DCP (sensitivity = 76%, specificity = 92%; AUC = 0.843) or AFP (sensitivity = 73%, specificity = 92%; AUC = 0.837) alone.

Serum monomeric laminin-γ2 as a novel biomarker for hepatocellular carcinoma.

The diagnosis of hepatocellular carcinoma (HCC) in the early stages is important for successful clinical management. Laminin (Ln)‐γ2 expression has been reported in various types of malignant carcinomas. We recently developed a highly sensitive method to measure serum monomeric Ln‐γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). Using our CLIA, we evaluated its diagnostic value in sera from patients with chronic liver disease (CLD) and patients with hepatocellular carcinoma (HCC). Serum alpha‐fetoprotein (AFP) and des‐gamma‐carboxy prothrombin (DCP) were also examined in these subjects. Median levels of Ln‐γ2 were significantly higher in patients with HCC (173.2 pg/mL; range: 39.5–986 pg/mL) compared with patients with CLD (76.7 pg/mL; range: 38.7–215.9 pg/mL) and with healthy volunteers (41.1 pg/mL; range: 10.9–79.0 pg/mL). The optimal cutoff value for Ln‐γ2 that allowed us to distinguish between HCC and nonmalignant CLD was 116.6 pg/mL. Elevated Ln‐γ2 levels were observed in 0% of healthy volunteers, 17% of patients with CLD, and 63% of patients with HCC. The positivity rate in patients with HCC for the combination of Ln‐γ2 and DCP was 89.5%, which was better than that for either of the two markers alone (63% and 68%, respectively). Among patients with early‐stage HCC (T1 or T2), the positivity rates for monomeric Ln‐γ2, AFP and DCP were 61%, 39% and 57%, respectively. Serum Ln‐γ2 may be a potential biomarker for HCC surveillance. The combination of Ln‐γ2 and DCP may be more sensitive for laboratory diagnosis of HCC than the combination of AFP and DCP.

Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma

Background and Aim: Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. Methods: Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. Results: The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. Conclusions: Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.

Effect of vitamin K deficiency or protein-induced protein II on the proliferation and metastasis of hepatoma cells and its mechanism

Objective To investigate the effect of vitamin K deficiency or protein-induced protein Ⅱ (PIVKA-Ⅱ) on the proliferation and metastasis of hepatoma cells and to explore its possible mechanism. Methods The SMMC-7721 cells were cultured with PIVKA-Ⅱ. The cell proliferation was assayed by cell counting kit-8 (CCK-8). The SMMC-7721 cell clone formation was assayed plate clone formation. The cell apoptosis was assayed by flow cytometry. The cell migration was assayed by transwell. The expression of transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blotting. Results The cell clone formation rate (1.94±0.14) and cell survival rate (1.53±0.21) in PIVKA-Ⅱ group was significantly higher than those in negative control group (1.12±0.08, 1.11±0.15) and blank control group (1.00±0.03, 1.00±0.04; F=68.254, 10.264, P=0.000, 0.000). The apoptosis rate of PIVKA-Ⅱ group(1.52±0.28) was lower than that of negative control group (3.13±0.36) and blank control group (3.45±0.43; F=48.436, P=0.000). The cell migration number (62.54±7.45) and mobility (2.13±0.21) of PIVKA-Ⅱ group was significantly higher than those in negative control group (31.34±6.03, 1.06±0.03) and blank control group (29.42±5.59, 1.00±0.05; F=46.241, 283.240, P=0.000, 0.000). The expression of TGF-α, bFGF, VEGF, MMP-2, MMP-9 (1.69±0.13, 1.30±0.04, 1.63±0.11) in PIVKA-Ⅱ group was higher than that in negative control group (1.11±0.07, 1.06±0.01, 1.08±0.02, 1.14±0.06, 1.17±0.04) and blank control group (1.00±0.01, 1.00±0.01, 1.00±0.02, 1.00±0.01, 1.00±0.01; F=2 014.254, 476.355, 1 845.364, 6.486, 7.045, P=0.000, 0.000, 0.000, 0.000, 0.000). Conclusion PIVKA-Ⅱ can promote the proliferation and migration of hepatocellular carcinoma cells and inhibit the apoptosis of hepatoma cells. The mechanism may be related to that PIVKA-Ⅱ can promote the expression of TGF-α, bFGF, VEGF, MMP-2 and MMP-9. Key words: Vitamin K deficiency or antagonist-Ⅱ-induced protein; Hepatocellular carcinoma; Proliferation

Glasgow Prognostic Score and Prognosis After Hepatectomy for Hepatocellular Carcinoma.

Systemic inflammation can promote tumor growth. The Glasgow Prognostic Score (GPS), a simple assessment of systemic inflammation status, could be useful to predict the prognosis of hepatocellular carcinoma (HCC) patients after hepatectomy. Consecutive HCC patients were enrolled following hepatectomy at our institution between 2005 and 2012. Univariate and multivariate analyses were performed to identify variables associated with overall survival (OS) and recurrence-free survival. Patients were stratified according to the GPS. To minimize selection bias, propensity score analysis was performed. An elevated GPS (1-2), des-gamma carboxyprothrombin levels (≥40 mAU/mL), and the presence of multiple tumors were significantly associated with a poor OS. Alpha-fetoprotein levels (≥20.0ng/mL), des-gamma carboxyprothrombin levels (≥40 mAU/mL), and the presence of multiple tumors were independent risk factors for a poor recurrence-free survival. After one-to-one matching, 92 patients each with a normal GPS (0) and an elevated GPS (1-2) had similar preoperative and operative characteristics. Poorer OS times in patients with an elevated GPS were confirmed by excluding possible misinterpretation. An elevated GPS was an independent prognostic indicator for OS after hepatectomy in HCC patients. The GPS, which employs inexpensive and readily available biomarkers, could be a novel tool for predicting survival in such patients.

Field deployable platform for prognostic hepatic cancer screening.

267 Background: Hepatocellular carcinoma (HCC) incidence rates are increasing in many parts of the world particularly in low- and middle-income countries (LMICs). Although treatable if identified in the early stages in many LMICs patients are identified later in disease. A low cost point of care system that could identify patients at a higher risk of having HCC could be used as a screening tool to identify patients at an earlier stage. We are currently developing a low cost point of care hand held platform that can simultaneously evaluate the blood levels of common HCC markers. We present our preliminary findings on the limit of detection (LOD) of our system. Methods: Our device combines the rapid but qualitative results of a vertical flow assay with the quantitative capabilities of surface enhanced Raman scattering spectroscopy. Our final product will be able to simultaneously measure the blood levels of the five most proven markers of HCC: alphafetoprotein (AFP), lens-culinaris agglutinin binding alphafetoprotein (AFP-L3), des-gamma-carboxy prothrombin (DCP), core antibodies to hepatitis B virus (HBV), and core antibodies to hepatitis C virus (HCV). In this pilot study we initially evaluated the LOD of our system by evaluating known concentrations of AFP in human serum samples. We then evaluated serum levels of AFP in HCC patient samples (n = 5) with our device and compared with values obtained using a standard ELISA. Results: The LOD of our system for the detection of AFP in spiked serum samples was 1 ng/mL. Our quantitative lateral flow assay measured AFP within 10% of the values of obtained by standard ELISA, with serum values ranging from 10-900 ng/mL. The quantitative vertical flow assay was able to provide results within 5 minutes at a cost of approximately $2/sample. Conclusions: Our inexpensive, portable and rapid test was able to measure serum levels of AFP at values comparable to standard clinical methods. Importantly, one blood marker would be insufficient to identify high risk patients, so further research will be required to expand the number of markers within the panel. The end goal will be to evaluate the ability of our point of care system to screen high risk population in LMICs, thereby identifying at risk patients earlier in disease progression.

Diagnostic utility of alpha-fetoprotein and des-gamma-carboxyprothrombin in nigerians with hepatocellular carcinoma.

Alpha-fetoprotein (AFP) and Des-gamma-carboxyprothrombin (DCP) have been extensively studied as biomarkers for the diagnosis of and prognostication in hepatocellular carcinoma (HCC). However there are only few reports on the clinical characteristics of hepatocellular carcinoma in relation to the combination of the two tumor markers in hepatitis B virus-related HCC. The aim of this study was to investigate the clinical characteristics of HBV-related HCC in relation to different sets of AFP and DCP values. Sixty-two patients with untreated HCC were studied. The positive value of AFP was set at 20 1U/L while DCP positive value was set at 150 mAU/ml. Patients were divided into three groups: Group 1(n=36) with AFP ≥ 20 IU/L and DCP ≥ 150 mAU/ml. Group 2(n=24) with AFP <20 1U/L and DCP ≥ 150 mAU/ml. Group 3 (n=2) with AFP < 20 1U/L and DCP < 150 mAU/ml. There were no patients in group 4 meant for those with AFP ≥ 20 1U/L and DCP < 150 mAU/ml. Clinical and laboratory variables were compared among the groups. Clinical and laboratory variables were comparable among the groups with the exception of gender and values of serum alanine aminotransferase (ALT). Males were significantly more than females among the groups (p<0.03). ALT values were significantly different among the groups (p<0.006). Paired comparisons between the groups showed the mean values of serum ALT were significantly higher in group 2 than in group 1(p<0.003). The mean serum ALT values were also higher in group 2 than in group 3 (p <0.014). There was no significant difference between group 1 and group 3 (P = 0.124). HCC patients who are sero-positive for DCP and sero-negative for AFP have significantly higher levels of serum ALT; serum ALT levels may be of diagnostic importance in AFP-negative, HBV-related HCC patients.

Prognostic Impact of Indocyanine Green Plasma Disappearance Rate in Hepatocellular Carcinoma Patients after Radiofrequency Ablation: A Prognostic Nomogram Study.

Objective Radiofrequency ablation has been used widely for the local ablation of hepatocellular carcinoma, particularly in its early stages. The study aim was to identify significant prognostic factors and develop a predictive nomogram for patients with hepatocellular carcinoma who have undergone radiofrequency ablation. We also developed the formula to predict the probability of 3- and 5-year overall survival based on clinical variables. Methods We retrospectively studied 96 consecutive patients with hepatocellular carcinoma who had undergone radiofrequency ablation as a first-line treatment. Independent and significant factors affecting the overall survival were selected using a Cox proportional hazards model, and a prognostic nomogram was developed based on these factors. The predictive accuracy of the nomogram was determined by Harrell's concordance index and compared with the Cancer of the Liver Italian Program score and Japan Integrated Staging score. Results A multivariate analysis revealed that age, indocyanine green plasma disappearance rate, and log(des-gamma-carboxy prothrombin) level were independent and significant factors influencing the overall survival. The nomogram was based on these three factors. The mean concordance index of the nomogram was 0.74±0.08, which was significantly better than that of conventional staging systems using the Cancer of the Liver Italian Program score (0.54±0.03) and Japan Integrated Staging score (0.59±0.07). Conclusion This study suggested that the indocyanine green plasma disappearance rate and age at radiofrequency ablation (RFA) and des-gamma-carboxy-prothrombin (DCP) are good predictors of the prognosis in hepatocellular carcinoma patients after radiofrequency ablation. We successfully developed a nomogram using obtainable variables before treatment.