Pin Nan Cheng

Lin28B Is an Oncofetal Circulating Cancer Stem Cell-Like Marker Associated with Recurrence of Hepatocellular Carcinoma

By using an expressed sequence tag bioinformatic algorithm, we identified that Lin28 homolog B (Lin28B) may have an oncofetal expression pattern which may facilitate detecting cancer cells in adults. It is also reported to be a potential marker for cancer stem cells. Therefore, we sought to verify oncofetal-stemness characters of Lin28B and test its potential as a circulating cancer stem cell-like marker in adult HCC patients. Lin28B mRNA was examined in a panel of fetal tissue, adult tissue and tumors. Lin28B was over-expressed or knocked down in HepG2 cells to evaluate its potential as a stem cell-like marker. RT-qPCR for Lin28B was performed in the peripheral blood mononuclear cells from patients with HCC receiving surgery (n=96) and non-HCC controls (n=60) and analyzed its clinical significance. Lin28B showed an oncofetal expression pattern. Its overexpression could upregulate stemness markers (OCT4, Nanog and SOX2) and enhance tumorsphere formation in vitro. Lin28B knockdown had opposite effects. Circulating Lin28B was detected in peripheral blood mononuclear cells in 3 cases (5%) of non-HCC controls and 32 cases (33.3%) of HCC patients. In HCC patients, circulating Lin28B was associated with high tumor grade (P=0.046), large size (P=0.005), high AJCC stage (P=0.044) and BCLC stage (P=0.017). Circulating Lin28B was significantly associated with decreased recurrence-free survival (P<0.001). Circulating Lin28B separated early stage HCC into 2 recurrence-free survival curves (P=0.003). In multivariate analysis, circulating Lin28B was an independent variable associated with early recurrence (P=0.045) and recurrence in early stage HCC (P=0.006). In conclusion, the oncofetal gene Lin28B is a potential oncofetal cancer-stem-cell-like circulating tumor cell marker that correlates with HCC recurrence after hepatectomy. Circulating Lin28B could refine early AJCC stages. Our finding supports the possible use of a TNMC (C for circulating tumor cells) staging system in HCC.

A reverse-transcription competitive PCR assay based on chemiluminescence hybridization for detection and quantification of hepatitis C virus RNA

A reverse-transcription competitive PCR (RT-cPCR) combined with chemiluminescence hybridization was designed for the detection and quantitative determination of serum hepatitis C virus (HCV) RNA. The concentration of HCV RNA was calculated based on an external standard curve that was generated by coamplification of internal competitor and target sequences in serial dilutions. The detection limit of the chemiluminescence RT-cPCR was 100 copies/ml (94 IU/ml). Meanwhile, the linear range for quantitation extended from 850 copies/ml (795 IU/ml) to 4.95x10(7) copies/ml. The performance of the current assay for measuring circulating HCV levels from 26 anti-HCV-antibody positive patients was compared with that of branched-chain DNA (bDNA) and nested RT-PCR assays. Eighteen patients had HCV RNA levels that exceeded the quantitation limit by the chemiluminescence RT-cPCR, but only 11 patients were quantitation-positive by the bDNA. A significant correlation of the quantitation values was found between the chemiluminescence RT-cPCR and the bDNA (R2=0.8391). Among the eight patients with HCV RNA titers below the quantitation limit, four remained positive by the chemiluminescence cRT-PCR, demonstrating the results in agreement with those using the nested RT-PCR. Furthermore, good linearity was revealed for the HCV genotypes 1b, 2a, 2b in 3-order magnitude diluted serum samples. In conclusion, the proposed chemiluminescence RT-cPCR method can detect quantitatively HCV RNA as accurately as the bDNA method and has sensitivity as high as nested RT-PCR.

Aeromonas Spontaneous Bacterial Peritonitis: A Highly Fatal Infectious Disease in Patients with Advanced Liver Cirrhosis

BACKGROUND/PURPOSEnAeromonas infections, rarely reported in Western countries, are not uncommon infectious diseases in Taiwan. The clinical manifestations and prognostic factors of Aeromonas spontaneous bacterial peritonitis (SBP) in patients with liver cirrhosis were investigated.nnnMETHODSnWe reviewed the medical charts and microbiological records of liver cirrhosis patients with Aeromonas SBP between January 1990 and December 2005, in a medical center in southern Taiwan.nnnRESULTSnThirty-one liver cirrhosis patients developed Aeromonas SBP within a 16-year period. The majority (26, 84%) had concurrent Aeromonas bacteremia. A. sobria (55%) and A. hydrophila (45%) were the causative species. The predominant clinical manifestations included fever (84%), abdominal pain (74%), hypotension on admission (48%), altered mental status (45%), and acute renal failure (42%). Gram-negative bacilli were found in Gram staining of ascitic fluids in 27% of 26 patients, while aeromonads were isolated from ascitic fluids in 55% of 31 patients. The yield rate of ascitic fluid cultures decreased greatly, if paracentesis was performed at > 3 hours after the administration of antimicrobial therapy. All but one patient received in-vitro-active antimicrobial agents within 48 hours, but the all-cause mortality rate was 56%. Initial high Pitts bacteremia score was independently associated with a fatal outcome in multivariate analysis.nnnCONCLUSIONnAeromonas SBP is a fatal disease, and must be included in the differential diagnosis of SBP in patients with advanced liver cirrhosis in endemic areas.

Rapid Prediction of Treatment Futility of Boceprevir with Peginterferon-Ribavirin for Taiwanese Treatment Experienced Hepatitis C Virus Genotype 1-Infected Patients.

The efficacy and safety of the boceprevir (BOC)-containing triple therapy in Taiwanese treatment-experienced patients remains elusive. After 4 weeks of peginterferon/ribavirin lead-in therapy, patients with cirrhosis or previous null-response received triple therapy for 44 weeks; whereas others received 32 weeks of triple therapy followed by 12 weeks of peginterferon/ribavirin therapy. Patients with HCV RNA > 100 IU/mL at week 12 or with detectable HCV RNA at week 24 of treatment were viewed as futile. A total of 123 patients received treatment. The rates of sustained virological response (SVR) and relapse were 66.7% and 8.9%, respectively by using intention-to-treat analysis. Multivariate analysis revealed that factors associated with SVR included HCV-1b (odds ratio [OR]/ 95% confidence intervals [CI]: 19.23/1.76–525.15, P = 0.01), BOC adherence (7.69/1.55–48.78, P = 0.01), serum albumin (OR/CI:6.25/1.14–40.07, P = 0.03) levels and HCV RNA levels (OR/CI:0.34/0.12–0.79, P = 0.01). Twenty-six (21.1%) patients experienced severe adverse events (SAEs). Multivariate analysis revealed that APRI > 1.5 was the single factor associated with occurring SAEs (OR/CI: 3.77/ 0.97–14.98, P = 0.05). Merging the cut-off values of HCV RNA > 7 log IU/mL at baseline and HCV RNA > 6 log IU/mL at week 4 provided the earliest and best combing viral kinetics in predicting week 12/24 futility with the PPV of 100% and accuracy of 93.5%. HCV-1 treatment experienced Taiwanese patients treated with boceprevir-containing triple therapy in real world had comparable efficacy and safety profiles with those reported in clinical trials. Early viral kinetics before week 4 of treatment highly predicted futility at week 12 or 24 of treatment.

Therapy with interferon-α and ribavirin for chronic hepatitis C virus infection upregulates membrane HLA-ABC, CD86, and CD28 on peripheral blood mononuclear cells

Multiple interferon‐stimulated genes (ISGs) involving T‐cell activation are upregulated during initial interferon‐α‐based therapy for chronic hepatitis C virus (HCV) infection. However, the long‐term impact on therapeutic outcome in patients remains unknown. In this study, the effects of anti‐HCV therapy on the surface ex‐pression of HLA‐ABC, CD86, and CD28 were longitudinally assessed. These proteins are integral membrane receptors of antigen presentation and triggering of costimulatory signals for activating CD8+ T cells. Peripheral blood mononuclear cells were collected at baseline and post‐treatment for 1 day, and 2, 4, 12, and 24 weeks, respectively. This treatment led to a time‐related elevation of membrane levels of HLA‐ABC and CD86 on B‐cells and monocytes in patients with a sustained response (nu2009=u200923), but not in those without (nu2009=u20098). Meanwhile, upregulation of CD28 on CD4+ and CD8+ T cells was comparable in both groups of sustained responders and non‐responders. Steady increases in the B cells surface and intracellular HLA‐ABC were observed, thus, the surface‐to‐intracellular ratios did not alter over the period of treatment. Furthermore, multivariate analysis shows that increased HLA‐ABC on monocytes by week 12 correlates significantly with sustained response (Pu2009=u20090.033). In conclusion, differential modulation of T‐cell activation ISGs, such as HLA‐ABC and CD86 might correlate with the outcome of interferon‐α‐based therapy in chronic hepatitis C patients. J. Med. Virol. 80:989–996, 2008.

Efficacy of entecavir in chronic hepatitis B patients with persistently normal alanine aminotransferase: Randomized, double-blind, placebo-controlled study

BACKGROUNDnIt is still inconclusive whether chronic hepatitis B (CHB) patients with persistently normal alanine aminotransferase (PNALT) should receive nucleoside/nucleotide analogues. This study is to evaluate the efficacy of entecavir in improving liver histology in CHB patients with PNALT.nnnMETHODSnIn this prospective randomized, double-blind, placebo-controlled study, 380 CHB patients with PNALT were screened, 82 patients received biopsy and 43 patients met the HBV DNA and histology criteria and were randomly assigned to either an entecavir or placebo group for 52 weeks, with 22 and 21 in each group, respectively. The primary objective was to evaluate histological improvement. The secondary objective is to evaluate virological efficacy.nnnRESULTSnA total of eight (38.1%) patients in the entecavir group and eight (44.4%) in the placebo group (P=0.752) showed histological improvement. The decrease in total Knodell scores (±sd) was 1.3 ±1.9 in the entecavir group and 1.5 ±2.2 in the placebo group (P=0.803). The subjects with undetectable HBV DNA at week 52 were 16/21 (76.2%) in the entecavir group and 0/18 (0%) in the placebo group (P<0.001). The mean HBV DNA reduction from baseline to week 52 was 4.73 ±0.83 in the entecavir and 0.25 ±0.81 in the placebo group (P<0.001).nnnCONCLUSIONSnCHB patients with PNALT receiving entecavir therapy for one year achieved virological efficacy, but not histological benefit. ClinicalTrials.gov number NCT01833611.

Clinical comparison of high-dose interferon-α2b with or without ribavirin for treatment of interferon-relapsed chronic hepatitis C

BACKGROUNDnInterferon a with ribavirin combination therapy is effective but still unsatisfactory in the treatment of patients with interferon-relapsed chronic hepatitis C.nnnAIMSnTo compare, in a randomized, double blind, placebo-controlled study, high-dose interferon-alpha2b with or without ribavirin in the treatment for interferon-relapsers.nnnPATIENTSnA total of 52 patients with interferon-relapsed chronic hepatitis C were randomly assigned to receive 24-week treatment with interferon-alpha2b (6 MU three times per week) combined with either ribavirin (1,000 to 1,200 mg per day) or a matched placebo and then followed for an additional 24 weeks.nnnMETHODSnHepatitis C virus RNA was detected by reverse-transcription polymerase chain reaction. For determining viral concentration, the commercial bDNA Quantiplex hepatitis C virus-RNA 2.0 assay was used. Genotyping was performed by reverse hybridization assaynnnRESULTSnAt the end of treatment, no detectable hepatitis C virus RNA levels were observed in 92% (24/26) of patients on interferon alpha2b/ribavirin and 81% (21/26) of patients on interferon alpha2b/placebo. At the end of the follow-up, a higher sustained virological response rate was seen in patients treated with interferon alpha2b/ribavirin than those treated with interferon alpha2b/placebo (69% vs 23%, p < 0.001). Patients with either initially high levels of viral concentration or with genotype 1 responded poorly. Patients who received interferon-alpha2b/ribavirin treatment and in whom no hepatitis C virus RNA was detected at 4th week after treatment had 90% chance to achieve sustained virological response.nnnCONCLUSIONSnHigh-dose interferon-alpha2b plus ribavirin treatment is highly effective in interferon-relapsed patients.

Progesterone receptor membrane component 1 as a potential prognostic biomarker for hepatocellular carcinoma

AIM To investigate the clinicopathological significance of progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 in hepatocellular carcinoma (HCC). METHODS We performed immunohistochemical staining to evaluate the estrogen receptor (ER), progesterone receptor (PR), PGRMC1, and PGRMC2 in a clinical cohort consisting of 89 paired HCC and non-tumor liver samples. We also analyzed HCC data (n = 373) from The Cancer Genome Atlas (TCGA). We correlated the expression status of PGRMC1 and PGRMC2 with clinicopathological indicators and the clinical outcomes of the HCC patients. We knocked down or overexpressed PGRMC1 in HCC cell lines to evaluate its biological significance in HCC cell proliferation, differentiation, migration, and invasion. RESULTS We found that few HCC cases expressed ER (5.6%) and PR (4.5%). In contrast, most HCC cases expressed PGRMC1 (89.9%) and PGRMC2 (100%). PGRMC1 and PGRMC2 exhibited significantly lower expression in tumor tissue than in non-tumor tissue (P < 0.001). Lower PGRMC1 expression in HCC was significantly associated with higher serum alpha-fetoprotein expression (P = 0.004), poorer tumor differentiation (P = 0.045) and liver capsule penetration (P = 0.038). Low PGRMC1 expression was an independent predictor for worse disease-free survival (P = 0.002, HR = 2.384, CI: 1.377-4.128) in our cases, as well as in the TCGA cohort (P < 0.001, HR = 2.857, CI: 1.781-4.584). The expression of PGRMC2 did not relate to patient outcome. PGRMC1 knockdown promoted a poorly differentiated phenotype and proliferation of HCC cells in vitro, while PGRMC1 overexpression caused the opposite effects. CONCLUSION PGRMC1 is a non-classical hormonal receptor that negatively regulates hepatocarcinogenesis. PGRMC1 down-regulation is associated with progression of HCC and is a poor prognostic indicator.

Combined Transarterial Embolization/Chemoembolization-Based Locoregional Treatment with Sorafenib Prolongs the Survival in Patients with Advanced Hepatocellular Carcinoma and Preserved Liver Function: A Propensity Score Matching Study

Background: Sorafenib is the standard treatment for patients with Barcelona Clinic Liver Cancer (BCLC) stage C hepatocellular carcinoma (HCC). However, the treatment outcome is not satisfactory. We retrospectively analyzed whether adding transarterial embolization/chemoembolization (TA(C)E)-based locoregional therapy to sorafenib can further improve treatment efficacy. Patients and Methods: We included 147 BCLC stage C HCC patients with Child-Turcotte-Pugh class A liver function and treated with sorafenib for analysis. Through propensity score matching, we divided patients into the combined treatment group (n = 63; patients received TA(C)E-based locoregional treatment and sorafenib) and the sorafenib monotherapy group (n = 63). We analyzed the effects of patients’ clinical and tumor-related factors on their overall survival (OS) and time to tumor progression. Results: The OS was better in the combined treatment group than in the sorafenib monotherapy group (419 vs. 223 days, p = 0.028). In the Cox regression model, combined treatment, a lower baseline α-fetoprotein (AFP) level < 400 ng/mL, tumors without main portal venous tumorous thrombosis, and age ≥60 years were identified as independent factors for OS. Subgroup analysis demonstrated that patients with a higher baseline AFP level > 400 ng/mL, age < 60 years, tumors with branched portal venous tumorous thrombosis only or without extrahepatic metastasis benefited the most from combined treatment. Conclusion: Combining TA(C)E-based locoregional treatment with sorafenib resulted in better OS in patients with BCLC stage C HCC compared with sorafenib alone. TA(C)E-based locoregional treatment can be an adjunctive treatment to sorafenib for patients with advanced HCC and a satisfactory liver functional reserve.