Beili Wang

MiR-1297 regulates the growth, migration and invasion of colorectal cancer cells by targeting cyclo-oxygenase-2.

Cyclo-oxygenase-2(Cox-2), a key regulator of inflammation-producing prostaglandins, promotes cell proliferation and growth. Therefore, a better understanding of the regulatory mechanisms of Cox-2 could lead to novel targeted cancer therapies. MicroRNAs are strongly implicated in colorectal cancer but their specific roles and functions have yet to be fully elucidated. MiR-1297 plays an important role in lung adenocarcinoma and laryngeal squamous cell carcinoma, but its significance in colorectal cancer (CRC) has yet to be reported. In our present study, we found miR-1297 to be down regulated in both CRC-derived cell lines and clinical CRC samples, when compared with normal tissues. Furthermore, miR-1297 could inhibit human colorectal cancer LOVO and HCT116 cell proliferation, migration, and invasion in vitro and tumorigenesis in vivo by targeting Cox-2. Moreover, miR-1297 directly binds to the 3`-UTR of Cox-2, and the expression level was drastically decreased in LOVO and HCT116 cells following overexpression of miR-1297. Additionally, Cox-2 expression levels are inversely correlated with miR-1297 expression in human colorectal cancer xenograft tissues. These results imply that miR-1297 has the potential to provide a new approach to colorectal cancer therapy by directly inhibiting Cox-2 expression.

Elevated PIVKA-II is Associated with Early Recurrence and Poor Prognosis in BCLC 0-A Hepatocellular Carcinomas

BACKGROUND To investigate the prognostic value of serum PIVKA-II (prothrombin induced by the absence of vitamin K or antagonist-II) in BCLC (Barcelona Clinic Liver Cancer) 0-A hepatocellular carcinoma (HCC) patients after curative resection. MATERIALS AND METHODS Preoperative sera were collected from 140 patients with BCLC 0-A HCCs undergoing curative resection during 2011-2012 in Zhongshan Hospital. Follow-up ended on November 2013. ELISA was used to detect the serum concentrations of preoperative PIVKA-II. The prognostic value of PIVKA-II and other clinicopathological factors was analyzed by the Kaplan-Meier method and the multivariate Cox proportional hazards model. RESULTS During follow-up, 39 of 140 patients suffered recurrence and the 1-year recurrence rate was 27.9%. The high-PIVKA-II expression group had lower 1-year time to progression (TTP) compared with the low-expression group (54.8% vs 20.2%, p<0.001). Patients with high preoperative PIVKA-II expression showed a relatively higher risk of developing postoperative recurrence than those with low expression in the low-recurrence-risk subgroups, including α-fetoprotein ≤400ng/mL (45.4% vs 16.7%; p=0.006), tumor size ≤5 cm (54.2% vs 18.1%; p<0.001), single tumor (56.0% vs 19.1%; p<0.001), absence of satellite lesions (53.3% vs 19.8%; p=0.001), absence of vascular invasion (52.6% vs 14.9%; p=0.002), and Edmondson stage I/II (60.9% vs 20.3%; p<0.001). PIVKA-II was the strongest independent prognostic factor for TTP (hazard ratio, 2.877; 95% CI 1.524-5.429; p=0.001). CONCLUSIONS Elevated PIVKA-II is associated with early recurrence of BCLC 0-A HCC after curative resection and can be considered a novel prognostic predictor.

Dynamic change of the systemic immune inflammation index predicts the prognosis of patients with hepatocellular carcinoma after curative resection.

Abstract Background: The aim of the study was to determine the utility of the dynamic change and serial monitoring of the systemic immune inflammation index (SII), which was based on the numbers of patients’ lymphocytes (L), platelets (P), neutrophils (N) and defined as P*N/L, for predicting prognosis of patients with hepatocellular carcinoma (HCC) after curative resection. Methods: We conducted a prospective study of 163 patients with HCC who underwent curative resection at Zhongshan Hospital from January 2012 to May 2013. SII was calculated using data acquired before and approximately 1 month after surgery. An optimal cutoff value stratified patients into groups with high or low SII. Patients were classified into unfavorable and favorable groups using the dynamic change of the SII. Two groups that were further divided into four categories within the entire cohort and the low-risk subgroups were serially monitored for ≥6 months. Prognostic values of the SII and other factors were determined using the Kaplan-Meier method, the Cox proportional hazards model, and the receiver operating characteristics (ROC) curve. Results: The favorable group was likely to have cirrhosis, and the unfavorable group was likely to have larger tumors and a higher recurrence rate. Multivariate analysis revealed that tumor size and dynamic change of the SII were independent risk factors for early recurrence. Moreover, the predictive value of the SII was retained in α-fetoprotein (AFP)-negative and HBeAg-negative-HBV-DNA <2000 IU/mL subgroups. Further, the serial changes of the SII for recurrence and no recurrence groups were statistically significant. Conclusions: The dynamic change and serial monitoring of the SII represent new indicators for predicting the early recurrence of HCC determining advance optimal therapy in advance.

Application of the albumin-bilirubin grade for predicting prognosis after curative resection of patients with early-stage hepatocellular carcinoma.

BACKGROUND Patients with Barcelona Clinic Liver Cancer (BCLC) 0+A are considered to have early-stage hepatocellular carcinoma (HCC). The albumin-bilirubin (ALBI) grade is a significant predictor of overall survival (OS) for HCC. However, data are lacking to support its significance for patients with early-HCC. METHODS We recruited 318 patients with early-HCC who underwent curative resection between January 2012 and August 2013. The Kaplan-Meier method and log-rank tests were used to compare OS of patients with different ALBI grades. Cox regression analysis was applied to evaluate ALBI grade as an independent predictor of OS. RESULTS Early-HCC patients with ALBI grade II experienced significantly shorter OS (p<0.001) and higher death rates. In the Child-Pugh (C-P) grade-A group, patients with ALBI grade I had a more favorable prognosis than those with grade II (p<0.001), while the C-P grade did not distinguish patients with poor prognosis from the entire group. Cox regression analysis demonstrated that ALBI grade was the most significant independent predictor of OS, and the ALBI grade retained its clinical significance in low α-fetoprotein subgroup. CONCLUSION ALBI grade predicted OS in patients with early-HCC. Reclassification of C-P grade according to ALBI grade might improve the management of HCC.

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

BACKGROUND The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. MATERIALS AND METHODS We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. RESULTS It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. CONCLUSIONS It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients.

Unlabeled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in pancreatic adenocarcinoma tissues.

Abstract Background: The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. Methods:We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. Results: It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. Conclusions: It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.

Serum IgG4:IgG Ratio Predicts Recurrence of Patients with Hepatocellular Carcinoma after Curative Resection

Aim: IgG4 is associated with a Th1-to-Th2 switch, which plays a vital role in metastasis, in patients with malignances; thus, we aimed to investigate its clinical significance in predicting hepatocellular carcinoma (HCC) recurrence in the present study. Methods: The correlation between serum IgG4:IgG ratio and recurrence was analyzed in a cohort of 195 patients undergoing curative resection in 2012. Another 100 patients were analyzed in a prospective independent cohort during 2012-2013 to validate the value of serum IgG4. Serum IgG4 and total IgG concentrations were measured with an automatic immune analyzer and the optimal cutoff value for serum IgG4 levels was determined by X-tile software. Results: Our data revealed that serum IgG4:IgG were significantly elevated in patients with tumor recurrence (P<0.05). A cutoff IgG:IgG4 ratio of 0.08 was set to stratify HCC patients into high (>0.08) and low (≤0.08) groups. High serum IgG4:IgG ratio correlated with significantly shorter time-to-recurrence (median 11.85 months vs. 39.20, P=0.005). Univariate and multivariate analyses demonstrated that serum IgG4:IgG ratio is an independent indicator of tumor recurrence and this retained its clinical significance even in conventional low-recurrence-risk subgroups, including patients with low α-fetoprotein and early-stage diseases. Conclusion: Our results demonstrated that elevated serum IgG4:IgG ratio is associated with poor clinical outcomes in HCC patients and therefore, and can serve as a novel prognostic predictor for HCC patients undergoing resection. Analyzing serum IgG4 would be useful to tailor individualized therapies for patients.

Determination of carbohydrate-deficient transferrin in a Han Chinese population

BackgroundCarbohydrate-deficient transferrin (CDT) is a widely used alcohol biomarker. Because of the high prevalence of chronic alcohol abuse in many countries, CDT plays an important role in the areas of traffic, clinical, and forensic medicine. However, CDT levels have not been determined in the Han Chinese population. Therefore, we investigated the frequency of genetic transferrin variants and the relationship between CDT levels and alcohol consumption in this population. From this data, we established a CDT cut-off for Han Chinese and evaluated the analytical performance of the CDT capillary zone electrophoresis system.ResultsThe prevalence of transferrin variants was 4.14%. The mean CDT level of the reference group was 0.73%. We recommended CDT level >1.5% as cut off standard of alcohol intake to ensuring the specificity was best. The CDT test total precision for 0.5%, 0.7%, and 1.55% was 14.4%, 11.5%, and 7.2%, respectively. The data showed good linearity in the studied range of 0.6% to 8.2%.ConclusionsThese results demonstrate that CDT is a useful marker to detect heavy daily alcohol consumption. We proposed and evaluated the first CDT cut-off for the Han Chinese population, and we showed that the CDT capillary zone electrophoresis system is a reliable analytic method.

Application of Serum Annexin A3 in Diagnosis, Outcome Prediction and Therapeutic Response Evaluation for Patients with Hepatocellular Carcinoma

PurposeAnnexin A3 (ANXA3) could induce progression of hepatocellular carcinoma (HCC) via promoting stem cell traits of CD133-positive cells. Moreover, serum ANXA3 showed preliminary diagnostic potential, however further validation was required. Meanwhile, the prognostic value of ANXA3 remained elusive. The present study aimed to validate diagnostic performance and further systematically investigate the prognostic value of serum ANXA3.MethodsSerum ANXA3 of 368 HCC patients was determined by enzyme-linked immunosorbent assay (ELISA); 295 of these patients underwent resection and 73 underwent transcatheter arterial chemoembolization (TACE). Diagnostic performance of ANXA3 was evaluated by receiver operating characteristic (ROC) analysis, and the prognostic value was evaluated by Cox regression and Kaplan–Meier analysis. To evaluate the relationship between serum ANXA3 and circulating CD133 mRNA-positive tumor cells (CD133mRNA+ CTCs), real-time polymerase chain reaction was conducted in 69 patients who underwent resection.ResultsSerum ANXA3 provided greater diagnostic performance than α-fetoprotein (area under the curve [AUC] 0.869 vs. 0.782), especially in early diagnosis (AUC 0.852 vs. 0.757) and discriminating HCC from patients at risk (0.832 vs. 0.736). Pretreatment ANXA3 was an independent predictor of tumor recurrence (hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26–2.76, p = 0.002)/progression (HR 1.88, 95% CI 1.04–3.43, p = 0.038) and survival (resectable: HR 2.26, 95% CI 1.44–3.56, p = 0.001; unresectable: HR 2.08, 95% CI 1.10–4.05, p = 0.025), and retained its performance in low-recurrence-risk subgroups. Specifically, dynamic changes of ANXA3-positive status was associated with worse prognosis. ANXA3 was positively correlated with CD133mRNA+ CTCs (r = 0.601, p < 0.001). In patients with detectable CD133mRNA+ CTC, high ANXA3 was positively associated with a higher risk of recurrence and shorter overall survival.ConclusionsSerum ANXA3 shows promise as a biomarker for diagnosis, outcome prediction, and therapeutic response evaluation in patients with HCC.