Systematic evaluation of circulating inflammatory markers for hepatocellular carcinoma.

BACKGROUNDS & AIMS: A number of circulating inflammatory factors are implicated in the pathogenesis and prognostication of hepatocellular carcinoma (HCC). We aim to evaluate the prognostication of multiple serum inflammatory factors simultaneously and develop an objective inflammatory score for HCC. METHODS: A prospective cohort of 555 patients with HCC with paired serum samples was accrued from 2009 to 2012. The blood levels of conventional inflammatory markers, namely C-reactive protein (CRP), albumin, neutrophils, lymphocytes and platelet, were determined, and 41 other exploratory markers were measured by a multiplex assay. The prognostication and interaction of markers were determined by univariate and multivarite analyses. RESULTS: The cohort was randomly divided into training cohort (n=139) and validation cohort (n=416). There were no differences in baseline characteristics between the two cohorts. In the training cohort, independent prognostic factors for overall survival included CRP (hazard ratio [HR] 1.107; P=.003), albumin (HR 0.953; P=.032) and interleukin-8 (HR=5.816; P<.001). We have modified the existing inflammation-based index (IBI) by adding serum interleukin-8 level. The modified IBI could stratify patients into four groups with distinct overall survival (P<.001). The results were also validated in the validation cohort. When compared with IBI and other conventional inflammatory markers, the modified IBI had better prognostic performance with higher c-index and homogeneity likelihood ratio chi-square. CONCLUSIONS: Among the conventional and exploratory circulating inflammatory markers, higher CRP, lower albumin and higher interleukin-8 were independent prognosticators. By combining these factors, a simple and accurate inflammatory index could be constructed.

Clinical utility of plasma Epstein-Barr virus DNA and ERCC1 single nucleotide polymorphism in nasopharyngeal carcinoma.

BACKGROUND: Single nucleotide polymorphism (SNP) of the excision repair cross-complementing group 1 (ERCC1) gene has been linked with sensitivity to platinum and radiation. The authors hypothesized that the ERCC1 genotype for the SNPs cytosine-to-thymine substitution at codon 118 (C118T) and cytosine-to-adenine substitution at codon 8092 (C8092A) is prognostic in patients with nasopharyngeal carcinoma (NPC) who receive either radiotherapy (RT) or cisplatin plus RT. METHODS: The authors tested their hypothesis using biomarker screening samples from the Hong Kong NPC Study Group 0502 trial, which was a prospective, multicenter clinical trial that used post-RT plasma Epstein-Bar virus (EBV) DNA (pEBV) levels to screen patients with high-risk NPC for adjuvant chemotherapy. RESULTS: ERCC1 SNPs were analyzed in 576 consecutive patients who were screened by pEBV. In the total biomarker population, there was no significant association of ERCC1 C118T or C8092A genotype with relapse-free survival (RFS) or overall survival (OS). There also was no correlation between ERCC1 genotype and ERCC1 protein or messenger RNA expression in a subset of patients who had available paired biopsies. Post-RT pEBV status was the only independent prognosticator for RFS and OS in multivariate analyses. However, there was a significant interaction between ERCC1 C118T genotype and post-RT pEBV status (RFS, P = .0106; OS, P = .0067). The ERCC1 C118T genotype was significantly associated with both RFS (hazard ratio, 1.67; 95% confidence interval, 1.07-2.61; P = .024) and OS (hazard ratio, 2.31; 95% confidence interval, 1.22-4.40; P = .0106) in the post-RT pEBV-negative population, but not in the pEBV-positive population. CONCLUSIONS: The current results prospectively validate pEBV as the most significant prognostic biomarker in NPC that can be used to select high-risk patients for adjuvant therapy. The ERCC1 C118T genotype may help to identify a favorable subgroup (approximately 7%) of pEBV-negative patients with NPC who have an excellent prognosis and can be spared the toxicities of further therapy.

Performance and Operational Feasibility of Epstein-Barr Virus-Based Screening for Detection of Nasopharyngeal Carcinoma: Direct Comparison of Two Alternative Approaches.

PURPOSE: Two Epstein-Barr virus (EBV)-based testing approaches have shown promise for early detection of nasopharyngeal carcinoma (NPC). Neither has been independently validated nor their performance compared. We compared their diagnostic performance in an independent population. METHODS: We tested blood samples from 819 incident Taiwanese NPC cases (213 early-stage, American Joint Committee on Cancer version 7 stages I and II) diagnosed from 2010 to 2014 and from 1,768 controls from the same region, frequency matched to cases on age and sex. We compared an EBV antibody score using immunoglobulin A antibodies measured by enzyme-linked immunosorbent assay (EBV antibody score) and plasma EBV DNA load measured by real-time PCR followed by next-generation sequencing (NGS) among EBV DNA-positive individuals (EBV DNA algorithm). RESULTS: EBV antibodies and DNA load were measured for 2,522 (802 cases; 1,720 controls) and 2,542 (797 cases; 1,745 controls) individuals, respectively. Of the 898 individuals positive for plasma EBV DNA and therefore eligible for NGS, we selected 442 (49%) for NGS testing. The EBV antibody score had a sensitivity of 88.4% (95% CI, 86.1 to 90.6) and a specificity of 94.9% (95% CI, 93.8 to 96.0) for NPC. The EBV DNA algorithm yielded significantly higher sensitivity (93.2%; 95% CI, 91.3 to 94.9; P = 1.33 × 10-4) and specificity (98.1%; 95% CI, 97.3 to 98.8; P = 3.53 × 10-7). For early-stage NPC, the sensitivities were 87.1% (95% CI, 82.7 to 92.4) for the EBV antibody score and 87.0% (95% CI, 81.9 to 91.5) for the EBV DNA algorithm (P = .514). For regions with a NPC incidence of 20-100/100,000 person-years (eg, residents in southern China and Hong Kong), these two approaches yielded similar numbers needed to screen (EBV antibody score: 5,656-1,131; EBV DNA algorithm: 5,365-1,073); positive predictive values ranged from 0.4% to 1.7% and 1.0% to 4.7%, respectively. CONCLUSION: We demonstrated high sensitivity and specificity of EBV antibody and plasma EBV DNA for NPC detection, with slightly inferior performance of the EBV antibody score. Cost-effectiveness studies are needed to guide screening implementation.

A study of circulating interleukin 10 in prognostication of unresectable hepatocellular carcinoma.

BACKGROUND: The level of circulating interleukin 10 (IL-10) is elevated in a proportion of patients with hepatocellular carcinoma (HCC). The objective of the current study was to evaluate the prognostic significance of serum the IL-10 level in patients with unresectable HCC. METHODS: Patients with unresectable HCC who provided serum at the time of diagnosis were enrolled prospectively in the study. The level of circulating IL-10 in serum samples was determined by enzyme-linked immunosorbent assay. The association of the IL-10 level with overall survival was evaluated in relation to sociodemographics, liver function, hepatitis B viral load, and tumor staging. RESULTS: In total, 222 patients were recruited; of these, 82.4% were positive for hepatitis B virus surface antigen, and 65.8% had Barcelona Clinic Liver Cancer stage C disease. The mean log IL-10 level was 1.1 pg/mL, and 146 patients had an IL-10 level >1 pg/mL (high IL-10 group). The high IL-10 group had worse overall survival than the low IL-10 group (5.0 months vs 14.9 months; hazard ratio, 2.192; P < .0001). The IL-10 level was associated with worse hepatic function and with a high alanine transaminase (ALT) level. The IL-10 level remained an independent prognostic factor (hazard ratio, 1.824; P = .0005) after adjustment for sociodemographics, tumor staging, treatment, Child-Pugh stage, and ALT level. The IL-10 level also subdivided patients into 2 populations with distinct survival (10.2 months vs 3.5 months; P = .0027). CONCLUSIONS: The serum IL-10 level was identified as an independent prognostic factor for unresectable HCC. The current findings suggested that an elevated IL-10 level may be related to hepatic injury caused by cirrhotic processes rather than tumor load. The authors concluded that the IL-10 level offers additional prognostic value to the existing tumor staging systems.

Efficacy, Safety, and Pharmacokinetics of Axitinib in Nasopharyngeal Carcinoma: A Preclinical and Phase II Correlative Study.

Purpose: We hypothesized that axitinib is active with an improved safety profile in nasopharyngeal carcinoma (NPC).Experimental Design: We evaluated axitinib in preclinical models of NPC and studied its efficacy in a phase II clinical trial in recurrent or metastatic NPC patients who progressed after at least one line of prior platinum-based chemotherapy. We excluded patients with local recurrence or vascular invasion. Axitinib was started at 5 mg twice daily in continuous 4-week cycles. Primary endpoint was clinical benefit rate (CBR), defined as the percentage of patients achieving complete response, partial response, or stable disease by RECIST criteria for more than 3 months.Results: We recruited 40 patients, who received a median of 3 lines of prior chemotherapy. Axitinib was administered for a mean of 5.6 cycles, with 16 patients (40%) receiving ≥6 cycles. Of 37 patients evaluable for response, CBR was 78.4% (95% CI, 65.6%-91.2%) at 3 months and 43.2% (30.4%-56.1%) at 6 months. Grade 3/4 toxicities were uncommon, including hypertension (8%), diarrhea (5%), weight loss (5%), and pain (5%). All hemorrhagic events were grade 1 (15%) or grade 2 (3%). Elevated diastolic blood pressure during the first 3 months of axitinib treatment was significantly associated with improved overall survival (HR, 0.29; 95% CI, 0.13-0.64, P = 0.0012). Patient-reported fatigue symptom was associated with hypothyroidism (P = 0.039). Axitinib PK parameters (Cmax and AUC(0-t)) were significantly correlated with tumor response, toxicity, and serum thyroid-stimulating hormone changes.Conclusions: Axitinib achieved durable disease control with a favorable safety profile in heavily pretreated NPC patients. Clin Cancer Res; 24(5); 1030-7. ©2018 AACR.

The effect of centrifugation on circulating mRNA quantitation opens up a new scenario in expression profiling from patients with metastatic colorectal cancer.

OBJECTIVES: To investigate the effect of centrifugation in circulating mRNA quantitation and to design a new, consistent protocol for it. DESIGN AND METHODS: The concentrations of beta-catenin mRNA, P-selectin mRNA, cytokeratin 20 mRNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the plasma, the mononuclear and red cells (MARC) from metastatic colorectal (CRC) patients whose blood samples were centrifuged with 2 different forces, were measured. A new protocol to measure expression profiles in MARC was studied. RESULTS: When the centrifugal force was increased: (1) the concentrations of all transcripts were dramatically decreased in the plasma samples; (2) only the concentrations of P-selectin mRNA and GAPDH mRNA were significantly increased in the mononuclear cell samples; and (3) their concentrations in the red cells did not change significantly. The new protocol found 9 differential expressed genes. CONCLUSIONS: Centrifugal force causes inconsistent mRNA quantitation in the plasma. The new protocol discovered 9 differential expressed genes for metastatic CRC.

Cyclooxygenase-2 expression in advanced nasopharyngeal carcinoma--a prognostic evaluation and correlation with hypoxia inducible factor 1alpha and vascular endothelial growth factor.

Cycloxygenase-2 (COX-2), hypoxia inducible factor 1-alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) can be induced by the Epstein-Barr virus oncoprotein latent membrane protein-1 (LMP-1) in nasopharyngeal cancer (NPC) cell lines. This study examined the prognostic relevance of COX-2 and its relationship with HIF-1alpha and VEGF expression in NPC biopsies. Primary tumor biopsies were obtained from 78 participants of a randomized trial who received radiotherapy (RT) with or without concurrent chemotherapy for locoregionally advanced NPC. These were analyzed for COX-2 expression and then correlated with age, sex, disease stage, treatment arm, survival and disease recurrence, VEGF and HIF-1alpha expression in a regression model. 83% of tumors expressed COX-2, 47% co-expressed COX-2 and VEGF, 38% co-expressed COX-2 and HIF-1alpha. On univariate analysis, COX-2 expression did not correlate with survival and recurrence, but moderate to high COX-2 expression was associated with advanced nodal stage (p=0.03). Although univariate analysis showed that COX-2-HIF-1alpha co-expression was associated with worse progression-free survival (p=0.046), time to local (p=0.004) and regional recurrence (p=0.007), multivariate analysis failed to confirm any correlation between COX-2-HIF-1alpha or COX-2-VEGF co-expression and survival or disease recurrence. Contrary to previous report, this study failed to demonstrate any prognostic significance of COX-2 expression alone or co-expression with HIF-1alpha or VEGF in advanced NPC.

Proteomic comparison of nasopharyngeal cancer cell lines C666-1 and NP69 identifies down-regulation of annexin II and beta2-tubulin for nasopharyngeal carcinoma.

CONTEXT: Nasopharyngeal carcinoma (NPC), common in southern China and North Africa, has a complex etiology involving interplay between viral, environmental, and hereditary factors and is almost constantly associated with the Epstein-Barr virus. Since the prognosis of locally advanced and metastatic diseases is poor, increased understanding of the pathogenesis of NPC would be important for discovering novel markers for patients' management. OBJECTIVES: To compare the proteomic expression profile between an Epstein-Barr virus-associated NPC cell line (C666-1) and a normal NP cell line (NP69). The proteins with differential expression were analyzed in 40 undifferentiated NPC paraffin-embedded specimens. DESIGN: Differentially expressed proteins discovered between the two cell lines were identified by mass spectrometry. After confirmation by immunocytochemical staining, their expression in patient samples was measured using 40 pairs of undifferentiated NPCs together with their adjacent normal epithelia. RESULTS: Proteomic findings indicated that adenosine triphosphate synthase alpha chain was up-regulated, whereas annexin II, annexin V, beta(2)-tubulin, and profilin 1 were down-regulated. After confirming the results in agar-processed cell lines, annexin II and beta(2)-tubulin expression were found to be lower in tumor cells than in adjacent normal epithelial cells in 100% and 90% of the patients' specimens, respectively. Finally, annexin II down-regulation was positively associated with lymph node metastasis, suggesting that it may be a prognostic factor in NPC. CONCLUSIONS: The results suggest that annexin II and beta(2)-tubulin down-regulation is important in NPC formation and may represent potential targets for further investigations.

The contribution of bifunctional SkipDewax pretreatment solution, rabbit monoclonal antibodies, and polymer detection systems in immunohistochemistry.

CONTEXT: In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems. OBJECTIVES: To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol. DESIGN: The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens. RESULTS: For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay. CONCLUSIONS: Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

Prognostic significance of tumor angiogenesis, Ki 67, p53 oncoprotein, epidermal growth factor receptor and HER2 receptor protein expression in undifferentiated nasopharyngeal carcinoma--a prospective study.

BACKGROUND: This study prospectively examines the prognostic role of p53 oncoprotein (p53), Ki67-antigen (Ki67), tumor angiogenesis (MVD), epidermal growth factor receptor (EGFR), and HER2 receptor protein (HER2) expression in Chinese with undifferentiated nasopharyngeal carcinoma (NPC). METHODS: Seventy-eight Chinese were recruited from October 1995 to July 1997 at the Prince of Wales Hospital, Hong Kong. Pretreatment immunohistochemical preparations of the primary tumor were made, and clinical data were collected prospectively until October 30, 2000. The markers were correlated with overall survival (OS), disease-free survival (DFS), time to progression (TTP), and UICC stage. RESULTS: On univariate analysis, EGFR expression correlated with poorer OS (p =.0001), DFS (p =.01), shorter TTP (p =.0001), and advanced T stage (p =.036). Strong EGFR expression, when compared with weak or moderate, was associated with poorer OS (p =.04) and shorter TTP in a subgroup of patients with UICC stage III-IV disease. HER2 expression was associated with advanced UICC stage (p =.006). The presence of p53 expression correlated with poorer DFS (p =.01) and a trend toward shorter TTP (p =.06). No correlation was found with Ki67-antigen or MVD. On multivariate analysis, only EGFR expression was significantly linked to shorter OS and TTP. CONCLUSIONS: EGFR expression in undifferentiated NPC is associated with a poor clinical outcome. A prognostic role of p53 and HER2 expression is suggestive but not consistently defined in this study. The relatively high prevalence of positive staining for EGFR supports the use of molecular targeted therapy in this disease.

Quantification and utility of monosialylated alpha-fetoprotein in the diagnosis of hepatocellular carcinoma with nondiagnostic serum total alpha-fetoprotein.

BACKGROUND: At concentrations <500 microg/L, serum alpha-fetoprotein (AFP) has low specificity in the diagnosis of hepatocellular carcinoma (HCC), but monosialylated AFP (msAFP) is more specific for HCC. We describe two strategies for quantitative analysis of msAFP and explore their diagnostic accuracy in cases of HCC with nondiagnostic serum total AFP concentrations. METHODS: We first used isoelectric focusing, Western blot, and densitometry (IEF-Western blot assay). We then developed a second assay, a novel glycosylation immunosorbent assay (GISA), based on the specificity of sialyltransferase and immunosorbent technology. Both assays were used to measure msAFP and msAFP percentage relative to total AFP in sera with nondiagnostic AFP concentrations from 36 patients with newly diagnosed HCC and from 18 patients with liver cirrhosis. RESULTS: The msAFP percentages and concentrations were significantly higher in the HCC patient group regardless of the quantification methods. The msAFP concentrations and msAFP percentages obtained by the two assays were highly correlated (r = 0.70 and 0.49, respectively). For discrimination of HCC with nondiagnostic serum total AFP from liver cirrhosis, the areas under the ROC curves were 0.81 (95% confidence interval, 0.70-0.92) for msAFP by IEF-Western blot assay, 0.73 (0.58-0.87) for msAFP by GISA, 0.89 (0.80-0.97) for msAFP percentage by IEF-Western blot assay, and 0.74 (0.59-0.89) for msAFP percentage by GISA. CONCLUSIONS: Both the serum concentration and percentage of msAFP are potential diagnostic markers for HCC with nondiagnostic AFP. GISA can quantify a specific glycoform of a serologic marker.