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Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China, and is highly sensitive to radiotherapy. The control region (D-loop) of mtDNA is a polymorphic region in which point mutations occur frequently. In this study, point mutation and common deletion (CD) mutations were investigated in 23 samples of NPC tumor tissue and in the radiation-treated NPC cell line CNE2. Polymorphisms at 72 (7.28%, 72/988) nucleotide positions in D-loop region and 6 (0.75%, 6/795) nucleotide positions in part of the functional gene encoding regions were detected in all NPC patients. Of the detected polymorphisms, 8 are novel. These variants are nonencoding transitions, including np292T-->C , np517G-del, np16038A-->G, np513G-del, np16242C-->A, np513G-del, np16242C-->A and np15787T-->C transition. A total of 39 point mutations in the D-loop region of mtDNA were detected in 43.5% (10/23) of the NPC patients. Three point mutations in the functional gene encoding regions of mtDNA were detected in only 8.7% (2/23) of NPC patients. The effect of he mutation at np709G-->A in the 12sRNA gene is unclear, and the A-->G substitution at np15769 in the cytochrome B gene is a synonymous mutation. The C-->T substitution at np15970 in the T Psi C loop of the tRNA(pro) gene could alter the position of the proline residue. After irradiation, the survival fraction of CNE2 cells decreased as X-ray dose increased. Moreover, X-ray radiation could induce apoptosis and the CD mutation in a time- and dose-dependent manner, but did not induce mtDNA point mutations. A positive correlation between the apoptosis index and the ratio of CD/WT mtDNA was observed in irradiated CNE2 cells. Our results suggest that CD mutation induced by irradiation is one of the late events after apoptosis of the cancer cells, and the mtDNA CD mutation may associated with the susceptibility of NPC cells to IR-induced apoptosis.

Angiogenesis plays a pivotal role in tumor growth, tissue invasion and metastasis. Endostatin is an angiogenesis inhibitor and has been shown to reduce tumor growth in animal models. However, therapy with recombinant endostatin protein was hampered by its short half-life and very-low yield of bioactive protein. We performed a phase I dose-escalation clinical trial using intratumoral injection of an adenovirus containing human endostatin gene (Ad-rhE; E10A; 10(10)-10(12) virus particles (vp)) in 15 patients with advanced solid tumors. We observed intratumoral injections of E10A without dose-limiting toxicity. Most frequently reported E10A-related adverse events were transient fever and local response. Distribution studies revealed that the vector was detected in the blood, throat and injection site, but rarely in the urine and stool. An increased endostatin expression was detected using enzyme immunoassay in serum in 13 of 14 treated patients throughout the period of treatment despite the presence of neutralizing antiadenovirus antibody. Median serum basic fibroblast growth factor levels decreased from 32.4 pg ml(-1) at baseline to 24.9 pg ml(-1) after 28 days of first treatment. Thus, direct intratumoral injection of up to 10(12) vp of E10A to patients is well tolerated and further studies are necessary to define and increase clinical efficacy.

The purpose of this study was to create a prognostic model for distant metastasis in patients with locally advanced NPC who accept concurrent chemotherapy combined with intensity-modulated radiotherapy (CCRT) to identify high-risk patients who may benefit from neoadjuvant chemotherapy (NACT). A total of 881 patients with newly-diagnosed, non-disseminated, biopsy-proven locoregionally advanced NPC were retrospectively reviewed; 411 (46.7%) accepted CCRT and 470 (53.3%) accepted NACT followed by CCRT. Multivariate analysis demonstrated N2–3 disease, plasma Epstein–Barr virus (EBV) DNA > 4000 copies/mL, serum albumin ≤46 g/L and platelet count >300 k/cc were independent prognostic factors for distant metastasis in the CCRT group. Using these four factors, a prognostic model was developed, as follows: 1) low-risk group: 0–1 risk factors; and 2) high-risk group: 2–4 risk factors. In the high-risk group, patients who accepted NACT + CCRT had significantly higher distant metastasis-free survival and progression-free survival rates than the CCRT group (P = 0.001; P = 0.011). This simple prognostic model for distant metastasis in locoregionally advanced NPC may facilitate with the selection of high-risk patients who may benefit from NACT prior to CCRT.

Background In the era of intensity-modulated radiotherapy (IMRT), the efficacy of additional neoadjuvant chemotherapy (NACT) to concurrent chemoradiotherapy (CCRT) in locoregionally advanced nasopharyngeal carcinoma (NPC) is currently being investigated in ongoing trials. Overall survival (OS) is the gold standard endpoint in NPC trials. We performed this analysis to identify surrogate endpoints for OS, which could shorten follow-up duration and speed up assessment of treatment effects. Methods We retrospectively analysed 208 matched-pair patients with locoregionally advanced NPC receiving NACT+CCRT or CCRT. Progression-free survival (PFS), failure-free survival (FFS), distant failure-free survival (D-FFS) and locoregional failure-free survival (LR-FFS) at 2 and 3 years were assessed as surrogates for 5-year OS according to Prentice’s criteria. The strength of the associations were assessed using Spearman’s rank correlation coefficient. Results No significant differences were observed between treatment arms for any surrogate endpoint at 2 years, which rejected Prentice’s second criterion. In contrast, 3-year LR-FFS, PFS, FFS and D-FFS were consistent with all four of Prentice’s criteria; the rank correlation coefficient (0.730) between 3-year PFS and 5-year OS was highest. Conclusions 3-year PFS, FFS and D-FFS could be valid surrogate endpoints for 5-year OS; 3-year PFS may be the most accurate.

Lysyl oxidase (LOX) is an extracellular matrix-remodeling enzyme that plays important roles in tumor development and progression. To evaluate the prognostic value of LOX levels in primary nasopharyngeal carcinoma, we performed an immunohistochemical analysis using 233 tissue biopsy specimens from as many patients. We found that the extent of immunohistochemical LOX staining correlated inversely with the clinicopathological features and survival. High LOX expression correlated with decreases in 5-year survival, overall survival, distant metastasis-free survival and disease-free survival (p < 0.05). Cox regression analysis confirmed that LOX was a significant prognostic indicator of increased risk of 5-year mortality for all patients (hazard ratio [HR], 1.670; 95% confidence interval [95% CI], 1.033-2.701 [p < .005]). Higher LOX expression was also an independent predictor of poor prognosis in patients with nasopharyngeal carcinoma. These findings suggest LOX may be a new biomarker predictive of NPC prognosis and may also be a useful treatment target.

Introduction The rearrangement of the anaplastic lymphoma kinase (ALK) gene accounts for approximately 1%–6% of lung adenocarcinoma cases and defines a molecular subgroup of tumors characterized by clinical sensitivity to ALK inhibitors such as crizotinib. This study aimed to identify the relationship between ALK rearrangement and the clinicopathologic characteristics of non-small cell lung cancer (NSCLC) and to analyze the therapeutic responses of crizotinib and conventional chemotherapy to ALK rearrangement in NSCLC patients. Methods A total of 487 lung cancer patients who underwent testing for ALK rearrangement in our department were included in this study. ALK rearrangement was examined by using fluorescence in situ hybridization (FISH) assay. Results Among the 487 patients, 44 (9.0%) were diagnosed with ALK rearrangement by using FISH assay. In 123 patients with adenocarcinoma who were non-smokers and of a young age (≤58 years old), the frequency of ALK rearrangement was 20.3% (25/123). Short overall survival (OS) was associated with non-adenocarcinoma tumor type (P = 0.006), poorly differentiated tumors (P = 0.001), advanced-stage tumors (P

// Lu Zhang 1, 2, * , Lin-Quan Tang 1, 2, * , Qiu-Yan Chen 1, 2 , Huai Liu 4, 5, 6 , Shan-Shan Guo 1, 2 , Li-Ting Liu 1, 2 , Ling Guo 1, 2 , Hao-Yuan Mo 1, 2 , Chong Zhao 1, 2 , Xiang Guo 1, 2 , Ka-Jia Cao 1, 2 , Chao-Nan Qian 1, 2 , Mu-Sheng Zeng 1 , Jian-Yong Shao 1, 7 , Ying Sun 1, 8 , Jun Ma 1, 8 , Ming-Huang Hong 1, 3 , Hai-Qiang Mai 1, 2 1 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P. R. China 2 Department of Nasopharyngeal Carcinoma, Sun Yat-Sen University Cancer Center, Guangzhou, P. R. China 3 GCP Center, Sun Yat-Sen University Cancer Center, Guangzhou, P. R. China 4 Department of Radiotherapy, Hunan Cancer Hospital, Changsha, P. R. China 5 Department of Radiotherapy, The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, P. R. China 6 Key Laboratory of Translational Radiation Oncology, Changsha, P. R. China 7 Department of Molecular Diagnostics, Sun Yat-Sen University Cancer Center, Guangzhou, P. R. China 8 Department of Radiation Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, P. R. China * These authors contributed equally to this work Correspondence to: Hai-Qiang Mai, e-mail: maihq@sysucc.org.cn Ming-Huang Hong, e-mail: hongmh@sysucc.org.cn Keywords: nasopharyngeal carcinoma, Epstein-Barr viral DNA, TNM staging, intensity-modulated radiotherapy, prognosis Received: July 31, 2015 Accepted: November 26, 2015 Published: December 24, 2015 ABSTRACT Background: The objective of this study is to verify the prognostic value of pretreatment plasma Epstein-Barr viral deoxyribonucleic acid (pEBV DNA) levels in nasopharyngeal carcinoma (NPC) patients to complement TNM classification based on the application of the intensity-modulated radiotherapy (IMRT) technique. Methods: In total, 1467 patients staged at I–IVa–b (M0) and treated with IMRT were retrospectively analyzed at our cancer center from January 2007 to December 2010. Patient survival among different stages and EBV DNA levels were compared. Results: Outcome analyses of different stages and EBV DNA levels revealed that patients in stages II–III with low EBV DNA levels had similar survival as that of patients in stages IVa–b with low EBV DNA (5-yr overall survival (OS), 94.7% vs. 92.9% ( P = 0.141), progression failure-free survival (PFS), 87.2% vs. 89.0% ( P = 0.685), distant metastasis failure-free survival (DMFS), 93.5% vs. 92.4% ( P = 0.394) and locoregional failure-free survival (LRFS), 93.8% vs. 96.3% ( P = 0.523)). Conversely, patients in stages II–III with high EBV DNA had better survival than patients in stages IVa–b with high EBV DNA (5-yr OS, 82.7% vs. 71.7% ( P = 0.001), PFS, 70.7% vs. 66.2% ( P = 0.047), DMFS, 79.6% vs. 74.8% ( P = 0.066) and LRFS, 89.3% vs. 87.6% ( P = 0.425)) but poorer survival than patients in stages IVa–b with low EBV DNA (5-yr OS, 82.7% vs. 92.9% ( P = 0.025), PFS, 70.7% vs. 89.0, ( P < 0.001), DMFS, 79.6% vs. 92.4%, ( P = 0.001), LRFS, 89.3% vs. 96.3%, ( P = 0.022)). Conclusion: pEBV DNA is a strong prognostic factor for patients with NPC when complemented with TNM staging in the era of IMRT application.

Anaplastic lymphoma kinase (ALK) rearrangement is present in approximately 5% of lung adenocarcinoma. Clinical trials on ALK inhibitor phase I to III have shown an interesting disease control rate and acceptable tolerability in ALK rearrangement patients. In clinical application, the precise diagnostic strategy for identifying ALK rearrangements remains to be determined. In this study, ALK rearrangement was screened by using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), direct sequencing, 2 fluorescence in situ hybridization (FISH) assays, and immunohistochemistry in 173 lung adenocarcinomas. We identified 18 cases (10.4%) with EML4-ALK fusion-positive by qRT-PCR, and all were positive for EML4-ALK fusion gene validated by direct sequencing. The result was consistent with that of other methods. Furthermore, of the 18 EML4-ALK fusion-positive cases, 16 (9.2%) were positive by using EML4-ALK fusion probe FISH, and 15 (8.7%) were positive by using ALK break-apart probe FISH and immunohistochemistry staining. Of the 18 ALK fusion-positive lung adenocarcinomas, 8 cases (44.4%) were histologically diagnosed as subtypes of cribriform adenocarcinoma, 7 cases (38.9%) as cribriform adenocarcinoma mixed with papillary and/or mucinous pattern, 2 cases (11.1%) as papillary adenocarcinoma, and 1 case (5.6%) as mucinous adenocarcinoma. In the present study, the ALK rearrangement frequency detected by qRT-PCR in Chinese NSCLC patients was higher than that in the western populations. QRT-PCR is a rapid, sensitive technology that could be used as a screening tool for identifying EML4-ALK fusion-positive NSCLC patients who would be sensitive for receiving ALK inhibitor therapy.

DNA methylation, the best known epigenetic marker, can be used as a prognostic biomarker in many cancers. We examined DNA methylation status and survival in nasopharyngeal carcinoma (NPC) patients. Aberrant DNA-methylated genes in 24 NPC tissues and 24 noncancer nasopharyngitis biopsy tissues (NCNBT) were identified using Illumina 450K BeadChip. Correlations between DNA methylation and clinical outcomes were evaluated using bisulfite pyrosequencing in 454 NPC patients. Genome-wide methylation analysis demonstrated that NPC tissues had distinct DNA methylation patterns compared with NCNBT. Among all significant CpG sites, 2,173 CpG sites with β change ≥ 0.2 (1,880 hypermethylated, 293 hypomethylated) were identified (P < 0.05). A methylation gene panel comprising six hypermethylated genes was constructed with the average Z-score method. Patients in the training cohort with high methylation had poorer disease-free survival [DFS, HR, 2.26; 95% confidence interval (CI), 1.28–4.01; P, 0.005] and overall survival (OS, HR, 2.47; 95% CI, 1.30–4.71; P, 0.006) than those with low methylation. There were similar results in the validation (DFS, HR, 2.07; 95% CI, 1.17–3.67; P, 0.013; OS, HR, 1.83; 95% CI, 1.01–3.31; P, 0.046) and independent cohorts (DFS, HR, 1.94; 95% CI, 1.08–3.47; P, 0.026; OS, HR, 2.09; 95% CI, 1.10–3.98; P, 0.022). Analysis indicated that the methylation gene panel was an independent prognostic factor. Furthermore, patients with low methylation had a favorable response to concurrent chemotherapy with an improved DFS (P = 0.045) and OS (P = 0.031), whereas patients with high methylation did not benefit from concurrent chemotherapy. The six–hypermethylated gene panel was associated with poor survival in patients with NPC, demonstrating its potential usefulness as a prognostic biomarker to clinicians in NPC management. Mol Cancer Ther; 14(12); 2864–73. ©2015 AACR.

Purpose To explore the prognostic value of the plasma load of Epstein-Barr viral (EBV) DNA and the tumor response to neoadjuvant chemotherapy (NACT) in advanced-stage nasopharyngeal carcinoma (NPC). Patients and Methods In all, 185 consecutive patients with stage III to IVb NPC treated with NACT followed by concurrent chemoradiation therapy (CCRT) were prospectively enrolled. The primary endpoint was progression-free survival (PFS), and the secondary endpoints included locoregional relapse–free survival (LRFS) and distant metastasis–free survival (DMFS). Results EBV DNA was detected in 165 (89%) patients before treatment but was undetectable in 127 (69%) patients after NACT. Detectable EBV DNA levels after NACT were correlated with poor prognosis (3-year PFS 71.8% vs 85.2%, P =.008 and 3-year DMFS 82.5% vs 92.3%, P =.013). An unsatisfactory tumor response (stable disease or disease progression) after NACT was also correlated with poor clinical outcome (3-year PFS 71.1% vs 85.9%, P =.005 and 3-year LRFS 82.7% vs 93.5%, P =.012). Multivariate analysis showed that the EBV DNA level after NACT (hazard ratio [HR] 2.31, 95% CI 1.18-4.54, P =.015) and the tumor response to NACT (HR 2.84, 95% CI 1.42-5.67, P =.003) were both significant prognostic factors for PFS. Multivariate analysis also showed that EBV DNA after NACT was the only significant predictor of DMFS (HR 2.99, 95% CI 1.25-7.15, P =.014) and that tumor response to NACT was the only significant predictor of LRFS (HR 3.31, 95% CI 1.21-9.07, P =.020). Conclusion Detectable EBV DNA levels and an unsatisfactory tumor response (stable disease or disease progression) after NACT serve as predictors of poor prognosis for patients with advanced-stage NPC. These findings will facilitate further risk stratification, early treatment modification, or both before CCRT.

To evaluate the frequency and clinicopathological features of ROS1 and RET rearrangements in N2 node positive stage IIIA (IIIA-N2) non-small cell lung cancer (NSCLC) patients, we retrospectively screened 204 cases with a tissue microarray (TMA) panel by fluorescent in situ hybridization (FISH), and confirmed by direct sequencing and immunohistochemistry (IHC). The relationship between ROS1 or RET rearrangements, clinicopathological features, and prognostic factors were analyzed in resected stage IIIA-N2 NSCLC. Of the 204 cases, 4 cases were confirmed with ROS1 rearrangement, but no RET rearrangement was detected. All 4 ROS1-rearranged cases were adenocarcinomas. The predominant pathological type was acinar pattern in ROS1-rearranged tumors, except for 1 case harboring a mixture acinar and mucous tumor cells. Variants of ROS1 rearrangement were SDC4-ROS1 (E2:E32), SDC4-ROS1 (E4:E32) and SDC4-ROS1 (E4:E34). There was no significant association between ROS1 rearrangement and clinicopathological characteristics. In this cohort, multivariate analysis for overall survival (OS) indicated that squamous cell carcinoma and lobectomy were independent predictors of poor prognosis; R0 surgical resection and non-pleural invasion were independent predictors of good prognosis. In resected stage IIIA-N2 NSCLC patients, ROS1-rearranged cases tended to occur in younger patients with adenocarcinomas. The prognosis of resected stage IIIA-N2 is generally considered poor, but patients with ROS1 rearrangement will benefit from the targeted therapy.

High frequency loss of 3p21.3 region is a common event in various kinds of tumors including nasopharyngeal carcinoma (NPC). RASSF1A has been identified as a putative tumor suppressor gene residing in this region. Chromosome alterations and epigenetic changes are commonly observed as mechanisms for inactivation of RASSF1A function. In this study, we applied the PCR-cloning-sequencing strategy to examine somatic mutations in RASSF1A in NPC tissues as compared with the sequences detected in the matched peripheral blood lymphocytes. Our results revealed a high incidence of RASSF1A mutation in primary tumor tissues of NPC. There are totally 35 mutations identified in 74% (17/23) of these NPC cases, including 30 transitions, three transversions and two deletions. Most of these mutations result in amino acid changes: three nonsense (stop codon) mutations, two-1 bp deletion (frameshift), 26 missense and the remaining four are synonymous (silent). No obvious 'hot-spot' mutations were observed in this study. A similarly high rate (74%) of promoter methylation of RASSF1A was also detected in the same group of NPC tissues, but no significant correlation between mutation and methylation was detected. Our results suggest various mechanisms involved in inactivation of RASSF1A function and indicate a critical role of RASSF1A in NPC development.

Nasopharyngeal carcinoma (NPC) is a malignancy with striking geographic and ethnic distribution, and familial clustering of NPC is frequently observed. Emigration and dialect studies also suggest the involvement of genetic factors in the development of NPC. Therefore, many investigations on susceptibility genes have been carried out and polymorphism or specific allele types of HLA, TCR, GSTM1, PIGR and cytochrome P450 were reported to be related to high risk of NPC. More importantly, a pedigree-based study provided evidence of linkage of NPC to chromosome 4p15.1-q12. Somatic genetic changes, such as allelic loss, chromosomal gains or losses, specific gene alterations, were reported to occur frequently in NPC. These findings mentioned above were summarized in this paper and a carcinogenesis model for NPC is proposed.

Tumor response to preoperative chemoradiotherapy and postoperative survival differs among patients with locally advanced rectal cancer. The objective was to find correlations of mutated oncogenes and clinical outcomes in locally advanced rectal cancer. A total of 70 patients with preoperative preoperative chemoradiotherapy followed by radical surgery at a single cancer center between 2006 and 2012 were enrolled. Pretreatment tumor biopsy samples were assayed for 238 mutation hotspots harboring 19 oncogenes by time-of-flight mass spectrometry and OncoCarta Array. Oncogene mutations were found in 48.6% of patients (34/70). KRAS was the most frequent driver mutation, found in 35.7% of patients (25/70), followed by PIK3CA (14.3%), NRAS (5.7%), FLT3 (2.9%), and BRAF (1.4%). Multiple gene mutations were observed in eight patients (11.4%). Tumors with KRAS mutations responded poorly to preoperative chemoradiotherapy (p = 0.044). Patients with oncogene mutations had worse 3-year disease-free survival than those without mutations (67.2% vs 94.2%, p = 0.010). Patients with KRAS or RAS mutations had lower 3-year disease-free survival (68% vs 88.3%, p = 0.016; 65.5% vs 92.3%, p = 0.004, respectively) and 3-year overall survival (88% vs 95.4%, p = 0.020; 89.7% vs 94.9%, p = 0.036, respectively) than those without KRAS or RAS mutations. Oncogene mutation status affected tumor response to treatment and long-term survival in locally advanced rectal cancer.

Background Pregnancy-associated nasopharyngeal carcinoma (PANPC) has been associated with poor survival. Recent advances in radiation technology and imaging techniques, and the introduction of chemotherapy have improved survival in nasopharyngeal carcinoma (NPC); however, it is not clear whether these changes have improved survival in PANPC. Therefore, the purpose of this study was to compare five-year maternal survival in patients with PANPC and non-pregnant patients with NPC. Methods After adjusting for age, stage and chemotherapy mode, we conducted a retrospective case-control study among 36 non-metastatic PANPC patients and 36 non-pregnant NPC patients (control group) who were treated at our institution between 2000 and 2010. Results The median age of both groups was 30years (range, 23–35years); median follow-up for all patients was 70months. Locoregionally-advanced disease accounted for 83.3% of all patients with PANPC and 92.9% of patients who developed NPC during pregnancy. In both the PANPC and control groups, 31 patients (86.1%) received chemotherapy and all patients received definitive radiotherapy. The five-year rates for overall survival (70% vs. 78%, p =0.72), distant metastasis-free survival (79% vs. 76%, p =0.77), loco-regional relapse-free survival (97% vs. 91%, p =0.69) and disease-free survival (69% vs. 74%, p =0.98) were not significantly different between the PANPC and control groups. Multivariate analysis using a Cox proportional hazards model revealed that only N-classification was significantly associated with five-year OS. Conclusion This study demonstrates that, in the modern treatment era, pregnancy itself may not negatively influence survival outcomes in patients with NPC; however, pregnancy may delay the diagnosis of NPC.

Background Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that acts as a prognostic marker for several human malignancies. In this study, we investigated the clinical significance of CIP2A and its function in nasopharyngeal carcinoma (NPC). Methods Quantitative RT-PCR, western blot, and immunohistochemistry analyses were used to quantify CIP2A expression in NPC cell lines and clinical samples. Kaplan-Meier curves were used to estimate the association between CIP2A expression and patient survival. The functional role of CIP2A in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation and xenograft growth. Results CIP2A levels were upregulated in NPC cell lines and clinical samples at both the mRNA and protein levels (P

Although microRNA-30a (miR-30a) has been shown to regulate cancer metastasis, the molecular mechanism has not yet been clearly elucidated in nasopharyngeal carcinoma (NPC). The present study was to investigate the miR-30a expression pattern and its potential functions and further to identify its target gene and corresponding clinical applications in NPC. MiR-30a was identified to be down-regulated in NPC primary tumors compared with metastatic tumors using quantitative real-time PCR. Furthermore, over-expression of miR-30a transfected with precursor increased the ability of metastasis and invasion of NPC tumor cells in vivo and in vitro. E-cadherin was screened as a putative target gene of miR-30a by computational algorithms. Luciferase reporter assays showed that over-expression of miR-30a directly reduced the activity of a luciferase transcript combined with the 3′-untranslated region (3′-UTR) of E-cadherin. Kaplan–Meier survival analysis and log-rank test were analyzed for 1077 NPC patients for overall survival, indicating that a high expression of E-cadherin was beneficial for NPC prognosis ( P = 0.001). Importantly, NPC patients with high expression of E-cadherin had much lower risk of poor prognosis (hazard ratio = 0.757, P = 0.017) using multivariate analysis. In conclusion, miR-30a could play an important role in regulating NPC metastasis and potentially provide useful guidelines for individualized therapy.

Zi-Chen Zhang,1,* Sha Fu,1,* Fang Wang,1 Hai-Yun Wang,1 Yi-Xin Zeng,2 Jian-Yong Shao11Department of Molecular Diagnostics, 2Department of Experimental Research, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, People's Republic of China *These authors contributed equally to this work Abstract: Nasopharyngeal carcinoma (NPC) is a common tumor in Southern China, but the oncogene mutational status of NPC patients has not been clarified. Using time-of-flight mass spectrometry, 238 mutation hotspots in 19 oncogenes were examined in 123 NPC patients. The relationships between mutational status and clinical data were assessed with a χ2 or Fisher's exact test. Survival analysis was performed using the Kaplan–Meier method with the log-rank test. In 123 patients, 21 (17.1%) NPC tumors were positive for mutations in eight oncogenes: six patients had PIK3CA mutations (4.9%), five NRAS mutations (4.1%), four KIT mutations (3.3%), two PDGFRA mutations (1.6%), two ABL mutations (1.6%), and one with simultaneous mutations in HRAS, EGFR, and BRAF (1%). Patients with mutations were more likely to relapse or develop metastasis than those with wild-type alleles (P=0.019). No differences or correlations were found in other clinical characteristics or in patient survival. No mutations were detected in oncogenes AKT1, AKT2, CDK, ERBB2, FGFR1, FGFR3, FLT3, JAK2, KRAS, MET, and RET. These results demonstrate an association between NPC and mutations in NRAS, KIT, PIK3CA, PDGFRA, and ABL, which are associated with patient relapse and metastasis. Keywords: NPC, oncogene, mutation

BACKGROUNDThe goal of this study was to establish a plasma microRNA profile by use of next-generation sequencing that could aid in assessment of patient prognosis in nasopharyngeal carcinoma (NPC).METHODSTwo panels of NPC patients and healthy controls (HCs) were recruited for this study. We used deep sequencing to screen plasma microRNAs. Differentially expressed microRNAs were verified by quantitative real-time PCR (qPCR). Kaplan–Meier survival analysis with the log-rank test was used to compare overall survival (OS) and progression-free survival (PFS) between groups.RESULTSTwenty-three plasma miRNAs with differential expression levels were selected for qPCR analysis on an independent set including 100 NPC patients and 55 HCs. NPC patients with low concentrations of miR-483–5p and miR-103 had better prognosis for 5-year OS than those with high concentrations (87.5% vs 55.8%, P < 0.001; 80.9% vs 62.3%, P = 0.031). Those with low concentrations of miR-29a and let-7c had poorer prognosis (54.8% vs 82.8%, P = 0.002; 56.3% vs 84.6%, P = 0.001). A 3-signature miRNA integrated with clinical stage was further identified in an independent set. We calculated a prognostic index score and classified patients into low-, medium-, and high-risk groups. Five-year OS among the 3 groups was significantly different (90.9%, 66.7%, and 23.8%; P < 0.001). By multivariate analysis, a high-risk score was the most significantly unfavorable prognostic factor independent of other clinical variables (P < 0.001, hazard ratio = 15.1, 95% CI = 5.2–43.9).CONCLUSIONSDifferentially expressed plasma miRNAs as identified by next-generation sequencing can be helpful for predicting survival in NPC patients.

The expression levels of nm23-H1 mRNA and its protein in human nasopharyngeal carcinoma (NPC) were detected to clarify the relationship between nm23-H1 and metastasis and prognosis of patients with NPC. nm23-H1 mRNA expression in fresh tissues from 78 patients with NPC was investigated by in situ hybridization and RT-PCR. Routine labeling streptavidin-biotin immuno-histochemistry with the nm23-H1 murine monoclonal antibody was employed to study the expression of nm23-H1 protein in paraffin-embedded specimens from 231 patients with NPC treated in our hospital. The clinical pathologic data and results of follow-up were collected. Comparisons between expression of nm23-H1 protein or mRNA and clinical outcome were performed using the chi2 test. Multivariate prognostic analyses were performed by the Cox regression model. We found that nm23-H1-negative tumors were associated with a higher incidence of lymph-node metastasis (84.2%) than nm23-H1-positive ones (32.8%, p < 0.01). The distant metastasis and loco-regional recurrence rates in the nm23-H1-negative group were 55.8% and 31.68%, respectively but only 17.2% and 11.5%, respectively, in the nm23-H1-positive group (p < 0.01). A significant association was found between expression of nm23-H1 protein and prognosis (p < 0.01). Expression of nm23-H1 protein indicated favorable prognosis, suggesting that the absence of nm23-H1 protein expression was significantly associated with lymph-node metastasis, recurrence and distant metastasis in NPC. Expression of the nm23-H1 gene may be valuable for assessing the prognosis of NPC.

There is no consensus in the salvage treatment for non-small-cell lung cancer (NSCLC) with acquired resistance to primary epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Fifty-one consecutive EGFR-mutated NSCLC patients with TKI retreatment after acquired resistance were enrolled in this study. The quantitation of mutation abundance was performed by real-time fluorescent quantitative PCR. The correlation between mutation abundance and outcomes of readministrated TKI was analyzed by survival analysis. Patients with high (H) mutation abundance (24/51) had a significantly (log-rank, P < 0.05) longer (5.27-2.53 months) median progression-free survival (PFS), compared with the low (L) abundance group (27/51), whereas the median overall survival showed no difference (21.00-18.20 months, log-rank P = .403) between the two groups. Objective response and disease control rates in group H and group L regarding the second round TKI treatment were 8.3, 70.8 and 0, 48.1 %, respectively. Groupings with different mutation abundances were significantly associated with PFS under multivariate Cox proportional hazards regression model [hazard ratio (HR) for group H vs. L, 0.527; P = .036]. Mutation abundance affects the efficacy of EGFR-TKIs readministration in NSCLC with acquired resistance. The quantitative mutation abundance of EGFR may be a potential predictor for selecting optimal patients to readministrate EGFR-TKIs after acquired resistance to primary TKI.

MicroRNA-214 (MiR-214) is aberrantly expressed in several human tumors such as ovarian cancer and breast cancer. However, the role of miR-214 in nasopharyngeal carcinoma (NPC) is still unknown. In this study, we report that miR-214 was overexpressed in NPC cell lines and tissues. Silencing of miR-214 by LNA-antimiR-214 in NPC cells resulted in promoting apoptosis and suppressing cell proliferation in vitro, and suppressed tumor growth in nude mice in vivo. Luciferase reporter assay was performed to identify Bim as a direct target of miR-214. Furthermore, this study showed that low Bim expression in NPC tissues correlated with poor survival of NPC patients. Taken together, our findings suggest that miR-214 plays an important role in NPC carcinogenesis.

Background This study was designed to determine whether advanced non-small-cell lung cancer (NSCLC) patients with high copy number of epidermal growth factor receptor (EGFR) can benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs). Methods EGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) and EGFR mutations was tested using Luminex xTAG technology in 502 TKI-treated NSCLC patients. The association between both biomarkers and clinical benefit from EGFR-TKI were analyzed. Results EGFR FISH + and EGFR mutations were significantly associated with higher response rates (37.2% and 43.7%, respectively), superior progression-free survival (PFS) (FISH+, 11.2 months; hazard ratio [HR], 0.51; 95% CI, 0.42 to 0.62; p vs. 2.0 months; HR, 0.56; 95% CI, 0.41 to 0.75; p vs. 2.0 months; HR, 0.43; 95% CI, 0.24 to 0.76; p = 0.003). However, FISH + had no association with improved PFS in EGFR-mutated patients (HR, 0.77; 95% CI, 0.57 to 1.03; p = 0.076). Conclusions A combined analysis of EGFR FISH and mutation is an effective predictor of EGFR-TKI therapy. Specifically, a high EGFR copy number may predict benefit from TKIs treatment for NSCLC patients with wild-type EGFR.

We evaluated whether DNA repair gene polymorphisms had an effect on clinical outcomes in metastatic/recurrent nasopharyngeal carcinoma (NPC) patients treated with cisplatin-based chemotherapy.Clinical data of 101 patients with metastatic/recurrent NPC between 2004 and 2011 were reviewed. Five potentially functional polymorphisms (ERCC1 Asn118Asn, ERCC1 C8092A, XPD Lys751Gln, XRCC1 Arg399Gln and XRCC1 Arg280His) were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.The ERCC1 C8092A polymorphism was an independent predictor of PFS in Chinese NPC patients treated with cisplatin-based chemotherapy. Compared to the patients carrying the C/C genotype, the patients with the C/A or A/A genotype had an increased risk of disease progression on cisplatin-based chemotherapy (7.9 vs. 9.3 months; HR 1.61; 95 % CI 1.08-2.61; p = 0.047). However, no association between the other polymorphisms, response rate, disease progression and survival was detected in metastatic/recurrent NPC patients.The ERCC1 C8092A polymorphism might be a useful predictive marker in metastatic/recurrent NPC patients treated with cisplatin-based chemotherapy. However, a large-scale prospective study is warranted to validate our findings.

Delta-like ligand 4 (DLL4), one of the transmembranous Notch ligands, is upregulated at the site of tumor growth, particularly during tumor angiogenesis. Expression pattern of DLL4 in nasopharyngeal carcinoma (NPC) and the clinical and prognostic significance remain unclear. In this study, immunohistochemical analysis (IHC) was used to examine the protein level of DLL4 in NPC tissues from two independent cohorts. In the testing cohort (311 cases), we applied the X-tile program software able to assess the optimal cutoff points for biomarkers in order to accurately classify patients according to clinical outcome. In the validation cohort (113 cases), the cutoff score derived from X-title analysis was investigated to determine the association of DLL4 expression with disease-specific survival (DFS). Our results showed that high expression of DLL4 was observed in 134 of 313 (42.8 %) in the testing cohort and 58 of 113 (43.6 %) in the validation cohort. High expression of DLL4 independently predicted poorer disease-specific survival, as evidenced by univariate and multivariate analysis (P < 0.05). Moreover, DLL4 expression was significantly elevated in distant NPC metastases relative to primary NPC tumors (P = 0.001). Importantly, we found a significant positive relationship between DLL4 and vascular endothelial growth factor (VEGF) (P < 0.001). Patients with dual elevated DLL4 and VEGF expression displayed a significant overall survival disadvantage compared to those with dual low expression (P < 0.05). These findings provide evidence that high expression of DLL4 serves as an independent predictor of poor prognosis in NPC patients.

Epidermal growth factor receptor (EGFR) gene mutation and copy number are useful predictive markers that guide the selection of non-small cell lung cancer (NSCLC) patients for EGFR-targeting therapy. This study aimed to investigate the correlation between EGFR gene mutation and copy number and clinicopathologic characteristics of Chinese patients with NSCLC. NSCLC specimens collected from 205 patients between November 2009 and January 2011 were selected to detect EGFR gene mutations with real-time polymerase chain reaction (RT-PCR) and to detect EGFR gene copy number with fluorescence in situ hybridization (FISH). EGFR mutations primarily occurred in females, non-smokers, and patients with adenocarinomas (all P < 0.001). Tissues from 128 (62%) patients were FISH-positive for EGFR, including 37 (18%) with gene amplification and 91 (44%) with high polysomy. EGFR gene mutation was correlated with FISH-positive status (R = 0.340, P < 0.001). Multivariate analysis showed that not smoking (OR = 5.910, 95% CI = 2.363-14.779, P < 0.001) and having adenocarcinoma (OR = 0.122, 95% CI = 0.026-0.581, P = 0.008) were favorable factors for EGFR gene mutation. These results show a high frequency of EGFR FISH positivity in NSCLC tissues from Chinese patients and a significant relevance between EGFR gene mutations and FISH-positive status. Among the FISH-positive samples, EGFR gene mutation occurred more frequently in samples with gene amplification compared to those with high polysomy, suggesting that EGFR mutation and gene amplification should be used as clinical decision parameters to predict response to EGFR-targeting therapy.

Background Heat shock protein 70, a stress protein, has been implicated in tumor progression. However, its role in nasopharyngeal carcinoma (NPC) progression has not yet been clearly investigated. Methods Immunohistochemistry (IHC) was employed to examine the expression patterns of Hsp70, human leukocyte antigen –A (HLA-A) in NPC tissue samples. Results The expression of Hsp70 exhibited different spatial patterns among nuclear, membrane and cytoplasm in 507 NPC tumor tissues. Kaplan-Meier survival analysis demonstrated that different Hsp70 expression patterns are correlated with different patient outcomes. High membranal and cytoplasmic levels of Hsp70 predicted good survival of patients. In contrast, high nuclear abundance of Hsp70 correlated with poor survival. Moreover, the membranal and cytoplasmic levels of Hsp70 were positively correlated with levels of the MHC I molecule HLA-A. Conclusions Different Hsp70 expression patterns had distinct predictive values. The different spatial abundance of Hsp70 may imply its important role in NPC development and provide insight for the development of novel therapeutic strategies involving immunotherapy for NPC.

Human leukocyte antigen G (HLA-G) has multiple immune regulatory functions including the induction of immune tolerance in malignancies. The roles of HLA-G have not been investigated in nasopharyngeal carcinoma (NPC). This study is aimed to evaluate the role of HLA-G as prognostic factor for NPC patients as well as its role in the immune regulation. Western assays showed high HLA-G expression in NPC cell lines, but low in the immortalized nasopharyngeal epithelial cell line NP69. HLA-G protein was further detected in 79.2% of 552 NPC specimens with immunohistochemistry (IHC), but not in normal nasopharyngeal epithelium tissue. Moreover, high expression of HLA-G predicted poor survival of NPC patients and positively correlated with tumor N classification and recurrence or metastasis. Multivariate analysis indicated that HLA-G was an independent and unfavorable prognostic factor. Furthermore, the presence of CD68+ macrophages and IL-10 were also examined, which are two prognostic markers of NPC and important factors for regulating immune surveillance. The correlations of HLA-G with these two immune factors were revealed in NPC tissues. Taken together, our results suggest that HLA-G is an independent biomarker for NPC prognosis, and HLA-G might contribute to NPC progression, which might jointly regulate immune surveillance in NPC together with macrophages and IL-10.

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.

The purpose of this study was to investigate the correlation between gene mutation and gene copy number and their association with the clinical profiles and pathological features in Chinese patients with non-small cell lung cancer (NSCLC).Surgical specimens of cancer tissue were collected from 118 NSCLC patients. Gene mutations in exon 19 and exon 21 were detected by real-time PCR and gene copy number was detected by fluorescence in situ hybridization (FISH). Chi-square (χ(2)) test was performed to analyze the correlation between EGFR mutation and gene copy number, and explore their association with clinicopathological features in the NSCLC patients.The mutation frequency in EGFR was 41.5% (49/118). EGFR mutations occured in 50.0% (48/96) of patients with adenocarinoma and 5.0% (1/20) of patients with squamous cell carcinoma. EGFR gene high copy number was detected in 70.3% (83/118)of the patients. The FISH-positive rate was 78.1% (75/96) in adenocarcinoma and 35.0% (7/20) in squamous cell carcinoma. EGFR mutation and high copy number mainly occurred in the adenocarcinoma, advanced stage, female gender, and non-smoking patients. There was a significant correlation between EGFR gene mutation and gene high copy number.EGFR gene mutation and gene high copy number are more common in Chinese NSCLC patients with adenocarcinomas, advanced stage, non-smokers and females. There is a significant correlation between gene mutation and gene high copy number. Combined analysis of EGFR mutation and gene copy number by FISH may provide a superior approach in selecting patients who may benefit from anti-EGFR target therapy.

To explore the relationship between the mutations of epidermal growth factor receptor (EGFR) gene and clinicopathological characteristics in patients with non-small cell lung cancers (NSCLC).Paraffin-embedded tissue specimens were obtained from 1444 patients with NSCLC. The genomic DNA was extracted. Mutations of EGFR gene (exons 19 and 21) were detected by real-time PCR.DNA was available in 1410 cases. Somatic mutations of the EGFR gene were identified in 401 cases (27.8%). Among patients with EGFR mutations, 41.4% (n=166) had del E746-A750 of exon19, 6.7% (n=27) had del L747-P753insS of exon 19, 50.3% (n=201) had L858R of exon 21, and 1.5% (n=6) had L861Q of exon 21. Woman, non-smoker and adenocarcinoma showed a higher percentage of EGFR mutation (43.2%, 37.6%, and 33.5%, respectively). However, there was no association among age, grades, lymph node metastasis, and TNM stages (P0.05). The mutation rate of BAC subtype (61.3%, 19/31) and adenocarcinoma with BAC features (48.0%, 12/25) was significantly higher than that of conventional adenocarcinoma (32.4%, 336/1038). A further assess of the smoking status found a trend that the more increased smoking exposure, the lower the incidence of EGFR mutations. A multivariable analysis revealed that adenocarcinoma, never smoking, and female were independently associated with EGFR mutations (odds rations=3.381, 2.393, and 1.727, respectively).The detection rate of EGFR mutation is higher in Chinese patients, especially in non-smoking female patients with adenocarcinoma. Real-time PCR is a sensitive and accurate method to detect the mutations of EGFR gene and can therefore provide useful information for clinical treatment.

Background The aim of the present study was to analyse the expression of Secreted protein acidic and rich in cysteine (SPARC) in nasopharyngeal carcinoma (NPC) specimens, and to evaluate its correlation with clinicopathologic features, including survival of patients with NPC Methods NPC tissue microarrays (TMAs) were constructed from Sun Yat-sen University Cancer Center (SYSUCC), another three centers on mainland China, Singapore and Hong Kong. Using quantitative RT-PCR and Western-blotting techniques, we detected mRNA and protein expression of SPARC in NPC cell lines and immortalized nasopharyngeal epithelial cells (NPECs) induced by Bmi-1 (NPEC2 Bmi-1). The difference of SPARC expression in the cell lines was tested using a t-test method. The relationship between the SPARC expression and clinicopathological data was assessed by chi-square. Survival analysis was estimated using the Kaplan-Meier approach with log-rank test. Univariate and multivariate analyses of clinical variables were performed using Cox proportional hazards regression models. Results The expression levels of SPARC mRNA and protein were markedly higher in NPC cell lines than in NPEC2 Bmi-1. Especially, the expression levels of SPARC mRNA and protein were much lower in the 6-10B than in the 5-8 F (P = 0.002, P = 0.001). SPARC immunostaining revealed cytoplasmic localization in NPC cells and no staining in the stroma and epithelium. In addition, high level of SPARC positively correlated with the status of distant metastasis (P = 0.001) and WHO histological classification (P = 0.023). NPC patients with high SPARC expression also had a significantly poorer prognosis than patients with low SPARC expression (log-rank test, P < 0.001), especially patients with advanced stage disease (log-rank, P < 0.001). Multivariate analysis suggested that the level of SPARC expression was an independent prognostic indicator for the overall survival of patients with NPC (P < 0.001). Conclusions SPARC expression is common in NPC patients. Our data shows that elevated SPARC expression is a potential unfavorable prognostic factor for patients with NPC.

The significance of SOX2 in nasopharyngeal carcinoma (NPC), a tumor associated with Epstein-Barr virus (EBV), remains unclear. Here, we reported that, in the univariate analysis, SOX2 expression was correlated with a poorer distant metastasis-free survival (DMFS). The EBV-VCA/IgA titers in patients with NPC were also associated with a poorer overall survival (OS). Patients with NPC, who had both strong staining of SOX2 and high titers of EBV-VCA/IgA, had the poorest prognoses. Therefore, SOX2 or the combination of the SOX2 expression level and EBV-VCA/IgA titers could be valuable to predict distant metastasis or the prognosis of NPC.

Currently, nasopharyngeal carcinoma (NPC) prognosis evaluation is based primarily on the TNM staging system. This study aims to identify prognostic markers for NPC.We detected expression of 18 biomarkers by immunohistochemistry in NPC tumors from 209 patients and evaluated the association between gene expression level and disease-specific survival (DSS). We used support vector machine (SVM)--based methods to develop a prognostic classifier for NPC (NPC-SVM classifier). Further validation of the NPC-SVM classifier was performed in an independent cohort of 1,059 patients.The NPC-SVM classifier integrated patient sex and the protein expression level of seven genes, including Epstein-Barr virus latency membrane protein 1, CD147, caveolin-1, phospho-P70S6 kinase, matrix metalloproteinase 11, survivin, and secreted protein acidic and rich in cysteine. The NPC-SVM classifier distinguished patients with NPC into low- and high-risk groups with significant differences in 5-year DSS in the evaluated patients (87% v 37.7%; P.001) in the validation cohort. In multivariate analysis adjusted for age, TNM stage, and histologic subtype, the NPC-SVM classifier was an independent predictor of 5-year DSS in the evaluated patients (hazard ratio, 4.9; 95% CI, 3.0 to 7.9) in the validation cohort.As a powerful predictor of 5-year DSS among patients with NPC, the newly developed NPC-SVM classifier based on tumor-associated biomarkers will facilitate patient counseling and individualize management of patients with NPC.

The role of microRNA-155 (miR-155) has been associated with oncogenesis of several human tumors. However the expression pattern of miR-155 has not been investigated in nasopharyngeal carcinoma (NPC). The present study was to assess miR-155 expression pattern and its possible function in NPC, to identify its targets and evaluate their clinical applications in NPC. MiR-155 was found to be upregulated in two Epstein-Barr virus (EBV) negative NPC derived cell lines CNE1 and TW03, as well as in NPC clinical samples by quantitative Real-time PCR and in situ hybridization detection. EBV encoded LMP1 and LMP2A could further enhance the expression of miR-155 in NPC CNE1 and TW03 cells. JMJD1A and BACH1 were identified as putative targets of miR-155 in a bioinformatics screen. Overexpression of miR-155 downregulated a luciferase transcript fused to the 3'UTR of JMJD1A and BACH1. MiR-155 mimic could downregulate the expression of JMJD1A and BACH1, while miR-155 inhibitor could upregulate JMJD1A expression in NPC cell lines. Moreover, downregulation of JMJD1A was significantly correlated with N stage in TNM classification (p = 0.023), a lower five-year survival rate (p = 0.021), and a lower five-year disease-free survival rate (p = 0.049) of NPC patients. Taken together, up-regulation of miR-155 in NPC is partly driven by LMP1 and LMP2A, and results in downregulation of JMJD1A, which is associated with N stage and poor prognosis of NPC patients. The potential of miR-155 and JMJD1A as therapeutic targets in NPC should be further investigated.

BACKGROUND: Plasma Epstein-Barr virus (EBV) DNA is widely used in screening, monitoring, and prediction of relapse in nonmetastatic nasopharyngeal carcinoma (NPC). However, data regarding utility of plasma EBV DNA in metastatic NPC are rare. The current study was to test the prognostic implication of plasma EBV DNA level in metastatic/recurrent NPC patients treated with palliative chemotherapy. METHODS: Plasma EBV DNA level was measured at baseline and thereafter at the start of each treatment cycle in 127 histologically proven metastatic/recurrent NPC patients treated with palliative chemotherapy. Correlations of pre-treatment and post-treatment plasma EBV DNA levels to survival and response were analyzed. RESULTS: Patients with a low pre-treatment plasma EBV DNA level (

Purpose: Metastatic colorectal cancer patients with low epidermal growth factor receptor (EGFR) gene copy number are unlikely to respond to anti-EGFR monoclonal antibody (mAb) treatment. The objective of this study was to investigate EGFR fluorescence in situ hybridization (FISH) patterns of chromosome 7 disomy with efficacy of cetuximab therapy in metastatic colorectal cancer patients. Experimental Design: We detected the EGFR FISH patterns and KRAS status in 74 tumors from cetuximab-treated metastatic colorectal cancer patients and analyzed with response rate (RR) and progression-free survival (PFS). Results: One of the 16 (6.25%) patients with chromosome 7 homogeneous disomy (defined as FISH negative) had objective response to cetuximab. A total of 53(76.8%) patients with chromosome 7 pattern of variable ratios of disomy versus polysomy (defined as FISH positive) had a significantly higher RR (37.7% versus 6.25%; P = 0.01), a trend towards longer PFS (4.5 versus 2.9 months; P = 0.07). Among 54 KRAS wild-type patients, EGFR FISH-positive patients had significantly higher RR (51.3% versus 9%; P = 0.01) and longer PFS (5.0 versus 2.3 months; P = 0.02) than EGFR FISH-negative patients. However, among 20 KRAS mutant-type patients, there was no difference in RR (0% versus 0%) and PFS (2.5 versus 3.8 months; P = 0.51) between EGFR FISH-positive and -negative patients. Conclusion: Our results show firstly that patients with EGFR FISH pattern of chromosome 7 disomy have a very low chance to benefit from cetuximab-based therapy. EGFR FISH pattern of chromosome 7 disomy may be as a negative predicative factor for cetuximab response in KRAS wild-type metastatic colorectal cancer patients. Clin Cancer Res; 17(2); 382–90. ©2011 AACR.

BackgroundLATS2, which encodes a novel serine/threonine kinase, is known to be important in centrosome duplication and in the maintenance of genomic stability. Recently, a potential role for LATS2 in cancer has been reported. In breast cancer and acute lymphoblastic leukemia (ALL), LATS2 mRNA is downregulated and has been suggested to be a tumor suppressor. However, the role of LATS2 in nasopharyngeal carcinoma has not been investigated. In this study, we aimed to investigate the expression pattern of LATS2 and its clinicopathological involvement in nasopharyngeal carcinoma to understand its effect on cell survival.MethodsUsing quantitative real time PCR and immunoblotting, the expression of LATS2 was detected in nasopharyngeal carcinoma cell lines and in the immortalized nasopharyngeal epithelial cell line NP69. Using immunohistochemistry, we analyzed LATS2 protein expression in 220 nasopharyngeal carcinoma cases. The association of LATS2 protein expression with the clinicopathological characteristics and the prognosis of nasopharyngeal carcinoma were subsequently assessed. Using methylation specific PCR, we detected the methylation status of the LATS2 promoter. RNA interference was performed by transfecting siRNA to specifically knock down LATS2 expression in 5-8F and CNE2.ResultsLATS2 protein was detected in 178 of 220 (80.91%) cases of nasopharyngeal carcinoma. LATS2 overexpression was a significant, independent prognosis predictor (P= 0.037) in nasopharyngeal carcinoma patients. Methylation specific PCR revealed that 36.7% (11/30) of nasopharyngeal carcinoma tissues and all of the chronic nasopharyngeal inflammation samples were methylated. Functional studies showed that the suppression of LATS2 expression in nasopharyngeal carcinoma (5-8F and CNE2) cell lines by using specific small interfering (siRNA) resulted in the inhibition of growth, induction of apoptosis and S-phase cell cycle increase. Overexpression of LATS2 in NP69 stimulated cell proliferation.ConclusionsOur results indicate that LATS2 might play a role in the tumorigenesis of nasopharyngeal carcinoma by promoting the growth of nasopharyngeal carcinoma cells. Transfection with specific siRNA might be feasible for the inhibition of growth, induction of apoptosis and S phase increase in nasopharyngeal carcinoma.

The FAS receptor/ligand system is a key regulator of apoptotic cell death and corruption of this signaling pathway has been shown to participate in carcinogenesis. Functional polymorphisms in the FAS (FAS −1377G/A) and FASL (FASL −844T/C) genes alter their transcriptional activity. Therefore, we examined the association between these polymorphisms and the risk of developing nasopharyngeal carcinoma (NPC). FAS −1377G/A and FASL −844T/C genotypes were determined by PCR-based RFLP analysis in 582 patients with NPC and 613 frequency-matched controls. We observed a significantly increased risk of NPC associated with the FAS −1377AA genotype [odds ratio (OR) = 1.69, 95% confidence interval (CI) = 1.21–2.35] compared with the FAS −1377 GG genotype. In addition, elevated NPC risk was also found among subjects carrying both FAS −1377AA and FASL −844CC genotypes compared with both FAS −1377GG and FASL −844CT or −844TT, the OR was 2.39 (95% CI = 1.50–3.79). After stratification by smoking status, heavy smokers (≥15 pack-years) carrying FAS −1377AA genotype had an increased risk of NPC compared with FAS −1377GG genotype (OR = 3.48, 95% CI = 1.66–7.30). Furthermore, we observed a statistically significant interaction between the two polymorphisms and heavy smoking status (OR = 5.92, 95% CI = 1.91–18.3). Our study provides the first evidence that functional FAS −1377 G/A and FASL −844 T/C polymorphisms are associated with the risk of NPC, and this association is especially noteworthy in tobacco smokers. Mol. Carcinog. © 2010 Wiley-Liss, Inc.

Methylenetetrahydrofolate reductase (MTHFR) plays a central role in converting folate to a compound which serves as a methyl donor for DNA methylation, an epigenetic modification known to be dysregulated in carcinogenesis. This case–control study assessed the contribution of MTHFR polymorphisms to the risk of nasopharyngeal carcinoma (NPC). MTHFR genotypes C677T and A1298C in 529 NPC patients and 577 frequency-matched controls were determined by PCR-based restriction fragment length polymorphism. We found a 1.57-fold increased risk of NPC in subjects with the MTHFR 1298AC genotype compared to subjects with the MTHFR 1298AA genotype. In addition, an elevated NPC risk was also found in subjects with both the MTHFR 677CT and 1298AC genotypes [odds ratio (OR) = 2.15, 95% confidence interval (CI) = 1.37–3.39] compared to subjects with the 677CC/1298AA genotypes. Furthermore, we observed an additive interaction between MTHFR polymorphisms and smoking status on the increased risk of NPC. The OR was 6.72 (95% CI = 1.85–24.48) among heavy smokers (pack-years ≥15) carrying 677TT compared with nonsmokers carrying the 677CC genotype. The OR was 7.23 (95% CI = 4.22–12.38) or 12.75 (95% CI = 2.74–59.3) among subjects carrying the 1298AC or 1298CC genotype in heavy smokers (pack-years ≥15) compared with 1298AA in nonsmokers. Our results provide the first molecular epidemiological evidence that MTHFR polymorphisms associate with the risk of NPC and this association is especially noteworthy in heavy smokers. © 2010 Wiley-Liss, Inc.

Our previous study suggested that melanoma nuclear protein 18 (Mel-18) acted as a tumor suppressor in human breast cancer. This study was designed to investigate the clinical and prognostic significance of Mel-18 in breast cancer patients.Mel-18 was detected by immunohistochemistry in paraffin-embedded tissues from 287 breast cancer patients, of which 287 were from primary cancer sites, 63 from matched adjacent noncancerous sites, and 35 from metastatic lymph nodes. Differences in Mel-18 expression and clinical characteristics were compared by χ² test. Prognostic outcomes correlated with Mel-18 were examined using Kaplan-Meier analysis and Cox proportional hazards model.The decreased Mel-18 expression is incremental depending upon the magnitude of cancer progression (P0.001). Mel-18 was conversely correlated with the pathological classifications (P0.001 for T, N, and M classifications, respectively), clinical staging (P0.001), and progesterone receptor (P = 0.030). Furthermore, patients with higher level of Mel-18 showed prolonged overall survivals (P0.001). The diminished Mel-18 expression may be a risk factor for the patients' survival (P0.001).Lower Mel-18 expression is correlated with advanced clinicopathologic classifications and a poor overall survival in breast cancer patients. These findings suggest that Mel-18 may serve as a useful marker in prognostic evaluation for patients.

Background Our recent cDNA microarray data showed that centromere protein F (CENP-F) is significantly upregulated in primary cultured nasopharyngeal carcinoma (NPC) tumor cells compared with normal nasopharyngeal epithelial cells. The goal of this study was to further investigate the levels of CENP-F expression in NPC cell lines and tissues to clarify the clinical significance of CENP-F expression in NPC as well as the potential therapeutic implications of CENP-F expression. Methods Real-time RT-PCR and western blotting were used to examine CENP-F expression levels in normal primary nasopharyngeal epithelial cells (NPEC), immortalized nasopharyngeal epithelial cells and NPC cell lines. Levels of CENP-F mRNA were determined by real-time RT-PCR in 23 freshly frozen nasopharyngeal biopsy tissues, and CENP-F protein levels were detected by immunohistochemistry in paraffin sections of 202 archival NPC tissues. Statistical analyses were applied to test for prognostic associations. The cytotoxicities of CENP-F potential target chemicals, zoledronic acid (ZOL) and FTI-277 alone, or in combination with cisplatin, in NPC cells were determined by the MTT assay. Results The levels of CENP-F mRNA and protein were higher in NPC cell lines than in normal and immortalized NPECs. CENP-F mRNA level was upregulated in nasopharyngeal carcinoma biopsy tissues compared with noncancerous tissues. By immunohistochemical analysis, CENP-F was highly expressed in 98 (48.5%) of 202 NPC tissues. Statistical analysis showed that high expression of CENP-F was positively correlated with T classification (P < 0.001), clinical stage (P < 0.001), skull-base invasion (P < 0.001) and distant metastasis (P = 0.012) inversely correlated with the overall survival time in NPC patients. Multivariate analysis showed that CENP-F expression was an independent prognostic indicator for the survival of the patient. Moreover, we found that ZOL or FTI-277 could significantly enhance the chemotherapeutic sensitivity of NPC cell lines (HONE1 and 6-10B) with high CENP-F expression to cisplatin, although ZOL or FTI-277 alone only exhibited a minor inhibitory effect to NPC cells. Conclusion Our data suggest that CENP-F protein is a valuable marker of NPC progression, and CENP-F expression is associated with poor overall survival of patients. In addition, our data indicate a potential benefit of combining ZOL or FTI-277 with cisplatin in NPC suggesting that CENP-F expression may have therapeutic implications.

Background The oncoprotein Epstain-Barr Virus (EBV)-encoded latent membrane protein1 (LMP1) modulates the pathological effects of the NF-κB, AP-1 and JAK/STAT pathways in nasopharyngeal carcinoma (NPC). Methods Microarray analysis was performed on the NPC cell line HONE1 stably transfected with a LMP1-expression plasmid or an empty vector. Based on assigned pathways analyzed using the KEGG database, the mTOR signaling pathway was selected for verification by quantitative RT-PCR. Western blot, RNA interference and immunofluorescence were used to determine the relationship between LMP1 and mTOR signing pathway genes, and their clinical significance to NPC. Results Our studies revealed that overexpression of LMP1 upregulated the mTOR signaling pathway, possibly through phosphorylation of AKT/mTOR/P70S6K/4EBP1 in the NPC cell lines HONE1 and 6-10B. Knockdown of LMP1 reduced expression of p-mTOR and p-4EBP1 in EBV-positive NPC cell line C666-1. In addition, LMP1 expression closely correlated with expression of p-mTOR, p-P70S6K and p-4EBP1 in NPC tumors. Expression of p-P70S6K, p-4EBP1 and LMP1, but not p-mTOR, significantly correlated with overall survival of NPC patients. However, only LMP1 was an independent prognostic factor. Conclusions These results suggest that the mTOR signaling pathway is regulated by LMP1 expression in NPC. LMP1 and the genes in the mTOR pathway such as p-P70S6K and p-4EBP1 may be potential prognostic biomarkers.

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial–mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC., Author Summary Epstein-Barr virus (EBV) infects about 90% of people worldwide and persists benignly as a latent infection. However, EBV is associated with different types of human cancer. Nasopharyngeal carcinoma (NPC) is the most commonly known EBV associated cancer and expresses a well defined set of latent viral genes, including LMP2A, which has been detected in the majority of NPC samples. Several studies indicated this latent viral protein drove cellular invasion and metastasis. For this study, enforced LMP2A expressing NPC cell lines were generated. We show here that LMP2A induces an Epithelial–Mesenchymal Transition and increases the Stem-like Cancer Cells in NPC. Our results suggest that LMP2A supports tumor initiation and recurrence of the infected nasopharyngeal epithelial cells. For the first time we report a virus protein that functions in the initiation and progression of cancer by inducing the cancer stem-like cells. These findings permit a more detailed understanding of function and contribution to viral pathogenesis and provide a novel therapeutic target for NPC therapy.

Purpose Approximately 30% of patients with stage IB non–small-cell lung cancer (NSCLC) die within 5 years after surgery. Current staging methods are inadequate for predicting the prognosis of this particular subgroup. This study identifies prognostic markers for NSCLC. Patients and Methods We used computer-generated random numbers to study 148 paraffin-embedded specimens for immunohistochemical analysis. We studied gene expression in paraffin-embedded specimens of lung cancer tissue from 73 randomly selected patients with stage IB NSCLC who had undergone radical surgical resection and evaluated the association between the level of expression and survival. We used support vector machines (SVM)–based methods to develop three immunomarker-SVM–based prognostic classifiers for stage IB NSCLC. For validation, we used randomly assigned specimens from 75 other patients. Results We devised three immunomarker-SVM–based prognostic classifiers, including SVM1, SVM2, and SVM3, to refine prognosis of stage IB NSCLC successfully. The SVM1 model integrates age, cancer cell type, and five markers, including CD34MVD, EMA, p21ras, p21WAF1, and tissue inhibitors of metalloproteinases (TIMP) –2. The SVM2 model integrates age, cancer cell type, and 19 markers, including BCL2, caspase-9, CD34MVD, low-molecular-weight cytokeratin, high-molecular-weight cytokeratin, cyclo-oxygenase-2, EMA, HER2, matrix metalloproteinases (MMP) –2, MMP-9, p16, p21ras, p21WAF1, p27kip1, p53, TIMP-1, TIMP-2, vascular endothelial growth factor (VEGF), and β-catenin. The SVM3 model consists of SVM1 and SVM2. The three models were independent predictors of overall survival. We validated the classifiers with data from an independent cohort of 75 patients with stage IB NSCLC. Conclusion The three immunomarker-SVM–based prognostic characteristics are closely associated with overall survival among patients with stage IB NSCLC.

Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis. [Cancer Res 2008;68(19):8137–45]

AIB1, a candidate oncogene in breast cancer, is commonly amplified and overexpressed in several types of human cancers. In this study, expression and amplification of AIB1 in nasopharyngeal carcinoma (NPC) were studied by immunohistochemical analysis and fluorescence in situ hybridization using tissue microarrays, including 80 specimens of NPC and 20 specimens of nonneoplastic nasopharyngeal mucosa. In this NPC cohort, overexpression and amplification of AIB1 was detected in 36 (51%) of 71 and 3 (7%) of 46 NPCs, respectively. Overexpression of AIB1 was observed more frequently in NPCs in late T stages (T3/T4, 24/35 [69%]) than in earlier stages (T1/T2, 12/36 [33%]; P < .05). In addition, 18 (72%) of 25 NPCs with lymph node metastasis (N1-3) showed overexpression of AIB1; the frequency was significantly higher than that in NPCs without node metastasis (N0, 18/49 [39%]; P < .05). These findings suggest that overexpression of AIB1 in NPCs may be important in the acquisition of an invasive and/or metastatic phenotype.

Background Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China. The China 1992 TNM staging system has been widely used for prognosis prediction of NPC patients in China. Although NPC patients can be classified according to their clinical stage in this system, their prognosis may vary significantly. Method 280 cases of NPC with clinical follow-up data were collected and expressions of survivin and VEGF in tumor tissues were investigated by immunohistochemistry (IHC). Apoptosis index (AI) in 100 cases of NPC was detected by the TUNEL method. Results Expression of survivin and VEGF were significantly associated with TNM stage, T-stage and metastasis of NPC. The patients with survivin and VEGF over-expression presented lower 5-year survival rate, as compared to those of low-expression (42.32% vs. 70.54%, 40.1% vs. 67.8%, respectively, P < 0.05), especially in advanced stage patients (36.51% vs. 73.41%, 35.03% vs. 65.22%, respectively, P < 0.05). The 5-year survival rate in NPC patients with survivin and VEGF dual over-expression was significantly lower than that of patients with dual low-expression (18.22% vs. 73.54%, respectively; P = 0.0003). Multivariate analysis indicated that both survivin and VEGF over-expression in NPC tumor tissues were strong independent factors of poor prognosis in NPC patients. The mean AI in the 39 survivin low-expression cases was 144.7 ± 39.9, which was significantly higher than that in 61 survivin over-expression cases (111.6 ± 39.8) (T test, P < 0.05). Conclusion Survivin and VEGF over-expression are independent prognostic factors for the patients with NPC. These results also suggest that tumor survivin and VEGF expressions are valuable prognostic markers for prognosis prediction in NPC patients.

To analyze the global gene expression profile of primary cultured nasopharyngeal carcinoma (NPC) cells using cDNA microarray techniques to screen new candidate genes related to the occurrence and progression of NPC.A NPC cell line C666 and primary cultured NPC cells from biopsy specimens in 5 cases were analyzed with microarray techniques in comparison with 3 normal nasopharyngeal epithelial (NPE) biopsy specimens. Several differentially expressed genes identified from the microarray results were verified by fluorescence real-time PCR (FQ-PCR) and immunohistochemistry (IHC).Primary cultured cells of both NPC and NPE were verified by cytokeratin IHC, EBER1 in situ hybridization and EBV-DNA real-time PCR. Compared with NPE cells, a total of 493 genes in at least 4/6 of the samples were identified to be differentially expressed in the primary cultured NPC cells, including 264 up-regulated and 229 down-regulated ones. Several differentially expressed genes according to the microarray results were confirmed by real-time PCR and IHC.cDNA microarray technique provides an effective and accurate means for global gene expression profiling of primary cultured NPC cells to screen the differentially expressed genes, which may serve as an important basis for studying the mechanism, classification and diagnosis of NPC at the molecular level.