Jingfeng Zong

Circulating Epstein-Barr virus microRNAs miR-BART7 and miR-BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment.

More than 75% of nasopharyngeal carcinoma (NPC) patients have already developed local or regional spread at diagnosis, which hampers effective treatment and results in a poor prognosis. It is essential to characterize more sensitive and specific biomarkers for screening of high risk individuals and assessment of NPC treatment effectiveness. NPC is an Epstein–Barr virus (EBV) associated tumor in which only a few viral proteins but more than 20 BamHI A rightward transcripts (BART) microRNAs are detected, at abundant levels. We hypothesized that these BART microRNAs may be novel biomarkers for NPC. Systematic analysis of EBV BART microRNA expression profiles in EBV latently infected Mutu I and Mutu III cell lines, EBV‐harboring NPC and noncancerous NP cells found that miR‐BART3, miR‐BART7 and miR‐BART13 microRNAs are highly expressed and regularly secreted into the extracellular environment of NPC cells. These BART microRNAs were evaluated for used as potential NPC biomarkers. Analysis of plasma specimens obtained from NPC patients (n = 89), and healthy (n = 28) and non‐NPC tumor patient controls (n = 18) found levels of both miR‐BART7 and miR‐BART13, but not miR‐BART3, to be distinctly presence among NPC patients, with elevated levels being particularly apparent among patients with advanced disease. Receiver operating characteristic curve analysis combining miR‐BART7 and miR‐BART13 levels produces a 90% predictive value for the presence of NPC. Analysis of 41 NPC patients before and after radiotherapy showed that miR‐BART7 and miR‐BART13, but not miR‐BART3, were diminished after treatment. These results indicate that EBV microRNAs, miR‐BART7 and miR‐BART13, may constitute useful new serological biomarkers for diagnosis of NPC and prediction of treatment efficacy.

A Comparison Between the Chinese 2008 and the 7th Edition Ajcc Staging Systems for Nasopharyngeal Carcinoma

Objectives:The Chinese 1992 staging system for nasopharyngeal carcinoma (NPC) was revised in 2008 and was renamed as the Chinese 2008 staging system. The seventh edition of the American Joint Committee on Cancer (AJCC) staging manual also defined new rules for classifying NPC in 2010. The purpose of the current study is to compare the 2 in terms of patient distribution and efficacy in predicting prognosis. Methods:A total of 816 patients with untreated nondisseminated NPC who underwent magnetic resonance imaging scan of the nasopharynx and neck were studied retrospectively. All magnetic resonance imaging scans were reevaluated independently by 2 radiologists specialized in head and neck cancers. All patients were restaged according to the Chinese 2008 staging system and the AJCC staging system of NPC. Results:Using the 2 staging systems, the consistency for patient distributions in T, N, and overall stages was found to be moderate, with the &kgr; value of 0.65, 0.54, and 0.46, respectively. According to the Chinese 2008 and the AJCC staging systems, the proportion of patients in stages I, II, III, and IV accounted for 2.3% versus 2.3%, 11.0% versus 23.7%, 39.4% versus 49.1%, and 47.3% versus 24.9%, respectively. The AJCC T classification was better in predicting the 5-year local relapse-free survival, whereas the Chinese 2008 N classification was superior in predicting the 5-year distant metastasis-free survival. However, survival curves for the 5-year overall survival were comparable in both systems. Conclusions:We revealed a slightly better patient distribution of overall stage with AJCC comparing with the Chinese 2008 staging system. The prognostic value of AJCC T classification was considered to be better, whereas that of Chinese 2008 N classification was superior.

Impact of intensity-modulated radiotherapy on nasopharyngeal carcinoma: Validation of the 7th edition AJCC staging system

BACKGROUND AND PURPOSE The purpose of this study was to evaluate the 7th edition UICC/AJCC staging system for nasopharyngeal carcinoma (NPC) patients who were treated with intensity modulated radiation therapy (IMRT). MATERIALS AND METHODS The clinical data of 1241 NPC patients with initial magnetic resonance imaging (MRI) scans were studied retrospectively. All MRIs were independently reevaluated and restaged according to the 7th edition by two radiologists specializing in head and neck cancers. Analysis of prognostic factors in local relapse-free survival (LRFS), distant metastasis-free survival (DMFS), disease-specific survival (DSS), and overall survival (OS) were performed. RESULTS The proportion of patients in Stage I, II, III, IVA and IVB were 4.8%, 26.2%, 45.4%, 18.4%, and 5.2%, respectively. The differences of LRFS between T1 and T2, and between T2 and T3 were not significant (P=0.055 and 0.605, respectively). Hazard ratios (HRs) for DSS and OS between T2 and T3 or between T3 and T4 differed significantly, but not between T1 and T2. The differences of DMFS between N0 and N1, between N1 and N2 were significant. However no significant difference was found in DMFS between N2 and N3a, or between N2 and N3b. For patients with T1-T3 disease, although skull base infiltration did not impact local failure, it was an independent prognostic factor for both distant failure and cancer death. CONCLUSION When treated with IMRT, the difference in the LRFS, DSS, and OS between T1 and T2 patients diminished, indicating that it is rational to merge T2 into T1. The prognostic value of the N classification of the current staging system had not changed much compared to the 6th edition.

NF-κB Signaling Regulates Expression of Epstein-Barr Virus BART MicroRNAs and Long Noncoding RNAs in Nasopharyngeal Carcinoma

ABSTRACT Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Suggestions for lymph node classification of UICC/AJCC staging system: a retrospective study based on 1197 nasopharyngeal carcinoma patients treated with intensity-modulated radiation therapy.

AbstractThis article provides suggestions for N classification of Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system of nasopharyngeal carcinoma (NPC), purely based on magnetic resonance imaging (MRI) in intensity-modulated radiation therapy (IMRT) era.A total of 1197 nonmetastatic NPC patients treated with IMRT were enrolled, and all were scanned by MRI at nasopharynx and neck before treatment. MRI-based nodal variables including level, laterality, maximal axial diameter (MAD), extracapsular spread (ECS), and necrosis were analyzed as potential prognostic factors. Modifications of N classification were then proposed and verified.Only nodal level and laterality were considered to be significant variables affecting the treatment outcome. N classification was thus proposed accordingly: N0, no regional lymph node (LN) metastasis; N1, retropharyngeal LNs involvement (regardless of laterality), and/or unilateral levels I, II, III, and/or Va involvement; N2, bilateral levels I, II, III, and/or Va involvement; and N3, levels IV, Vb, and Vc involvement. This proposal showed significant predicting value in multivariate analysis. N3 patients indicated relatively inferior overall survival (OS) and distant metastasis-free survival (DMFS) than N2 patients; however, the difference showed no statistical significance (P = 0.673 and 0.265 for OS and DMFS, respectively), and this was considered to be correlated with the small sample sizes of N3 patients (79 patients, 6.6%).Nodal level and laterality, but not MAD, ECS, and necrosis, were considered to be significant predicting factors for NPC. The proposed N classification was proved to be powerfully predictive in our cohort; however, treatment outcome of the proposed N2 and N3 patients could not differ significantly from each other. This insignificance may be because of the small sample sizes of N3 patients. Our results are based on a single-center data, to develop a new N classification that is universally acceptable; further verification by data from multicenter is warranted.

Multimodality Treatment May Improve the Survival Rate of Patients with Metastatic Nasopharyngeal Carcinoma with Good Performance Status

The aim of the present study was to evaluate the benefit of chemotherapy, combined with palliative radiotherapy (PRT) and other local treatments to the metastatic sites, for patients with metastatic nasopharyngeal carcinoma (NPC) who had a performance status 0–2. We conducted a retrospective review of available data from 197 biopsy-proven NPC patients who developed metastasis after their initial definitive treatment. These patients were grouped into three categories according to the different treatment paths that were followed: the best supportive care (64 patients), chemotherapy alone (55 patients), and multimodality treatment with chemotherapy combined with PRT and other local treatments to metastatic sites (78 patients). The 2-year metastatic survival rate of patients in the multimodality treatment group was 57.7%, which was significantly better than that of the patients in both the chemotherapy alone group and the best supportive care group (32.7% and 1.6%, respectively). The independent significant factors affecting survival were the disease-free interval prior to the detection of metastatic disease, the number of metastases, the number of chemotherapy cycles and the biological effective dose of PRT. In conclusion, multimodality treatment may improve survival of select patients with recurrent NPC with distant metastases.

Long-term survival of nasopharyngeal carcinoma patients with Stage II in intensity-modulated radiation therapy era

OBJECTIVES To evaluate the long-term survival and the role of chemotherapy in nasopharyngeal carcinoma (NPC) patients in Stage II treated by intensity-modulated radiation therapy (IMRT). METHODS Three hundred and eleven NPC patients in Stage II were reviewed. All were treated with IMRT with or without chemotherapy, with 191, 20 and 100 patients being defined as T1N1M0, T2N0M0 and T2N1M0 stage, respectively. RESULTS At a median follow-up of 57 months, the 5-year overall survival, disease-specific survival, distant metastasis-free survival, loco-regional relapse-free survival (LRRFS) and progression-free survival were 91.1, 93.5, 90.6, 95.9 and 87.6%, respectively. T2N1 patients had significant poorer survival outcomes than T1N1 patients, with T2N0 patients in between. Further analysis showed that the addition of chemotherapy could only improve LRRFS [hazard ratio (HR) 0.263, 95% confidence interval (CI) 0.083-0.839, P = 0.024], especially for T1N1 patients (HR 0.209, 95% CI 0.046-0.954, P = 0.043). For those in the T2N1M0 group, chemotherapy, as used in our series, added no benefit to any endpoint. CONCLUSIONS IMRT in NPC patients in Stage II was quite therapeutic; however, different subgroups have distinct survival outcomes. Distant metastasis was the main failure pattern, especially for those with T2N1 disease, and the chemotherapy currently in use failed to treat subclinical metastatic foci effectively. Further prospective study is warranted to find out the role and the optimal schedule of chemotherapy in this subgroup of patients.

The prognosis of nasopharyngeal carcinoma involving masticatory muscles: a retrospective analysis for revising T subclassifications.

AbstractThis work is a retrospective study of magnetic resonance imaging (MRI) and T-stage subclassifications of nasopharyngeal carcinoma (NPC) involving the masticatory muscles (MMs). We examined how involvement of MMs influences the clinical T-stage classifications and the survival outcomes of NPC patients.MRI data as well as the medical records from 816 NPC patients were analyzed retrospectively. All cases were restaged according to the seventh edition of American Joint Committee on Cancer staging system criteria. The overall survival (OS), local relapse-free survival (LRFS), and distant metastasis-free survival (DMFS) were analyzed by the Kaplan–Meier method, and their survival outcomes between different degrees of MM involvement and different T classifications were compared by using the log-rank test. All statistical analyses were conducted on SPSS 18.0 software. P > 0.05 was considered significant.Of the 816 NPC patients analyzed, 283 (34.68%) had tumors that involved MMs. All of those 283 patients involved the medial pterygoid muscle, and 125 cases (15.32%) involved the lateral pterygoid muscle. Multivariate analysis identified MM involvement as an independent prognostic factor for patients OS (P = 0.007) and LRFS (P = 0.024). MM involvement significantly correlated with a lower OS and LRFS (P < 0.01). In addition, compared with concurrent involvement of the medial and lateral pterygoid muscle, the medial pterygoid muscle involvement correlated with a higher OS and LRFS (P < 0.05). Among NPC patients, T-classifications 1 to 4 usually predicted the ultimate OS, LRFS, and DMFS (P > 0.1), unless the cancer involved the lateral pterygoid muscle.NPC involving the lateral pterygoid muscle presents a worse survival outcome than that involving the medial pterygoid muscle. Any cancer involving the lateral pterygoid muscle should be classified in a higher T-stage subclassification.

Decreased expression of the NKG2D ligand ULBP4 may be an indicator of poor prognosis in patients with nasopharyngeal carcinoma

U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) receptor on NK cells and subsets of T cells, is thought to activate anticancer immune responses. However, the expression pattern and prognostic effect of ULBP4 in nasopharyngeal carcinoma (NPC) has not been investigated. We first compared ULBP4 expression between archival 15 NPC tissues and 8 normal nasopharynx (NP) tissues using qPCR. Then ULBP4 expression among 111 NPC specimens was validated on immunohistochemical examination. In addition, the association of ULBP4 expression with clinical characteristics and survival outcomes was analyzed. Furthermore, the impact of ULBP4 expression in NPC cells on the cytotoxic activity of NK cells was investigated. Both mRNA and protein ULBP4 expressions of NPC tissues were significantly lower than those in normal NP tissues. However, no association of ULBP4 expression with clinical characteristics was observed. Patients with NPC having decreased expression of UBLP4 had significantly poorer overall survival (OS), progression-free survival (PFS), and distant metastasis-free survival (DMFS) than those with preserved levels of ULBP4. On multivariate analyses, low expression of ULBP4 was of borderline significance for OS, PFS, and DMFS (P = 0.060, 0.053, and 0.076, respectively). Further, LDH analysis demonstrated that the cytotoxic activitity of NK cells against C666-1 or 5-8F NPC cells with lenti-ULBP4 was considerably increased as compared to those with lenti-vector at various E/T ratios. Hence, restoration of ULBP4 expression may be a novel therapeutic strategy for treatment of NPC. However, further study is required to confirm these findings.

Parotid area lymph node metastases from preliminarily diagnosed patients with nasopharyngeal carcinoma: report on tumor characteristics and oncologic outcomes

The parotid area lymph node (PLN) is an uncommon site of metastasis originating from nasopharyngeal carcinoma (NPC). The study aimed to investigate clinical characteristics and outcomes of patients with preliminarily diagnosed NPC with PLN metastases. Here we retrospectively reviewed Magnetic resonance imaging (MRI) scans of 2221 patients with untreated nonmetastatic NPC who received intensity-modulated radiation therapy (IMRT). Finally, 64 (2.9%) patients were identified with PLN metastases, of which, 34 received PLN-sparing IMRT and 30 received PLN-radical IMRT. We also found that 42.2% had N3 disease and 95.3% had stages III-IVb. PLN metastases on MRI were characterized by ipsilateral retropharyngeal lymph node (RLN) or level II nodal extracapsular spread (ECS), ipsilateral giant cervical nodes, ipsilateral parapharyngeal extension, or solitary parotid metastasis. The 5-year overall survival, distant metastasis-free survival, regional relapse-free survival, and parotid relapse-free survival rates were 70.4%, 64.3%, 76.7%, and 87.9%, respectively. Distant metastases were the main cause of treatment failure and death. Using PLN-sparing IMRT, sparing PLN with minimal axial diameter of <10 mm, could increase the risk of parotid recurrence. However, it was not an independent prognostic factor. N classification and concurrent-based chemotherapy were almost statistically significant for distant failure and death. Overall, we demonstrated that the PLN metastases might be derived from RLN or level II nodal ECS, giant cervical nodes in a retrograde fashion, or parapharyngeal extension. Sparing PLN of <10 mm by IMRT should consider the risk of parotid recurrence. Distant metastases remained the dominant treatment failure. Further effective systemic chemotherapy should be explored.