Detection Of Nasopharyngeal Carcinoma Research Articles

An Anchoring and Steering Device for Wireless In Vivo Surveillance of Nasopharyngeal Carcinoma1

An Anchoring and Steering Device for Wireless In Vivo Surveillance of Nasopharyngeal Carcinoma1

Identification of candidate biomarkers for the early detection of nasopharyngeal carcinoma by quantitative proteomic analysis.

NPC is a lethal malignancy that is most prevalent in Southeast Asia, and early detection and treatment are essential for the survival and good prognosis of NPC patients. In the present work, we identified 26 differentially expressed proteins in NNET, AH and different stages of NPC tissues by using 2D-DIGE combined with MALDI-TOF/TOF analysis. Of these proteins, the down-regulation of ENO1 and over-expression of CYPA were confirmed with a high-throughput tissue microarray that included various stages of NPC tissues via an IHC assay, and the results indicated that the combination of ENO1 and CYPA can serve as a potential molecular marker for the early detection of NPC.

Epstein-Barr virus serology as a potential screening marker for nasopharyngeal carcinoma among high-risk individuals from multiplex families in Taiwan.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer that is highly treatable when diagnosed early, with 5-year disease-free survival of approximately 90%. However, NPC is typically diagnosed at advanced stages, in which disease-free survival is <50%. There is, therefore, a need for clinical tools to assist in early NPC detection, particularly among high-risk individuals. We evaluated the ability of anti-EBV IgA antibodies to detect incident NPC among high-risk Taiwanese individuals. NPC cases (N = 21) and age- and sex-matched controls (N = 84) were selected. Serum collected before NPC diagnosis was tested for ELISA-based IgA antibodies against the following EBV peptides: EBNA1, VCAp18, EAp138, Ead_p47, and VCAp18 + EBNA1 peptide mixture. The sensitivity, specificity, and screening program parameters were calculated. EBNA1 IgA had the best performance characteristics. At an optimized threshold value, EBNA1 IgA measured at baseline identified 80% of the high-risk individuals who developed NPC during follow-up (80% sensitivity). However, approximately 40% of high-risk individuals who did not develop NPC also tested positive (false positives). Application of EBNA1 IgA as a biomarker to detect incident NPC in a previously unscreened, high-risk population revealed that 164 individuals needed to be screened to detect 1 NPC and that 69 individuals tested positive per case detected. EBNA1 IgA proved to be a sensitive biomarker for identifying incident NPC, but future work is warranted to develop more specific screening tools to decrease the number of false positives. Results from this study could inform decisions about screening biomarkers and referral thresholds for future NPC early-detection program evaluations.

Combination of autoantibodies against NY-ESO-1 and viral capsid antigen immunoglobulin A for improved detection of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China and Southeast Asia, and early detection remains a challenge. Autoantibodies have been found to precede the manifestations of symptomatic cancer by several months to years, making their identification of particular relevance for early detection. In the present study, the diagnostic value of serum autoantibodies against NY-ESO-1 in NPC patients was evaluated. The study included 112 patients with NPC and 138 normal controls. Serum levels of autoantibodies against NY-ESO-1 and classical Epstein-Barr virus marker, viral capsid antigen immunoglobulin A (VCA-IgA), were measured by enzyme-linked immunosorbent assay. Measurement of autoantibodies against NY-ESO-1 and VCA-IgA demonstrated a sensitivity/specificity of 42.9/94.9% [95% confidence interval (CI), 33.7–52.6/89.4–97.8%] and 55.4/95.7% (95% CI, 45.7–64.7/90.4–98.2%), respectively. The area under receiver operating characteristic curve for autoantibodies against NY-ESO-1 (0.821; 95% CI, 0.771–0.871) was marginally lower than that for VCA-IgA (0.860; 95% CI, 0.810–0.910) in NPC. The combination of autoantibodies against NY-ESO-1 and VCA-IgA yielded an enhanced sensitivity of 80.4% (95% CI, 71.6–87.0%) and a specificity of 90.6% (95% CI, 84.1–94.7%). Moreover, detection of autoantibodies against NY-ESO-1 could differentiate early-stage NPC patients from normal controls. Our results suggest that autoantibodies against NY-ESO-1 may serve as a potential biomarker, as a supplement to VCA-IgA, for the screening and diagnosis of NPC.

Aberrant CpG island methylation of PTEN is an early event in nasopharyngeal carcinoma and a potential diagnostic biomarker

The inactivation of phosphatase and tensin homolog (PTEN) due to its CpG island hypermethylation has been observed in some types of tumors except nasopharyngeal carcinoma (NPC). In the present study, we focused on the aberrant methylation of PTEN CpG islands in NPC. The mRNA expression of PTEN was detected by quantitative PCR in 45 NPC and 22 non-tumor nasopharyngeal epithelial (NP) tissues. The methylation status of PTEN was examined by methylation-specific polymerase chain reaction and sequencing. The mRNA expression of PTEN in three NPC cell lines treated with 5-aza-2'-deoxycytidine (5-aza-dC) was also examined. PTEN was downregulated in both NPC tissues and NPC cell lines and a relatively higher methylation level of PTEN was found in NPC specimens (82.2%) relative to NP tissues (5.3%). The PTEN mRNA expression was restored in NPC cell lines by treatment with 5-aza-dC. These results first reveal an epigenetic alteration, aberrant methylation of PTEN, in NPC, which is probably an early event and may be regarded as a novel candidate biomarker for early stage of NPC detection and prevention.

Methods and matrices: approaches to identifying miRNAs for Nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC.MethodsWe tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set.ResultsBoth microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating “unknown” miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC.ConclusionsWe determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.

Identification of patients with nasopharyngeal carcinoma by serum protein profiling using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

As diagnosis of nasopharyngeal carcinoma at an early disease stage is important, we attempted to distinguish between patients with nasopharyngeal carcinoma and noncancer controls by using serum protein profiles. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and CM10 protein chip were used to detect the serum proteomic patterns of 65 patients with nasopharyngeal carcinoma before radiotherapy and 93 noncancer controls. Proteomic spectra of serum samples from 50 nasopharyngeal carcinoma patients and 60 noncancer controls were used as a training set. The validity of the classification tree was then challenged with a blind test set which included another 15 patients with nasopharyngeal carcinoma and 33 noncancer controls. Biomarker Wizard 3.01 and Biomarker Pattern 5.01 were used in combination to analyze the data and to develop diagnostic models. 21 protein peaks were significantly different between nasopharyngeal carcinoma and controls. 4 mass peaks (M4182, M5343, M5913 and M8702 mass/charge ratio) were chosen automatically to construct a classification tree. The classification tree correctly determined 93.8% (45/48) of the test samples with 93.3% (14/15) of the nasopharyngeal carcinoma samples and 93.9% (31/33) of the noncancer samples. Using a combination of serum protein profiles and Epstein-Barr viral capsid antigen immunoglobulin A antibody tests, the diagnostic sensitivity and specificity were increased to 100 and 97%, respectively. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry could correctly distinguish nasopharyngeal carcinoma from noncancer individuals and showed great potential for the development of a screening test for the detection of nasopharyngeal carcinoma.

Promoter hypermethylation of tumor suppressor genes in serum as potential biomarker for the diagnosis of nasopharyngeal carcinoma

Purpose: Promoter hypermethylation of tumor suppressor genes may serve as a promising biomarker for the diagnosis of cancer. Cell-free circulating DNA (cf-DNA) shares hypermethylation status with primary tumors. This study investigated promoter hypermethylation of five tumor suppressor genes as markers in the detection of nasopharyngeal carcinoma (NPC) in serum samples. Methods: cf-DNA was extracted from serum collected from 40 NPC patients and 41 age- and sex-matched healthy subjects. The promoter hypermethylation status of the five genes (RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1) was assessed by methylation-specific PCR after sodium bisulfite conversion. Differences in the methylation status of these five genes between NPC patients and healthy subjects were compared. Results: The concentration of cf-DNA in the serum of NPC patients was significantly higher than that in normal controls. The five tumor suppressor genes – RASSF1, CDKN2A, DLEC1, DAPK1 and UCHL1 – were found to be methylated in 17.5%, 22.5%, 25.0%, 51.4% and 64.9% of patients, respectively. The combination of four-gene marker – CDKN2A, DLEC1, DAPK1 and UCHL1 – had the highest sensitivity and specificity in predicting NPC. Conclusion: Screening DNA hypermethylation of tumor suppressor genes in serum was a promising approach for the diagnosis of NPC.

Sonographic Findings of Nasopharyngeal Carcinoma and Its Involvement in the Parapharyngeal Space

The purpose of this study was to determine the sonographic performance in pathologically proven cases of nasopharyngeal carcinoma and its involvement in the parapharyngeal space. The study included 58 patients being treated for suspected nasopharyngeal carcinoma detected by routine nasopharyngoscopy who underwent pathologic biopsy. Sonography was performed immediately thereafter with a convex array transducer in both the B-mode and color mode. Forty-five of the 58 patients (90 parapharyngeal spaces) in whom nasopharyngeal carcinoma was proved by both sonography and pathologic biopsy underwent preradiotherapy magnetic resonance imaging (MRI). The sonographic findings were compared to the pathologic findings. The sonographic findings of parapharyngeal space involvement were correlated with the MRI findings. The normal anatomy of the nasopharynx and parapharyngeal space, nasopharyngeal carcinoma, and its relationship with the parapharyngeal space were well shown on sonography. The sensitivity of sonography for detection of nasopharyngeal carcinoma was 97.8%, and the specificity was 41.7%. The sonographic findings of parapharyngeal space involvement had a high degree of agreement with MRI (κ = 0.757; P < .001). These promising initial data indicate that sonography may be a useful tool for diagnosing nasopharyngeal carcinoma and defining the relationship between the tumor and the parapharyngeal space.

A blood-based epigenetic test for early detection of nasopharyngeal carcinoma (NPC).

6063 Background: NPC is highly curable in early stages but 70% of NPC patients are diagnosed with advanced disease due to lack of effective screening. Genetic and epigenetic alterations involved in the pathogenesis of NPC are known. The higher order chromosomal structures reflecting aberrant transcriptional states of these genes can be measured via techniques such as chromosome conformation capture. Detection of these changes in peripheral blood may provide an accurate test for the early cancer detection. Methods: Blood samples have been collected from 84 patients with histologically confirmed NPC and 100 matched controls. Samples from 45 NPC patients and 68 controls have been analyzed. Fourteen genes known to be dysregulated in NPC were investigated. Potential higher order juxtaposition sites in the candidate genes were predicted using pattern recognition software. PCR primer sets were designed around the chosen sites to screen potential markers. Twenty-two markers showing predictability between NPC and control samples were analysed for optimal reproducibility using alternative primer sets. The optimal sets of markers were then tested amongst the complete set of samples. The dataset was processed by re-sampling using the synthetic minority oversampling technique. The overall sample was split into two groups (66% training set and 34% test set) in the classification. Results: Sixteen markers from 7 candidate genes were found to be optimal in differentiating between NPC and control samples in the first 103 samples. Using the multilayer perceptron (MLP) classification, the following results were obtained: Sensitivity 88.9%, 95% CI (79.2% - 98.6%); Specificity 72.7%, 95% CI (58.9% - 86.5%); PPV 72.7%, 95% CI (58.9% - 86.5%); NPV 88.9%, 95% CI (79.2% - 98.6%). The accuracy of the test was similar in detection of stage I and II NPC versus that of stage III or IV NPC. Conclusions: Using a PCR-based method to detect alterations in the cancer epigenome, the feasibility of developing a blood test of potential utility in early diagnosis of NPC was demonstrated. Analysis of larger numbers of patient samples and optimization of markers are ongoing. The performance characteristics of the test in the total population of 184 samples will be presented.

Abstract 3555: Identification of candidate epigenetic markers for early detection of nasopharyngeal carcinoma.

Abstract Nasopharyngeal carcinoma (NPC) is a human malignancy that is particularly prevalent in Southern China and Southeast Asia, and is strongly related to Epstein-Barr virus (EBV) infection. Since early detection of NPC will greatly improve the overall survival, screening becomes important for early NPC diagnosis. In this study, the methylation sensitive-high resolution melting (MS-HRM) method was established for detection of promoter DNA methylation using a panel of five tumor suppressor genes (RASSF1A, WIF1, DAPK1, RARβ2, p16), which were top candidates selected from 13 genes that had been tested in seven NPC cell lines and one immortalized NP cell line and which, showed highly different methylation status between cancer and immortalized cells. Biopsy samples from 52 NPC patients showed methylation for individual genes ranging from 30% to 96%. The combination of WIF1-RASSF1A, WIF1-DAPK1, and WIF1-RARβ2 all reached detection rates of 98% or above in biopsy samples, validating the frequent methylation status of these genes in NPC patients. WIF1, an important secreted Wnt inhibitor, is the most frequently methylated gene in our panel, as reported in many other cancers. In addition, the WIF1-RASSF1A combination showed the highest detection rate, reflecting that both early genetic events in chromosome 3p and the Wnt pathway play important roles in NPC development. To develop non-invasive methods for NPC high-risk screening, early diagnosis and prognosis, more NP brushings and cell-free plasma samples from NPC patients and healthy individuals have been tested for their methylation status of these candidate markers. Preliminary data indicate that the sensitivity and specificity reached more than 90% in certain gene combinations with NP brushings from patients of early NPC stage. The detection rate of methylation in cell-free plasma from NPC patients is even higher than the traditional EBV DNA test, which together show great potential for non-invasive high-risk screening for early NPC and early detection of local recurrence after therapy. In conclusion, a MS-HRM panel of multiple methylation markers is proposed as a complementary test for NPC early detection in combination with more established EBV-based markers. Citation Format: Xuesong Yang, Ying Ying Szeto, Yue Cheng, Dora Lai-wan Kwong, Maria Li Lung. Identification of candidate epigenetic markers for early detection of nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3555. doi:10.1158/1538-7445.AM2013-3555

Early detection of nasopharyngeal carcinoma by plasma Epstein‐Barr virus DNA analysis in a surveillance program

Nasopharyngeal carcinoma (NPC) is prevalent in Southeast Asia. Over the last decade, plasma Epstein-Barr virus (EBV) DNA has been developed as a tumor marker for NPC. In this study, the authors investigated whether plasma EBV DNA analysis is useful for NPC surveillance. In total, 1318 volunteers ages 40 to 60 years were prospectively recruited. Plasma EBV DNA and serology for viral capsid antigen immunoglobulin A (IgA) were measured. Participants who had detectable plasma EBV DNA or positive IgA serology underwent nasal endoscopic examination and a follow-up plasma EBV DNA analysis in approximately 2 weeks. All participants were followed for 2 years to record the development of NPC. Three individuals with NPC were identified at enrolment. All of them were positive for EBV DNA and remained positive in follow-up analysis. Only 1 of those patients was positive for EBV serology. In 1 patient who had NPC with a small tumor confined to the mucosa, the tumor was not detectable on endoscopic examination. Because of a 2-fold increase in plasma EBV DNA on the follow-up analysis, that patient underwent magnetic resonance imaging, which revealed the tumor. Among the participants who did not have NPC but had initially positive plasma EBV DNA results, approximately 66% had negative EBV DNA results after a median of 2 weeks. Plasma EBV DNA analysis proved useful for detecting early NPC in individuals without a clinical suspicion of NPC. Repeating the test in those who had initially positive results differentiated those with NPC from those who had false-positive results. Cancer 2013. © 2013 American Cancer Society.

Developing Genetic Epidemiological Models to Predict Risk for Nasopharyngeal Carcinoma in High-Risk Population of China

To date, the only established model for assessing risk for nasopharyngeal carcinoma (NPC) relies on the sero-status of the Epstein-Barr virus (EBV). By contrast, the risk assessment models proposed here include environmental risk factors, family history of NPC, and information on genetic variants. The models were developed using epidemiological and genetic data from a large case-control study, which included 1,387 subjects with NPC and 1,459 controls of Cantonese origin. The predictive accuracy of the models were then assessed by calculating the area under the receiver-operating characteristic curves (AUC). To compare the discriminatory improvement of models with and without genetic information, we estimated the net reclassification improvement (NRI) and integrated discrimination index (IDI). Well-established environmental risk factors for NPC include consumption of salted fish and preserved vegetables and cigarette smoking (in pack years). The environmental model alone shows modest discriminatory ability (AUC = 0.68; 95% CI: 0.66, 0.70), which is only slightly increased by the addition of data on family history of NPC (AUC = 0.70; 95% CI: 0.68, 0.72). With the addition of data on genetic variants, however, our model’s discriminatory ability rises to 0.74 (95% CI: 0.72, 0.76). The improvements in NRI and IDI also suggest the potential usefulness of considering genetic variants when screening for NPC in endemic areas. If these findings are confirmed in larger cohort and population-based case-control studies, use of the new models to analyse data from NPC-endemic areas could well lead to earlier detection of NPC.

Comparison of narrow-band imaging and conventional nasopharyngoscopy for the screening of unaffected members of families with nasopharyngeal carcinoma

Familial aggregation of nasopharyngeal carcinoma (NPC) has been widely reported. The excess risk is about 4-8-fold among first-degree relatives of NPC patients compared with those without a family history of the disease. We used nasopharyngoscopy and a narrow-band image system (NBI) to screen NPC high-risk patients and identify a good tool for the early detection of NPC in these high-risk groups. We recruited all available, affected blood relations of the patients. When NPC patients were more distant relatives, such as cousins, we recruited their shared second-degree relatives, such as unaffected aunts and uncles, to genetically connect the NPC cases. We performed transnasal endoscopy, first in white-light mode, then under the NBI system. There were two NBI patterns in NPC: microvascular proliferation and engorged blood vessels. The NBI pattern in normal nasopharyngeal mucosa was a regular cobblestone pattern. A prospective study included 211 asymptomatic members from 154 NPC families. We found four cases of NPC, all with a tumor stage of T1. In one patient (1/4), MRI revealed a 2-cm-diameter neck lymphadenopathy (N1). The correlation between conventional nasopharyngoscopy and NBI was very high (κ = 0.798, P = 0.000). In conclusions, NBI is not superior to conventional nasopharyngoscopy for the early detection of NPC in unaffected members of families with NPC history. The long-term follow-up is necessary in high-risk NPC patients. Further studies will be needed to determine which screening tool-conventional nasopharyngoscopy, NBI, or EB virus titer-is most effective.

Use of narrow-band imaging in detection of nasopharyngeal carcinoma

To compare narrow-band images of nasopharyngeal carcinoma with those of normal adenoidal tissue. Patients with a nasopharyngeal mass were evaluated using both conventional white light and narrow-band light. Biopsies were performed and Epstein-Barr viral serology was tested for all patients. Thirty consecutive patients were recruited. Twenty-one patients had normal adenoidal tissue and seven had nasopharyngeal carcinoma. One patient with papillary adenocarcinoma was excluded. The features of narrow-band imaging in normal adenoidal tissue were: (1) a regularly arranged follicular pattern, and (2) each 'follicle' comprising a pale centre with surrounding dark periphery. The features of narrow-band imaging in nasopharyngeal carcinoma were: (1) absence of surface patterns (n = 7), and/or (2) 'reverse', haphazard follicular pattern comprising a dark brown centre and pale periphery (n = 3). Narrow-band imaging of the surface of adenoidal tissue and nasopharyngeal carcinoma appears to identify distinct, characteristic features as described. Narrow-band imaging may be a useful adjunct in differentiating normal adenoidal tissue from malignancy. Further studies are needed to evaluate its diagnostic accuracy.

Two Epstein-Barr Virus-Related Serologic Antibody Tests in Nasopharyngeal Carcinoma Screening: Results From the Initial Phase of a Cluster Randomized Controlled Trial in Southern China

A nasopharyngeal carcinoma (NPC) mass screening trial using a combination of immunoglobulin A antibodies to Epstein-Barr virus capsid antigen and nuclear antigen-1 by enzyme-linked immunosorbent assay in addition to indirect mirror examination in the nasopharynx and/or lymphatic palpation (IMLP) was conducted in southern China. Cantonese aged 30-59 years residing in 2 cities randomly selected by cluster sampling, Sihui and Zhongshan, were invited to participate in this screening from May 2008 through May 2010. Participants were offered fiberoptic endoscopy examination and/or pathologic biopsy if their serologic tests reached our predefined level of high risk or if results from the physical examination indicated possible cancer (i.e., were IMLP positive). A total of 28,688 individuals were voluntarily screened in the initial round. The overall NPC detection rate was 0.14% (41/28,688) with an early diagnosis rate of 68.3% (28/41) during the first year of follow-up. Thirty-eight of 41 cases (92.7%) were detected among the high-risk group, and 7 of 41 cases (17.1%) were detected among the IMLP-positive group. The 2 Epstein-Barr virus serologic tests by enzyme-linked immunosorbent assay could be a feasible alternative for NPC screening in endemic areas. Further follow-up is needed to examine whether screening has an effect on decreasing mortality from NPC in these areas.

The Diagnostic Value of Narrow-Band Imaging for the Detection of Nasopharyngeal Carcinoma

Objectives: To evaluate the diagnostic value of narrow-band imaging (NBI) for the detection of nasopharyngeal carcinoma (NPC). Study Design: Prospective study. Setting: Tertiary medical center. Subjects and Methods: Between December 2009 and June 2010, a total of 1,854 patients were examined by means of an electronic nasopharyngolaryngoscope equipped with conventional white light (WL) and an NBI system. The sensitivity, specificity and positive/negative predictive values for detecting NPC were calculated and compared. Results: Of these patients, 62 cases (3.34%) were pathologically confirmed as NPC. The sensitivity, specificity, positive predictive value and negative predictive value for detecting NPC significantly increased from 90.3, 75.4, 11.3 and 99.6% with WL up to 100, 99.2, 81.6 and 100% with NBI, respectively. Conclusion: Our findings suggested that NBI endoscopy might serve as an ideal tool in the detection of NPC.

221P - Methylation Marker has a Complementary Value to Epstein-Barr Virus-Based Tests in Early Detection of Nasopharyngeal Carcinoma

ABSTRACT Undifferentiated nasopharyngeal carcinoma (NPC) is strongly linked to Epstein-Barr virus (EBV) infection. The presence of aberrant antibodies against EBV and high viral DNA load can be predictive for the disease and have been proposed for screening tools. Beside the EBV-based markers, analysis of aberrant methylation in gene promoter of tumor suppressor gene (TSG) may have potential value in identifying NPC at early stage. This study determined methylation status of multiple TSGs and evaluated whether it may improve NPC early detection using EBV-based assays. Association between EBV-encoded latent membrane protein 1 (LMP1) and methylation status was also tested. Nasopharyngeal brushings and sera were obtained from 53 NPC cases, 22 high risk individuals and 25 healthy EBV carriers. In all sera the detection of antibody against EBV was performed using EBV-IgA ELISA. EBV DNA load was measured by a quantitative light-cycler PCR on nasopharyngeal brushing samples. For methylation analysis, DNA was modified using bisulfite treatment and amplified by methylation-specific PCR (MSP) or quantitative MSP to target region within promoter of eleven TSGs. The expression of LMP1 was tested using immunohistochemistry. NPC samples demonstrated frequent methylation for individual TSGs (DAPK1 79%, CDH13 77%, DLC1 77%, RASSF1A 76%, CADM1 70%, p16 66%, WIF1 61%, CHFR 59%, RIZ1 57% and RASSF2A 29%). High risk individuals showed high frequency of four methylated genes, low methylation of two TSGs and undetectable methylation of another four TSGs. Healthy subjects had similar pattern as high risk individuals. Because not all of methylated genes allowed good discrimination between NPC and healthy individuals, a panel of markers (RASSF1A, p16, WIF1, CHFR and RIZ1) was determined and combined analysis revealed a detection rate of 98%. Quantitative analysis of MAL gene, TSG that has never been tested in NPC, showed promising result for NPC marker. The expression of LMP1 was observed to associate with methylated RASSF1A. In conclusion, methylation analysis has a complementary value to EBV-based assays for NPC risk assessment. The association of EBV oncogene to methylation status underlined the role of EBV in epigenetic event during oncogenesis. Disclosure All authors have declared no conflicts of interest.

Abstract 3998: The role of serum amyloid A1 (SAA1) in nasopharyngeal carcinoma (NPC)

Abstract Background and aims: Nasopharyngeal carcinoma (NPC) is a cancer which occurs in high frequency in Southern China. The identification of diagnostic and prognostic markers will be highly beneficial for early stage NPC detection. Previous microcell-mediated chromosome transfer (MMCT) studies showed that the transfer of an intact human chromosome 11 suppresses the in vivo tumor growth of a NPC cell line (HONE1) in nude mouse tumorigenicity assays. We aim to identify candidate genes which are associated with tumor suppression in NPC. Methods: Differential expression analysis of 19K genes was performed in oligonucleotide microarray hybridization for the chromosome 11 microcell hybrids (MCHs)/ tumor segregants (TSs) pairs to hunt for some other unknown NPC genes. The gene expression of the candidate gene was examined in the NPC cell lines and tumor tissues. Functional analysis of restoration of the candidate gene expression was studied. Results: Using oligonucleotide microarray analysis, Serum Amyloid A 1 (SAA1), mapping close to 11p15.1, was identified as showing consistent down-regulated expression in the TSs, as compared to their parental tumor-suppressing MCHs. Gene expression and protein analyses show that SAA1 was not expressed in the NPC HONE1 recipient cells, tumor segregants, and other NPC cell lines; SAA1 was exclusively expressed in the non-tumorigenic MCHs. The mechanism of SAA1 gene inactivation in these NPC cell lines was attributed to hypermethylation. The clinical relevance of SAA1 in NPC was examined by RT-PCR; 54.8% (23/42) of NPC specimens showed either down-regulation or loss of SAA1 gene expression. After transfection of SAA1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. Conclusions: These findings suggest that SAA1 is a candidate tumor suppressor gene in NPC. Acknowledgments Grant support: Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China grant HKU772309 (H.L. Lung) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3998. doi:1538-7445.AM2012-3998

Short-Term Effect of Different Teaching Methods on Nasopharyngeal Carcinoma for General Practitioners in Jakarta, Indonesia

In Indonesia, Nasopharyngeal Carcinoma (NPC) is the most frequent cancer of the head and neck region. At first presentation in the hospital most patients already have advanced NPC. Our previous study showed that general practitioners (GPs) working in Yogyakarta, Indonesia lack the knowledge necessary for early detection of NPC. By providing training on early symptoms of NPC we hope that the diagnosis and referral will occur at an earlier stage. Here we assess the current NPC knowledge levels of GPs in Jakarta, evaluate improvement after training, compare the effectiveness of two training formats, and estimate the loss of recall over a two week period.MethodsTwo Indonesian GPs visited 31 Primary Health Care Centres (PHCCs) and provided a lecture on NPC. The alternative format consisted of a symposium at the Universitas Indonesia, Jakarta, presented by local head and neck surgeons, with all GPs in the region being invited. To evaluate the effect of both formats a questionnaire was conducted before and after.ResultsThe lecture in the PHCCs was attended by 130 GPs. Sixty-six GPs attended the training in the university hospital and 40 GPs attended both. Pre training the NPC knowledge level was poor with an average of 1.6 symptoms being correctly identified out of a potential maximum of 12, this was increased to 4.9 post training (p<0.0001). GPs attending the PHCC course recorded a greater increase in correct symptoms than those attending the symposium (3.8 vs. 2.8; p = 0.01). After a two week period the knowledge levels had declined slightly from 5.5 correctly identified symptoms to 4.2 (p = 0.25).ConclusionThese results confirm our findings regarding GPs insufficient knowledge of NPC. Lectures in the PHCC and a symposium have both been proven to be effective training tools in the education of GPs.