Proteomic networks associated with tumor-educated macrophage polarization and cytotoxicity potentiated by heat-killed tuberculosis.

Local administration of attenuated mycobacterium has been used as a cancer treatment adjuvant to re-boost patient immune responses with variable clinical outcomes. We aimed to clarify the impact of attenuated heat-killed tuberculosis (HKTB) on tumor-associated macrophages which play critical roles in shaping immunological regulation in the tumor microenvironment. Upon HKTB stimulation, both primary macrophages derived from the peripheral blood of healthy subjects and from lung cancer patients as well as THP1-derived classically activated macrophages (Ms) and tumor-educated macrophages (TEMs) were polarized into the proinflammatory phenotype, as characterized by increased expression cluster of differentiation 86. A quantitative proteomic analysis revealed that stimulated TEMs were unable to activate the toll-like receptor 2, signal transducer and activator of transcription 1, or nuclear factor-κB signaling. Instead, they showed distinct intercellular adhesion molecule 1 signaling, impaired cell adhesion, and mitochondrial dysfunction. These molecular mechanisms might contribute to lower cytotoxicity of HKTB-stimulated TEMs against A549 cells via the release of distinct inflammatory cytokines compared to HKTB-stimulated Ms. Our study provides an unbiased and systematic interpretation of cellular and molecular alterations of HKTB-reeducated macrophages which should help illuminate potential strategies of HKTB-stimulated macrophage-based combination therapy for cancer treatment.

The Potential of hsa-mir-106b-5p as Liquid Biomarker in Prostate Cancer Patients in Indonesia.

PURPOSE: This study aims to explore the potential of hsa-mir-106b-5p as a new liquid biomarker for prostate cancer sufferers in Indonesia. METHODS: Analysis of hsa-mir-106b-5p expression of two tissue samples from BPH patients and two PCa patients used NanoString nCounter Expression Assay then validated by qRT-PCR using 10 patient urine samples for prostate cancer and BPH. Furthermore, analysis of the role of hsa-mir-106b-5p in prostate cancer was carried out bioinformatically. RESULTS: The results of this study indicated that the expression of hsa-mir-106b-5p in prostate cancer tissue was 1.23 times higher than that of BPH and urine of Indonesian patients (1.72 times). Moreover, this miRNA was upregulated in prostate cancer cells compared to normal cells 1.37 times. The hsa-mir-106b-5p appeared to be involved in the development of prostate cancer through the binding of genes involved in endoplasmic reticulum stress pathways and tumor suppressor genes. CONCLUSION: hsa-mir-106b-5p could modulate prostate cancer by interfering with the endoplasmic reticulum stress repair pathways and decreasing the expression of tumor suppressor genes involved in many biological processes. These updates our understanding of the role of hsa-mir-106b-5p in cancer and its potential as a candidate of a biomarker for clinical diagnosis of prostate cancer.

The Effects of Combination of Mimic miR-155-5p and Antagonist miR-324-5p Encapsulated Chitosan in Ovarian Cancer SKOV3.

OBJECTIVE: Ovarian cancer is a malignant tumor that attacks reproductive organs of women. MicroRNA is known to have an involvement in the prognosis of ovarian cancer. One of them is miR-155-5p which is down regulated and miR-324-5p which is up regulated. Chitosan is used as microRNA delivery system. The aims of this study is to find out the effects of combination microRNA encapsulated chitosan in cell line SKOV3. METHODS: Cell line SKOV3 obtained from Stem Cell and Cancer Institute (Kalbe). Mimic miR-155-5p and Antagonist miR-324-5p formulated with chitosan. Total RNA was extracted from nine samples (three as control and six as treatment), and prepared for cDNA synthesis. Expression of RNA and mRNA target was measured using q-PCR Biorad CFX96 C.100 and Gen Ex 7 software. Statistics analysis was measured using SPSS 16.0. RESULTS: The administration of combination microRNA encapsulated with chitosan affect the expression of miR-155-5p and miR-324-5p endogen (p <0.05). The expression of mRNA target HIF1α and GLI1 was down regulated after treatment. The correlation between expression of microRNA and mRNA target was strongly (p <0.05). CONCLUSION: This study successfully presented effects of combination of mimic miR-155-5p and antagonist miR-324-5p encapsulated chitosan which be considered as a potential therapy targets for ovarium cancer.

Mutational heterogeneity between different regional tumour grades of clear cell renal cell carcinoma.

Only a limited number of studies have explored the possible associations between tumour grade and mutated genes in clear cell renal cell carcinoma (ccRCC), and we set out to investigate this further using a multiple sampling and next generation sequencing (NGS) approach in a series of ccRCCs. Multiple regions were sampled from formalin-fixated paraffin-embedded ccRCC tumour blocks from seven patients. In 27 samples from six patients, we performed targeted NGS using a custom 42-gene panel based on the most frequently mutated genes in ccRCC reported in public databases. In four samples from the seventh patient, we performed whole exome sequencing (WES) and array comparative genomic hybridisation for detection of copy number variants (CNVs). Mutated genes and the tumour grades of the samples in which they had been identified were compared both within and between all individual tumours. CNVs were compared across all samples from patient 7. We identified clear genetic heterogeneity within and across tumours, but VHL mutations were seen in all patients. Looking across all samples, we identified eleven genes that were only mutated in samples with one particular tumour grade. However, these genes were never mutated in all samples with that tumour grade. Increasing chromosomal instability corresponded with increasing tumour grade, but we observed minimal association between tumour grade and total mutational load in the WES data. Our study confirms the genetic heterogeneity and tumour grade heterogeneity of ccRCC. Although a relatively small number of samples was analysed, genes were identified that could potentially be specific, though insensitive, markers of higher ccRCC tumour grades.

[Serum MiRNA as Predictive and Prognosis Biomarker in Advanced Stage Non-small Cell Lung Cancer in Indonesia].

BACKGROUND: Lung cancer is the most common cause of death in men in the world and in Indonesia where non-small cell carcinoma lung cancer (NSCLC) constitutes 85% of all lung cancer cases. The high mortality rate is due to a poor prognosis and is often diagnosed as having advanced stages. If it is known at the initial stage, the prognosis of lung cancer will be better. Prognosis can be predicted with a marker of prognostic biology, one of which is micro RNA (miRNA). This study aims to prove that serum miRNA can be predictive biological marker and prognosis in NSCLC patients in Indonesia. METHODS: This study was cohort retrospective among 52 subjects in "Dharmais" Hospital National Cancer Center. Sample was obtained from patients' serum. MiR-34, miR-148, miR-155 and miR-222 serum are measured through Real-Time PCR (qPCR). Data were analyzed and interpreted with descriptive analysis, bivariate analysis (Mann Whitney-U for two type of variables or Kruskal-Wallis for more than two type of variables. Kaplan-Meier analysis was used to know association between characteristic which are sociodemographic, performance status, clinico-pathology, and survival rate in miRNA expression. RESULTS: From this study, miRNA expression: miR-34 (46.15%), miR-148 (23.08%), miR-155 (40.38%) and miR-222 (32.69%). Performance status score was statistically significant correlation with miR-148 (P=0.049) and miR-222 (P=0.018). High miR-34 is associated with multiple M1b metastatic type (P=0.020), cancer cell type (adenocarcinoma, P=0.009) and adenocarcinoma epidermal growth factor receptor (EGFR) mutation (negative, P=0.031). There was a significant correlation between the high miR-222 as a poor prognosis in advanced stage NSCLC with M1b metastasis (Median Survival/MS: 27 d, P=0.049) and positive EGFR mutations (MS: 74 d, P=0.049) and correlation of miR-155 with adenocarcinoma (MS: 69 d, P=0.034) and positive EGFR gene mutations (MS: 58 d, P=0.023). CONCLUSIONS: High miR-34 expression in advanced stage NSCLC is the predictive factor for multiple metastatic, adenocarcinoma cell type and adenocarcinoma negative EGFR mutation. High expression of miR-155 and miR-222 are poor prognoses, especially high miR-222 found in metastasis M1b and positive EGFR mutation and miR-155 found in adenocarcinoma and positive EGFR gene mutations. Further studies regarding correlation between miRNA and survival rate are needed.

Effectiveness of a multicentre nasopharyngeal carcinoma awareness programme in Indonesia.

OBJECTIVE: To evaluate the effectiveness of a nasopharyngeal carcinoma (NPC) awareness programme on the short-term and long-term improvement of knowledge and referral of patients with NPC by primary healthcare centres (PHCCs) staff in Indonesia. DESIGN: The NPC awareness programme consisted of 12 symposia including a Train-The-Trainer component, containing lectures about early symptoms and risk factors of NPC, practical examination and the referral system for NPC suspects. Before and after training participants completed a questionnaire. The Indonesian Doctors Association accredited all activities. PARTICIPANTS: 1 representative general practitioner (GP) from each PHCC attended an NPC awareness symposium. On the basis of the Train-The-Trainer principle, GPs received training material and were obligated to train their colleagues in the PHCC. RESULTS: 703 GPs attended the symposia and trained 1349 staff members: 314 other GPs, 685 nurses and 350 midwives. After the training, respondents' average score regarding the knowledge of NPC symptoms increased from 47 points (of the 100) to 74 points (p<0.001); this increase was similar between symposium and Train-The-Trainer component (p=0.88). At 1½ years after the training, this knowledge remained significantly increased at 59 points (p<0.001). CONCLUSIONS: The initial results of this NPC awareness programme indicate that the programme effectively increases NPC knowledge in the short and long term and therefore should be continued. Effects of the improved knowledge on the stage at diagnoses of the patients with NPC will still need to be scrutinised. This awareness programme can serve as a blueprint for other cancer types in Indonesia and for other developing countries.

Primary treatment results of Nasopharyngeal Carcinoma (NPC) in Yogyakarta, Indonesia.

INTRODUCTION: Nasopharyngeal Carcinoma (NPC) is a major health problem in southern and eastern Asia. In Indonesia NPC is the most frequent cancer in the head and neck area. NPC is very sensitive to radiotherapy resulting in 3-year disease-free and overall survival of approximately 70% and 80%, respectively. Here we present routine treatment results in a prospective study on NPC in a top referral; university hospital in Indonesia. METHODS: All NPC patients presenting from September 2008 till January 2011 at the ear, nose and throat (ENT) department of the Dr. Sardjito General Hospital, Universitas Gadjah Mada, Yogyakarta, Indonesia, were possible candidates. Patients were included if the biopsy was a histological proven NPC without distant metastasis and were assessed during counselling sessions prior to treatment, as being able to complete the entire treatment. RESULTS: In total 78 patients were included for treatment analysis. The median time between diagnosis and start of radiotherapy is 120 days. Forty-eight (62%) patients eventually finished all fractions of radiotherapy. The median duration of the radiotherapy is 62 days for 66 Gy. Median overall survival is 21 months (95% CI 18-35) from day of diagnosis. CONCLUSION: The results presented here reveal that currently the treatment of NPC at an Indonesian hospital is not sufficient and cannot be compared to the treatment results in literature. Main reasons for these poor treatment results are (1) a long waiting time prior to the start of radiotherapy, (2) the extended overall duration of radiotherapy and (3) the advanced stage of disease at presentation.

Development of a non-invasive method, multiplex methylation specific PCR (MMSP), for early diagnosis of nasopharyngeal carcinoma.

Increasing evidence demonstrated that inactivation of tumor suppressor genes (TSGs) by aberrant promoter methylation is an early event during carcinogenesis. Aiming at developing early diagnostic or prognostic tools for various tumors, we took an EBV-associated tumor, nasopharyngeal carcinoma (NPC), as a model and developed a powerful assay based on "multiplex methylation specific-PCR (MMSP)". The MMSP assay was designed to detect tumor-specific methylation status of several NPC-related genes and was capable of acquiring multiplex information simultaneously through a single PCR reaction with the tiny tumor DNA derived from the direct body fluid close to the primary tumor. In this study, we collected paired nasopharyngeal (NP) swabs and NPC biopsies from 49 NPC patients and twenty noncancerous controls. A panel of markers including two EBV, and two cellular TSG markers were applied in this NPC-specific-MMSP assay. We optimized the working condition of MMSP so that it provides information equal to that from the corresponding separate PCRs. The results showed that MMSP patterns of NPC swab were largely consistent with those of corresponding biopsies and significantly distinguished themselves from those of 20 noncancerous volunteers. Among the 69 samples (49 NPCs and 20 normal controls), the sensitivity of detecting NPC from NP swabs is 98%. The specificity is as high as 100%. In conclusion, being characterized by its noninvasiveness, high reproducibility and informativeness, MMSP assay is a reliable and potential diagnostic tool for NPC. It paves the way for the development of population screening and early diagnosis approaches for various tumor types.

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population.

BACKGROUND: Undifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection. METHODS: Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied. RESULTS: NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%. CONCLUSION: Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

Interleukin-4 and interferon- γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma.

BACKGROUND: Cytokines have been suggested to participate in the pathogenesis of infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC). METHODS: Serum levels and gene expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) were assessed by immunologic and PCR assays, respectively in patients with Epstein-Barr virus (EBV)-associated IM and NPC and EBV nega-tive controls. RESULTS: The serum levels of IFN- γ were elevated, but those of IL-4 were decreased in IM and NPC patients as compared with those of the control group (p < 0.05). CONCLUSIONS: These results suggest that serum levels of IFN-γ may be predominant over those of IL-4 during the course of IM and NPC.

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls.

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.

Concentration of serum HER-2/neu as a prognostic factor in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC).

AIM: to determine whether serum HER-2/neu level could be used as a prognostic factor in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC). METHODS: a prospective cohort study was done in LABC and MBC patients in dr. Sardjito Hospital Yogyakarta from April 2006 to March 2008. Serum concentration of HER-2/neu was measured by ELISA done before and after chemotherapy. HER-2/neu expression tissue examination was done by immunohistochemistry. The clinical responses on therapy, survival and progression were recorded. RESULTS: twenty seven cases were obtained. Average concentration of serum HER-2/neu was 21.02 ± 7.1 ng/ml. The level of serum HER-2/neu in LABC was lower than MBC (17.21 ng/ml vs 28.64 ng/ml; p=0.32). Average concentration of serum HER-2/neu in partial responders was 13.20 ng/ml (95% Cl 0.142 - 26.25), stable responders was 19.42 ng/ml (95% Cl -0.255 - 39.09) and 29.35 ng/ml(95% Cl 1.95 - 56.74) in progressors (p=0.468). Patients with better clinical response had a lower average HER-2/neu serum level (16.12 ng/ml vs 29.35 ng/ml; p=0.247). HER-2/neu over expression was found in 40.7% of the tissues, 44% of LABC and 33.3% of MBC tissues (p=0.692). Negative HER-2/neu tissue protein expression had better clinical response (75% vs 45.5% p=0.224), and longer survival (p=0.08). CONCLUSION: neither the expression of HER-2/neu in the tissue nor the level of serum HER-2/neu can be used as clinical prognosis factor on advanced stage breast cancer in our study population.

Relationship of angiotensin converting enzyme gene polymorphism and hypertension in Yogyakarta, Indonesia.

AIM: To evaluate the association between ACE gene polymorphism I/D and hypertension in Yogyakarta population. METHODS: This study is a cross-sectional. Sample was taken by random sampling method from hypertensive, prehypertensve and normotensive subjects; from that were obtained 125 subjects, 97 subjects and 108 subjects, consecutively. ACE gene polymorphism I/D was examined by PCR. Genotype was classified as II, ID, or DD based on positive or negative insertion/delation allele. RESULTS: This study shows significant differences of three groups (ages, body mass index (BMI), and family history of hypertension) and total cholesterol level in blood, which tends to have greater value in the hypertension group. Frequency of genotype II, ID, DD are 85 (68%), 39 (31.2%), 1 (0.8%) in hypertension, 66 (61.1%), 38 (35.2%), 4 (3.7%) in normo-tension and 56 (57.7%), 37 (38.1%), 4 (4.1%) in pre-hypertension subject, consecutively. Chi-square analysis shows statistically significant association between ID+DD vs. II genotype and hypertension. Multiple logistic regression analysis shows four variables that significantly influence to hypertension, namely ages, family history of hypertension, BMI, and ACE gene polymorphism. CONCLUSION: ACE ID+DD genotype has significant relationship with hypertension in Melati population, Sleman, Yogyakarta, Indonesia.

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma.

BACKGROUND: BamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing. RESULTS: Most NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence. CONCLUSION: The sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence.

Combination of Epstein-Barr virus scaffold (BdRF1/VCA-p40) and small capsid protein (BFRF3/VCA-p18) into a single molecule for improved serodiagnosis of acute and malignant EBV-driven disease.

Current single Epstein-Barr virus (EBV) markers fail to reach 100% sensitivity for serodiagnosis of acute and malignant diseases associated with EBV infection. Previous study had identified immunodominant epitopes of VCA-p40 and VCA-p18, and indicated that these two VCA antigens may have diagnostic value for EBV-related diseases. A recombinant protein of the full-length BdRF1 fused to the immunodominant domain of BFRF3 as 6-his tagged protein in Escherichia coli was developed. The recombinant protein was extracted in 8M urea solution and purified by metal-affinity chromatography yielding a 55 kDa product (VCA-p40+18). VCA-p40+18 blot-strips examined for IgM reactivity in infectious mononucleosis samples yielded 100% sensitivity and specificity, with improved reactivity compared with IgM/VCA-p18-ELISAs. A recent study described a synthetic peptide-based IgA/[EBNA1+VCA-p18]-ELISA (IgA/EBV-ELISA), with a sensitivity of 90% for diagnosing nasopharyngeal carcinoma. Immunoblot analysis of biopsy-confirmed nasopharyngeal carcinoma cases with low or negative IgA/EBV-ELISA showed 100% IgG reactivity to VCA-p40 and VCA-p18 proteins. Evaluation of VCA-p40+18 as an additional marker for screening and diagnosis of nasopharyngeal carcinoma was carried out. The data showed positive IgA/VCA-p40+18 reactivity by ELISA for 63.6% (14 of 22) nasopharyngeal carcinoma samples that were missed by peptide-based IgA/EBV-ELISA, suggested VCA-p40+18 as an improved marker for nasopharyngeal carcinoma serodiagnosis. The VCA-p40+18 may be combined with an EBNA1 synthetic peptide as an antigen mixture in one or separate IgA ELISA for improved nasopharyngeal carcinoma serodiagnosis.

Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carcinoma.

Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.

Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma.

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.

Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.

Selection pressure-driven evolution of the Epstein-Barr virus-encoded oncogene LMP1 in virus isolates from Southeast Asia.

The geographically constrained distribution of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in southeast Asian populations suggests that both viral and host genetics may influence disease risk. Although susceptibility loci have been mapped within the human genome, the role of viral genetics in the focal distribution of NPC remains an enigma. Here we report a molecular phylogenetic analysis of an NPC-associated viral oncogene, LMP1, in a large panel of EBV isolates from southeast Asia and from Papua New Guinea, Africa, and Australia, regions of the world where NPC is and is not endemic, respectively. This analysis revealed that LMP1 sequences show a distinct geographic structure, indicating that the southeast Asian isolates have evolved as a lineage distinct from those of Papua New Guinea, African, and Australian isolates. Furthermore, a likelihood ratio test revealed that the C termini of the LMP1 sequences of the southeast Asian lineage are under significant positive selection pressure, particularly at some sites within the C-terminal activator regions. We also present evidence that although the N terminus and transmembrane region of LMP1 have undergone recombination, the C-terminal region of the gene has evolved without any history of recombination. Based on these observations, we speculate that selection pressure may be driving the LMP1 sequences in virus isolates from southeast Asia towards a more malignant phenotype, thereby influencing the endemic distribution of NPC in this region.

Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects.

Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.