Noninvasive diagnosis of nasopharyngeal carcinoma: nasopharyngeal brushings reveal high Epstein-Barr virus DNA load and carcinoma-specific viral BARF1 mRNA

Nasopharyngeal carcinoma (NPC) is the most prevalent ENT-tumour in Indonesia. We investigated the primary diagnostic value of Epstein-Barr virus (EBV) DNA load and mRNA detection in noninvasive nasopharyngeal (NP) brushings, obtained prospectively from consecutive Indonesian ENT-patients with suspected NPC (N=106) and controls. A subsequent routine NP biopsy was taken for pathological examination and EBER-RISH, yielding 85 confirmed NPC and 21 non-NPC tumour patients. EBV DNA and human DNA load were quantified by real-time PCR. NP brushings from NPC patients contained extremely high EBV DNA loads compared to the 88 non-NPC controls (p<0.0001). Using mean EBV DNA load in controls plus 3 SD as cut-off value, specificity, sensitivity, positive and negative predictive values were 98, 90, 97 and 91%, respectively. Epstein-Barr nuclear antigen 1 (EBNA1) and the carcinoma-specific BARF1 mRNA were detected by nucleic acid sequence based amplification and found in 86 and 74% of NP brushings, confirming NPC tumour cell presence. EBV RNA positivity was even higher in fresh samples stored at -80 degrees C until RNA expression analyses (88% for both EBNA1 and BARF1). EBV RNA-negative NP brushings from proven NPC cases had the lowest EBV DNA loads, indicating erroneous sampling. No EBV mRNA was detected in NP brushings from healthy donors and non-NPC patients. In conclusion, EBV DNA load measurement combined with detection of BARF1 mRNA in simple NP brushings allows noninvasive NPC diagnosis. It reflects carcinoma-specific EBV involvement at the anatomical site of tumour development and reduces the need for invasive biopsies. This procedure may be useful for confirmatory diagnosis in large serological NPC screening programs and has potential as prognostic tool.     

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.     

Changes of lactate dehydrogenase in corneal edema after cataract surgery treated with trans-corneal oxygenation therapy

AIM: To investigate the changes in levels of the lactate dehydrogenase (LDH) enzyme in corneal edema after cataract surgery with trans-corneal oxygenation therapy. METHODS: This pre-post design study design conducted on 15 patients with corneal edema after cataract surgery and receiving trans-corneal oxygenation therapy. Tear sample (using Schirmer paper, from the inferior fornix of the conjunctiva) was carried out prior to trans-corneal oxygenation therapy, on the day 2 (D2) and day 5 (D5) postoperatively before and after trans-corneal oxygenation therapy. Visual acuity [VA (LogMAR)], corneal endothelial density, central corneal thickness (CCT), and coefficient of variation corneal endothelial (CoV) were recorded. The value of LDH was measured using ELISA. The difference in mean LDH value before and after trans-corneal oxygenation therapy, between two groups were analyzed using Wilcoxon signed rank test. RESULTS: There was a decrease in LDH tear concentration at D2 (pre vs post: 1127.54±497.09 vs 696.91±489.49; P=0.002) and D5 (pre vs post: 1064.17±677.77 vs 780.28±428.95; P=0.027) after trans-corneal oxygenation therapy as well as decrease in LDH concentration on the D2 compared to D5 (P=0.041). The mean CCT was decreased significantly after the administration of trans-corneal oxygenation (pre vs post: 632.10±25.66 vs 563.90±51.54; P=0.005). The mean VA and CoV increased significantly after the administration of trans-corneal oxygenation (P=0.001 and P=0.028, respectively). However, there was no difference in mean of corneal endothelial density (P=0.814). CONCLUSION: Trans-corneal oxygenation therapy is associated with significant decrease of tears LDH levels in post cataract surgery with corneal edema. It is accompanied by clinical improvement such as significant reduction of CCT.     

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.Nasopharyngeal carcinoma (NPC) is a common cancer in China and Southeast Asia and closely associated with Epstein-Barr Virus (EBV) (26). In Indonesia, especially in the southern part of central Java, undifferentiated carcinoma (WHO type III) is the most common head and neck cancer and among the five most prevalent cancers overall. Due to unspecific symptoms and the hidden localization of the primary tumor at the early stage, more than 80% of the patients come to the hospital at a late stage (III or IV), when they already have metastasis in the cervical lymph node. Whereas late-stage disease has a poor prognosis and requires combined chemo-radiotherapy, early-stage NPC may reach complete remission by radiotherapy only (17). Therefore, screening for early-stage NPC among the population is important and clinically relevant. For developing countries, such an approach should be economical, employing standardization methods suited for mass screening.Patients with NPC have high-level broad-spectrum anti-EBV antibodies, especially immunoglobulin A (IgA), compared to regional healthy carriers and patients with other head and neck diseases (13, 14). Our group recently demonstrated that the molecular diversity underlying anti-EBV IgG and IgA responses in NPC patients was different, requiring multiple EBV antigens for complete serological coverage (7). Prior studies in China and Taiwan have shown the feasibility of using IgA serology for population screening (2, 15, 27). However, in these studies laborious and poorly standardized cell-based serological techniques were used. Nevertheless, these studies revealed the appearance of serological abnormalities, i.e., positive EBV IgA responses 2 to 3 years prior to onset of NPC (2, 15), which clearly demonstrated the opportunity of using EBV serology for early-stage detection of NPC. This particularly applies for screening in high-risk groups, such as family members of NPC patients and patients with suspicious head and neck symptoms (18, 21).For NPC serodiagnosis, cell-based indirect immunofluorescent assay (IFA) methods are still widely considered the gold standard. IFA involves the separate analysis of antibody responses to viral capsid antigen (VCA), early antigen (EA), and nuclear antigens (EBNA), each comprising multiple proteins and requiring different cell lines for specific analysis (10, 12, 13). However, this method shows considerable variation among laboratories and is time-consuming, subjective, and not suitable for large-scale automatic handling. Enzyme-linked immunosorbent assay (ELISA) techniques are increasingly used and have shown a better sensitivity and specificity compared to IFA and are suitable for large-scale application (4, 10, 11, 16, 20, 21).Recently, we developed an EBV IgA ELISA based on a combination of VCA p18- and EBNA1-derived synthetic peptides which is routinely used as an NPC diagnostic test in our local hospital (Sardjito Hospital, Yogyakarta, Indonesia). This EBV IgA ELISA combines the separate features of IgA VCA and IgA EBNA1, each of which has its value in NPC diagnosis. The combination of these markers in a single assay provided sensitivity and specificity of 85.4% and 90.1%, respectively (8). The presence of NPC-related serological abnormalities can be confirmed by immunoblotting to reveal the spectrum of antibody responses, which has diagnostic value by itself (5, 7, 16, 25). The combined EBV IgA ELISA and immunoblot assay showed increased sensitivity and specificity and positive predictive value (PPV) and negative predictive value (NPV) of more than 95% (7, 8). Because immunoblot studies revealed a diagnostic value of multiple EBV proteins, in particular certain EBV-EA markers, we recently developed a separate IgA EA ELISA using native EA proteins (22). In addition to their role in primary diagnosis, anti-EBV IgA responses, in particular the IgA EA response, also have a distinct role for posttreatment follow-up monitoring, as declining responses correlate with a good prognosis and increasing responses are related to persistence of relapsing tumor (6, 16, 24). The availability of two distinct and biochemically well-defined EBV IgA ELISA systems addressing responses to different EBV antigens for NPC-specific serology may add to further standardization. In this study we evaluate the combination of these two tests for primary diagnosis of NPC in a high-incidence population in Indonesia.     

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.     

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.     

Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening

Dried-blood (DB) samples on filter paper are considered clinical specimens for diagnostic use because of the ease of collection, storage, and transport. We recently developed a synthetic-peptide-based immunoglobulin A (IgA) (EBNA1 plus viral capsid antigen [VCA]-p18) enzyme-linked immunosorbent assay (ELISA) for nasopharyngeal carcinoma (NPC) screening. Here, we evaluate the use of two filter papers for DB sampling, i.e., Schleicher & Schuell (S&S) no. 903 and Whatman no. 3; the DB samples were either taken directly from a finger prick or spotted from a Vacutainer blood collector. The elution of DB samples on filter paper was optimized and tested for IgG and IgA reactivity by ELISA (EBNA1 plus VCA-p18) and compared to simultaneously collected plasma samples. The results showed that both types of filter paper can be used for sample collection in NPC diagnosis by using either finger prick or blood spot sampling. Both DB sampling methods produced comparable ELISA (EBNA1 plus VCA-p18) results for IgG and IgA reactivity in 1:100-diluted plasma samples. DB samples of whole blood or finger prick blood show correlation coefficients (r(2)) of 0.825 to 0.954 for IgA on S&S no. 903 filter paper, 0.9133 to 0.946 for IgA on Whatman no. 3 filter paper, 0.807 to 0.886 for IgG on S&S no. 903 filter paper, and 0.819 to 0.934 for IgG on Whatman no. 3 filter paper. Using plasma IgA as a reference, DB sampling showed sensitivities and specificities of 75.0 to 96.0% and 93.5 to 100%, respectively. DB samples could be stored at 37 degrees C for 1 to 4 weeks on S&S no. 903 filter paper and 1 to 6 weeks on Whatman no. 3 filter paper without a significant loss of reactivity, with provision of transport options for tropical conditions. IgA proved to be more stable than IgG. Whatman no. 3 filter paper is a more economical yet diagnostically comparable alternative to S&S no. 903 filter paper. Finger prick DB sampling is proposed for NPC diagnosis, particularly for remote hospitals and field screening studies.     

Tobacco consumption and genetic susceptibility to nasopharyngeal carcinoma (NPC) in Thailand

Background

The incidence of nasopharyngeal carcinoma (NPC) varies substantially worldwide, with an endemic pocket in Southeast Asia.

Method

We assessed lifestyle and genetic factors in relation to NPC risk among 681 NPC cases and 1,078 controls from Thailand. Evaluated lifestyle factors included traditionally preserved foods, tobacco smoking, betel quid chewing, and alcohol consumption. Genetic factors included six variants implicated in a previous a genome-wide association study (GWAS) of NPC and three variants residing near the CHRNA3 and TERT genes that were linked to lung cancer risk in Asian populations. Odds ratios (OR) and 95?% confidence intervals (95?% CI) were estimated using unconditional logistic regression.

Results

Frequent consumption of fermented vegetables was associated with increased NPC risk (OR of consumption ≥weekly vs. ≤rare 1.78, 95?% CI 1.24–2.55, p _trend?=?0.005), as was tobacco smoking (p _trend?<?0.001), former and current smokers displaying OR of 1.57 (95?% CI 1.10–2.30) and 2.00 (95?% CI 1.48–2.71) compared to never smokers, respectively. Four out of six genetic variants implicated in the recent NPC GWAS were associated with NPC risk (p _trend?≤?0.03), as well as two variants (rs402710 and rs2736098) on the TERT locus at 5p15.33 (p?=?0.004 and p?=?0.04, respectively).

Conclusions

These results strengthen our previous observation that tobacco smoking is an important risk factor of NPC in this population. Four out of six genetic variants identified in a recent NPC GWAS were confirmed, and the association noted with variants on 5p15.33 suggests that this locus is involved in NPC susceptibility, representing a novel finding in NPC epidemiology.     

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.     

Nitrate in drinking water and risk of colorectal cancer in Yogyakarta,Indonesia

Nitrate concentration in well water in Yogyakarta, Indonesia, and its surroundings tended to increase rapidly from time to time, and it may be associated with an elevated risk for several types of cancer. The purpose of this study was to examine the association between nitrate in drinking water and colorectal cancer (CRC) risk occurrence. A case-control study was conducted in Yogyakarta Special Province. Pathologically confirmed 75 CRC patients and 75 controls were consulted and their individual well water was sampled and examined for nitrate concentrations. Logistic regression analysis was conducted to establish the association between nitrate and CRC risk development. There was a significant correlation between nitrate in drinking water and CRC occurrence, and this value was relatively stable after being adjusted for protein intake, smoking history, age, and family history of cancer. These findings demonstrated that the risk of CRC development was fourfold among those with >10 years of nitrate exposure from well water compared with those with ≤10 years of nitrate exposure. Consequently, a significant association between nitrate in drinking water and occurrence of CRC in Yogyakarta was established.