Bambang Hariwiyanto

Single-Assay Combination of Epstein-Barr Virus (EBV) EBNA1- and Viral Capsid Antigen-p18-Derived Synthetic Peptides for Measuring Anti-EBV Immunoglobulin G (IgG) and IgA Antibody Levels in Sera from Nasopharyngeal Carcinoma Patients: Options for Field Screening

ABSTRACT Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.

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.

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°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.

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

ABSTRACT 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 (Spearmans 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.

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.

Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome

Poor prognosis in nasopharyngeal carcinoma patients may result from resistance to the apoptosis-inducing effect of radio- and/or chemotherapy. Apoptosis depends on proper activation of caspase 3, resulting in cleavage of key proteins like PARP-1. To investigate whether disruption of the apoptosis pathway results in therapy-resistant tumour cells, we investigated whether absence of caspase 3 activation in tumour biopsies of nasopharyngeal carcinoma patients is related to poor clinical outcome. Moreover, we investigated whether absence of caspase 3 activation is related to loss of procaspase 3 expression or expression of the apoptosis regulators p53, bcl-2 and XIAP. We studied 36 Indonesian nasopharyngeal carcinoma patients without evidence of distant metastases who were treated with curative intent by radiotherapy only. Activation of caspase 3 and expression of the different markers were determined using specific antibodies. Levels of caspase 3 activation were determined by quantifying positively staining tumour cells. Nasopharyngeal carcinoma-derived C15 and C17 tumour cells were used as control. Absence of caspase 3 activation was strongly related to a poor clinical response to radiotherapy and to a higher T and N stage, resulting in a particularly poor clinical outcome with regard to progression-free (P<0.0001) and overall survival time (P<0.0001). Absence of caspase 3 activation was significantly correlated to loss of expression of procaspase 3 (P=0.04). In nasopharyngeal carcinoma patients treated with curative intent, absence of active caspase 3-positive neoplastic cells predicts rapid fatal outcome, and is associated with poor response to radiotherapy and high T and N stage at time of presentation.

Photodynamic therapy as salvage therapy for patients with nasopharyngeal carcinoma experiencing local failures following definitive radiotherapy

BACKGROUND Treating local failures of nasopharyngeal carcinoma (NPC) is a challenge. This study evaluates photodynamic therapy (PDT) in the treatment of residual and recurrent NPC. METHOD In this phase II study, patients with local recurrent or residual NPC after curative intent (chemo-) radiation could be included. Exclusion criterion was a tumour depth more than 10mm. Foscan® 0.15mg/kg was administered intravenously. After 96h, the illumination was performed under local anaesthesia with a nasopharyngeal light applicator. Tumour response was measured 10 weeks after illumination by endoscopy, biopsy and CT-scan. Kaplan-Meier method was used for survival analysis. RESULTS Twenty-one patients were included. Fourteen patients were treated for residual disease (67%), and two for recurrent (10%). For five patients this distinction could not be made, due to uncertainty about complete response after initial treatment. The median follow-up time was 32 months. Twenty patients (95%) had a complete response 10 weeks post-treatment. Two patients had recurrent local disease at 5 and 7 months post-PDT. They received another course of PDT, one with success. The 2-year local control rate was 75%, progression free survival was 49% and overall survival was 65%. Nine patients (43%) had no evidence of disease and were in a good clinical condition (ECOG Performance Scale 0) at the end of the study period. No serious adverse events were observed. CONCLUSION This study showed that PDT is effective in treating local failures of NPC with a depth of less than 10mm. The treatment was easy to perform under local anaesthesia. Especially in regions were other modalities like radiation and surgery are limited PDT can be a good alternative treatment.

Remarkable response to photodynamic therapy in residual T4N0M0 nasopharyngeal carcinoma: A case report

Local treatment of residual or recurrent nasopharyngeal carcinoma (NPC) is a challenge. Photodynamic therapy (PDT) is an established treatment modality for incurable head and neck carcinoma. Several studies reported induction of an immune response after PDT. We present a patient with residual T4N0M0 NPC who was treated with PDT for residual disease after initial treatment with neo-adjuvant chemotherapy and radiotherapy. Five years after PDT, the tumor did not progress and the patient is still in good condition. We discuss this remarkable long-term response to PDT and speculate on possible mechanisms.

Seroreactivity against Epstein-Barr virus (EBV) among first-degree relatives of sporadic EBV-associated nasopharyngeal carcinoma in Indonesia

Epstein–Barr virus (EBV) infection and family history are significant risk factors associated with undifferentiated nasopharyngeal carcinoma. The presence of aberrant immunoglobulin A (IgA) antibodies against specific EBV antigens in healthy individuals can be predictive of the disease. Very limited reports explored the EBV IgA antibody presence within families of sporadic cases of nasopharyngeal carcinoma. This study aimed to determine whether EBV IgA was observed more frequently among family members of sporadic cases of nasopharyngeal carcinoma compared to community controls and evaluated the non‐viral factors as determinants of antibody level. First‐degree relatives of nasopharyngeal carcinoma patients (n = 520) and case‐matched community controls (n = 86) were recruited. Sera from all individuals were tested in standardized peptide‐based EBV IgA ELISA. Data on demographic variables and other exogenous factors were collected using a questionnaire through face‐to‐face interviews. A similar frequency of EBV IgA (cut‐off value/CoV 0.354) was observed in the first‐degree relatives of cases and in community controls (41.2% vs. 39.5%, P = 0.770). However, with a higher antibody level (OD450 = 1.000; about three times standard CoV), the relatives showed significantly higher frequency (36.9% vs. 14.7%, P = 0.011). When adjusted for all exogenous factors, the strongest factors associated with seropositivity are being a father (odds ratio/OR = 4.36; 95% confidence interval/CI = 1.56–12.21) or a sibling (OR = 1.89; 95% CI = 1.06–3.38) of a case of nasopharyngeal carcinoma. The higher level of EBV IgA seroreactivity in first‐degree relatives of sporadic cases of nasopharyngeal carcinoma compared to the general population supports the use of EBV IgA ELISA for screening among family members. J. Med. Virol. 84:768–776, 2012.

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.