Sofia Mubarika Haryana

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.

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.

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

BackgroundUndifferentiated 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.MethodsNasopharyngeal 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.ResultsNPC 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%.ConclusionMultiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.

Promiscuous CTL Recognition of Viral Epitopes on Multiple Human Leukocyte Antigens: Biological Validation of the Proposed HLA A24 Supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301+ and A*2402+ individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

Selection Pressure-Driven Evolution of the Epstein-Barr Virus-Encoded Oncogene LMP1 in Virus Isolates from Southeast Asia

ABSTRACT 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.

Dried-Blood Sampling for Epstein-Barr Virus Immunoglobulin G (IgG) and IgA Serology in Nasopharyngeal Carcinoma Screening

ABSTRACT 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 (r2) 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°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.

Two-Step Epstein-Barr Virus Immunoglobulin A Enzyme-Linked Immunosorbent Assay System for Serological Screening and Confirmation of Nasopharyngeal Carcinoma

ABSTRACT 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.

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.

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. J. Med. Virol. 83:665–678, 2011.

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.