Susanna Hilda Hutajulu

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.

Therapeutic implications of Epstein–Barr virus infection for the treatment of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is highly endemic in certain regions including the People’s Republic of China and Southeast Asia. Its etiology is unique and multifactorial, involving genetic background, epigenetic, and environment factors, including Epstein–Barr virus (EBV) infection. The presence of EBV in all tumor cells, aberrant pattern of antibodies against EBV antigens in patient sera, and elevated viral DNA in patient circulation as well as nasopharyngeal site underline the role of EBV during NPC development. In NPC tumors, EBV expresses latency type II, where three EBV-encoded proteins, Epstein–Barr nuclear antigen 1, latent membrane protein 1 and 2 (LMP1, 2), are expressed along with BamH1-A rightward reading frame 1, Epstein–Barr virus-encoded small nuclear RNAs, and BamH1-A rightward transcripts. Among all encoded proteins, LMP1 plays a central role in the propagation of NPC. Standard treatment of NPC consists of radiotherapy with or without chemotherapy for early stage, concurrent chemoradiotherapy in locally advanced tumors, and palliative systemic chemotherapy in metastatic disease. However, this standard care has limitations, allowing recurrences and disease progression in a certain proportion of cases. Although the pathophysiological link and molecular process of EBV-induced oncogenesis are not fully understood, therapeutic approaches targeting the virus may increase the cure rate and add clinical benefit. The promising results of early phase clinical trials on EBV-specific immunotherapy, epigenetic therapy, and treatment with viral lytic induction offer new options for treating NPC.

Purified hexameric Epstein-Barr virus encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients.

ABSTRACT WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n = 155) compared to healthy EBV-positive (n = 56) and EBV-negative (n = 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P = 0.015) than in regional Indonesian controls or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.

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.

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

BackgroundBamHI-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.ResultsMost 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.ConclusionThe 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.

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.

Determinants of cancer screening awareness and participation among Indonesian women

BackgroundCancer screening awareness and participation may be lower in low- and middle-income countries that lack established national screening programmes compared with those that do. We evaluated potential determinants of awareness about and participation in breast and cervical cancer screening, and breast self-examination (BSE) in women using survey data from Indonesia.MethodsFrom the fifth Indonesian Family Life Survey (2014–2015), a total of 5397 women aged 40 and older without any history of cancer who responded to questionnaires concerning Pap smears, mammography, and BSE were included. Multilevel modelling was used to assess potential determinants in relation to awareness about Pap smears and mammography, and participation in Pap smears and BSE practice. Multivariable analyses were performed to identify independent predictors of cancer screening.ResultsOf the 5397 respondents, 1058 (20%) women were aware of Pap smears, of which 297 had never had the procedure. Only 251 (5%) participants were aware of mammography. A total of 605 (12%) of women reported they performed BSE. Higher education and household expenditure were consistently associated with higher odds of awareness about Pap smears and mammography (e.g. odds ratio [OR] of being aware of Pap smear and mammography: 7.82 (95% CI: 6.30–9.70) and 7.70 (6.19–9.58), respectively, for high school graduates compared to women with less educational attainment in the multivariable models), and participation in Pap smears and BSE. We also identified enabling factors linked with greater cancer screening awareness and participation, including health insurance, shorter distance to health services, and social participation.ConclusionThere are socioeconomic disparities in cancer screening awareness and participation among Indonesian women. Our findings may help inform targeted health promotion and screening for cancer in the presence of limited resources.

Seroprevalence of IgA anti Epstein-Barr virus is high among family members of nasopharyngeal cancer patients and individuals presenting with chronic complaints in head and neck area

Epstein-Barr (EBV) infection and presence of a nasopharyngeal cancer (NPC) case in the family increases the risk of developing NPC. Aberrant anti-EBV immunoglobulin A (IgA) antibodies (EBV-IgA) may be present in the sera of non-cancer individuals and predict NPC. Limited studies report the presence of EBV-IgA antibodies within non-cancer individuals in Indonesia where the disease is prevalent. This study aimed at exploring whether EBV-IgA was found more frequently among first degree relatives of NPC patients and individuals presenting with chronic symptoms in the head and neck area compared to healthy controls. A total of 967 non-cancer subjects were recruited, including 509 family members of NPC cases, 196 individuals having chronic complaints in the head and neck region, and 262 healthy donors of the local blood bank. Sera were analyzed using a standardized peptide-based EBV-IgA ELISA. Overall, 61.6% of all individuals had anti-EBV IgA reactivity equal to or below cut off value (CoV). Seroreactivity above CoV was significantly higher in females (38.7%) compared to males (28.7%) (p = 0.001). Older individuals had more seroreactivity above CoV (42.5%) than the younger ones (26.4%) (p< 0.001). Seroprevalence was significantly higher in family members of NPC patients (41.7%), compared to 32.7% of individuals with chronic head and neck problems (p = 0.028) and 16.4% healthy blood donors (p< 0.001). As conclusion, this study showed a significant higher seroprevalence in healthy family members of NPC cases and subjects presenting with chronic symptoms in the head and neck area compared to healthy individuals from the general community. This finding indicates that both groups have elevated risk of developing NPC and may serve as targets for a regional NPC screening program.

HES-1 and CEBPA mRNA in Chronic and Late Phase (accelerated and blastcrisis) of Chronic Myeloid Leukemia (CML) Patients

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder of hematopoietic , characterized by Philadelphia chromosome containing BCR-ABL fusion gene. The gene encodes protein with constitutive tyrosine kinase activity result in myeloid proliferation and leads to form an early phase of CML called chronic phase. Unsuccessful treatment will lead to progression of the disease into late phase (accelerated and blast crisis). The mechanisms involving disease progression are still poorly understood. It is assumed that additional genetic event involves in differentiation blocking of myeloid progenitor cells, such as Hes-1 overexpression and CEBPA down regulation. However, study on the expression of these genes in CML patient’s samples is still limited. This study aims to measure Hes-1 and CEBPA mRNA in chronic and late phase of CML patients. The peripheral blood mRNA level of Hes-1 was measured in CML patient’s sample with BCR-ABL positive both in chronic phase (n=61) and late phase (n=17) using qRT-PCR with GAPDH as internal control. Hes-1 mRNA was statistically higher (p value=0.0) in the chronic phase (mean ± SD=97.8 ± 236.6) compared to those in late phase (mean ± SD=8.5 ± 30.7). In addition, even though CEBPA expression in chronic and late phase were not statistically different (p value=0.1), those in chronic phase (mean ± SD=5.2 ± 16.0) were generally higher compared to those in late phase (mean ± SD=1.7 ± 2.4). Hes-1 expression upregulated in 70.5% of chronic phase patients and in 17.6% of late phase patients, whereas CEBPA expression down regulated in 42.6% of chronic phase patients and in 47.1% in late phase patients. High standard deviation, particularly in mRNA Hes-1 gene expression measurement of the chronic phase, indicated the presence of individual variations in the sample that might be influenced by other genetic factors. This study found that Hes-1 mRNA is significantly higher in peripheral blood of chronic phase than blast crisis CML, whereas CEBPA mRNA is not different.

New Genetic Variation in BCR gene of Major B3a2 Breakpoint BCR-ABL Fusion Gene in Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia

Background: Polymorphic bases in several exons of the BCR gene have been found in several studies of the BCR-ABL fusion gene. Most of the polymorphisms do not have any implications for the primary structure of the BCR-ABL protein. Nucleotide changes are often located in the area close to the fusion region, and therefore may influence primer annealing. Our previous work failed to amplify 15 of 200 samples from BCR-ABL positive chronic myelogenous leukemia (CML) patients using multiplex PCR, the standard method to detect BCR-ABL transcripts used in our institution. The failure was considered due to problems in primer annealing caused by sequence variations. Sequence analysis of BCR-ABL fusion gene breakpoint types in CML patients has never been hitherto performed in Indonesia. Therefore, the aim of this study was to perform sequence analysis of several samples that did not show amplification using the standard method. Methods: Fifteen samples were qualitatively amplified by two-step PCR using inner primers in the 2nd PCR to determine the breakpoint type of the BCR-ABL fusion gene. The 2nd PCR products were used as templates to perform sequence analysis, and the results were compared to those in genbank. Result: Seven and 5 of 15 samples were confirmed as major b3a2 and major b2a2, respectively. One sample featured a combination of b3a2 and b2a2, and 2 samples a combination of b3a2 and b2a2 with an additional fragment at 500bp. Sequence analysis showed 3 sequence variations in the major b3a2 breakpoint. One had been reported earlier (c.3296T>C) but the others (c.3245C>T and c.3359T>C) were novel. Fragments at 500bp were confirmed as b3a2 and similar sequence b3a2 in genbank. Conclusion: This study found two new genetic variations in the BCR gene in BCR-ABL fusion cases.