Paul Vey Hong Lim

A genome-wide association study identifies ITGA9 conferring risk of nasopharyngeal carcinoma

To identify a gene(s) susceptible to nasopharyngeal carcinoma (NPC), we carried out a genome-wide association study (GWAS) through genotyping of more than 500 000 tag single-nucleotide polymorphisms (SNPs), using an initial sample set of 111 unrelated NPC patients and 260 controls of a Malaysian Chinese population. We further evaluated the top 200 SNPs showing the smallest P-values, using a replication sample set that consisted of 168 cases and 252 controls. The combined analysis of the two sets of samples found an SNP in intron 3 of the ITGA9 (integrin-α 9) gene, rs2212020, to be strongly associated with NPC (P=8.27 × 10−7, odds ratio (OR)=2.24, 95% confidence intervals (CI)=1.59–3.15). The gene is located at 3p21 which is commonly deleted in NPC cells. We subsequently genotyped additional 19 tag SNPs within a 40-kb linkage disequilibrium (LD) block surrounding this landmark SNP. Among them, SNP rs189897 showed the strongest association with a P-value of 6.85 × 10−8 (OR=3.18, 95% CI=1.94–5.21), suggesting that a genetic variation(s) in ITGA9 may influence susceptibility to NPC in the Malaysian Chinese population.

The ATM tumour suppressor gene is down-regulated in EBV-associated nasopharyngeal carcinoma

A micro‐array analysis using biopsies from patients with EBV‐positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer‐free controls revealed down‐regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q‐PCR confirmed down‐regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down‐regulated only in the EBV‐positive line, C666.1, and in none of five EBV‐negative lines. In vitro infection of EBV‐negative NPC cell lines with a recombinant EBV was followed by the down‐regulation of ATM mRNA and protein, and only EBV‐positive cells showed a defective DNA damage response following γ‐irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease. Copyright

HLA-A SNPs and amino acid variants are associated with nasopharyngeal carcinoma in Malaysian Chinese

Nasopharyngeal carcinoma (NPC) arises from the mucosal epithelium of the nasopharynx and is constantly associated with Epstein–Barr virus type 1 (EBV‐1) infection. We carried out a genome‐wide association study (GWAS) of 575,247 autosomal SNPs in 184 NPC patients and 236 healthy controls of Malaysian Chinese ethnicity. Potential association signals were replicated in a separate cohort of 260 NPC patients and 245 healthy controls. We confirmed the association of HLA‐A to NPC with the strongest signal detected in rs3869062 (p = 1.73 × 10−9). HLA‐A fine mapping revealed associations in the amino acid variants as well as its corresponding SNPs in the antigen peptide binding groove (pHLA‐A‐aa‐site‐99 = 3.79 × 10−8, prs1136697 = 3.79 × 10−8) and T‐cell receptor binding site (pHLA‐A‐aa‐site‐145 = 1.41 × 10−4, prs1059520 = 1.41 × 10−4) of the HLA‐A. We also detected strong association signals in the 5′‐UTR region with predicted active promoter states (prs41545520 = 7.91 × 10−8). SNP rs41545520 is a potential binding site for repressor ATF3, with increased binding affinity for rs41545520‐G correlated with reduced HLA‐A expression. Multivariate logistic regression diminished the effects of HLA‐A amino acid variants and SNPs, indicating a correlation with the effects of HLA‐A*11:01, and to a lesser extent HLA‐A*02:07. We report the strong genetic influence of HLA‐A on NPC susceptibility in the Malaysian Chinese.

Identification of a functional variant in SPLUNC1 associated with nasopharyngeal carcinoma susceptibility among Malaysian Chinese

Nasopharyngeal carcinoma (NPC) is a multifactorial and polygenic disease with high incidence in Asian countries. Epstein–Barr virus infection, environmental and genetic factors are believed to be involved in the tumorigenesis of NPC. The association of single nucleotide polymorphisms (SNPs) in LPLUNC1 and SPLUNC1 genes with NPC was investigated by performing a two‐stage case control association study in a Malaysian Chinese population. The initial screening consisted of 81 NPC patients and 147 healthy controls while the replication study consisted of 366 NPC patients and 340 healthy controls. The combined analysis showed that a SNP (rs2752903) of SPLUNC1 was significantly associated with the risk of NPC (combined P = 0.00032, odds ratio = 1.62, 95% confidence interval = 1.25–2.11). In the subsequent dense fine mapping of SPLUNC1 locus, 36 SNPs in strong linkage disequilibrium with rs2752903 (r2 ≥ 0.85) were associated with NPC susceptibility. Screening of these variants by electrophoretic mobility shift and luciferase reporter assays showed that rs1407019 located in intron 3 (r2 = 0.994 with rs2752903) caused allelic difference in the binding of specificity protein 1 (Sp1) transcription factor and affected luciferase activity. This SNP may consequently alter the expression of SPLUNC1 in the epithelial cells. In summary, our study suggested that rs1407019 in intronic enhancer of SPLUNC1 is associated with NPC susceptibility in which its A allele confers an increased risk of NPC in the Malaysian Chinese population.

Exome Sequencing Identifies Potentially Druggable Mutations in Nasopharyngeal Carcinoma

In this study, we first performed whole exome sequencing of DNA from 10 untreated and clinically annotated fresh frozen nasopharyngeal carcinoma (NPC) biopsies and matched bloods to identify somatically mutated genes that may be amenable to targeted therapeutic strategies. We identified a total of 323 mutations which were either non-synonymous (n = 238) or synonymous (n = 85). Furthermore, our analysis revealed genes in key cancer pathways (DNA repair, cell cycle regulation, apoptosis, immune response, lipid signaling) were mutated, of which those in the lipid-signaling pathway were the most enriched. We next extended our analysis on a prioritized sub-set of 37 mutated genes plus top 5 mutated cancer genes listed in COSMIC using a custom designed HaloPlex target enrichment panel with an additional 88 NPC samples. Our analysis identified 160 additional non-synonymous mutations in 37/42 genes in 66/88 samples. Of these, 99/160 mutations within potentially druggable pathways were further selected for validation. Sanger sequencing revealed that 77/99 variants were true positives, giving an accuracy of 78%. Taken together, our study indicated that ~72% (n = 71/98) of NPC samples harbored mutations in one of the four cancer pathways (EGFR-PI3K-Akt-mTOR, NOTCH, NF-κB, DNA repair) which may be potentially useful as predictive biomarkers of response to matched targeted therapies.

Oncogenic effects of WNT5A in Epstein-Barr virus‑associated nasopharyngeal carcinoma.

The molecular events that drive the progression of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) are still to be elucidated. Here, we report for the first time the pathogenic significance of an NPC-associated gene, wingless-type MMTV integration site family, member 5A (WNT5A) and the contribution of EBV to its expression. WNT5A is a representative Wnt protein that activates non-canonical Wnt signalling. With regard to its role in carcinogenesis, there is conflicting evidence as to whether WNT5A has a tumour-promoting or tumour-suppressive role. We show that WNT5A is upregulated in primary NPC tissue samples. We also demonstrate that WNT5A expression was dramatically increased in NPC cell lines expressing the EBV-encoded LMP2A gene, suggesting that this EBV-encoded latent gene is responsible for upregulating WNT5A in NPC. In addition, in vitro WNT5A overexpression promotes the proliferation, migration and invasion of NPC cells. Our results not only reveal pro-tumorigenic effects of WNT5A in NPC but also suggest that WNT5A could be an important therapeutic target in patients with EBV-associated disease.

Integrated pathway analysis of nasopharyngeal carcinoma implicates the axonemal dynein complex in the Malaysian cohort

Nasopharyngeal carcinoma (NPC) is an epithelial squamous cell carcinoma on the mucosal lining of the nasopharynx. The etiology of NPC remains elusive despite many reported studies. Most studies employ a single platform approach, neglecting the cumulative influence of both the genome and transcriptome toward NPC development. We aim to employ an integrated pathway approach to identify dysregulated pathways linked to NPC. Our approach combines imputation NPC GWAS data from a Malaysian cohort as well as published expression data GSE12452 from both NPC and non‐NPC nasopharynx tissues. Pathway association for GWAS data was performed using MAGENTA while for expression data, GSA‐SNP was used with gene p values derived from differential expression values from GEO2R. Our study identified NPC association in the gene ontology (GO) axonemal dynein complex pathway (pGWAS-GSEA = 1.98 × 10−2; pExpr‐GSEA = 1.27 × 10−24; pBonf‐Combined = 4.15 × 10−21). This association was replicated in a separate cohort using gene expression data from NPC and non‐NPC nasopharynx tissues (pAmpliSeq‐GSEA = 6.56 × 10−4). Loss of function in the axonemal dynein complex causes impaired cilia function, leading to poor mucociliary clearance and subsequently upper or lower respiratory tract infection, the former of which includes the nasopharynx. Our approach illustrates the potential use of integrated pathway analysis in detecting gene sets involved in the development of NPC in the Malaysian cohort.

In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients

ABSTRACT Peptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9–11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients’ T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.

Abstract 4263: Aberrant phospholipid signalling in EBV-associated nasopharyngeal carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy that is particularly prevalent in Asia. The main challenges in the management of NPC include late presentation and severe side effects of chemo-radiotherapies due to the close proximity of tumours to vital structures. Two bioactive lipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), which signal through G protein-coupled receptors, regulate many biological processes associated with tumour development and progression. Therefore, LPA and S1P have emerged as potential targets in cancer therapies, but the roles of these lipid molecules in the pathogenesis of NPC have not been investigated. Microarray analyses revealed differential expression of several key regulators of LPA and S1P metabolism in NPC cells compared to non-malignant nasopharyngeal epithelium. Imunohistochemical studies showed that autotaxin and SPHK1, the key enzymes involved in the production of LPA and S1P, respectively, were expressed by the tumour cells in primary NPC tissues. QPCR analyses demonstrated that an EBV-positive NPC cell line, C666-1, expressed higher levels of autotaxin and SPHK1 as compared to six EBV-negative NPC cell lines, suggesting that EBV infection might contribute to the de-regulation of phospholipid signalling in NPC. The microarray data showed that LPA receptor 5 (LPA5) was significantly down-regulated in primary NPC tissues and, similarly, LPA5 protein expression in tumour cells was weak or absent in 17/22 primary NPC cases. Consistent with these findings, LPA5 mRNA levels were significantly lower in a panel of six EBV-infected cell lines than in non-infected controls. By treating NPC cell lines with exogenous LPA, we showed that LPA enhanced the migration of NPC cells in vitro. Given that NPC is characterised by prominent lymphocyte infiltration, the effect of LPA on cells within the NPC tumour microenvironment was examined by treating several clones of EBV-specific cytotoxic T lymphocytes (CTLs) in vitro. LPA inhibited the secretion of IFN-γ from peptide stimulated CTLs in a dose-dependent manner, indicating that LPA could inhibit CTL function. Collectively, our results demonstrate that LPA and S1P signalling are de-regulated in NPC. Aberrant LPA production may promote a more aggressive phenotype in NPC and EBV infection might contribute to the aberrant LPA signalling by down-regulating LPA5. Targeting LPA and/or its signalling pathways might represent a novel therapeutic strategy with which to improve the efficiency of EBV-based CTL therapy in NPC patients. Citation Format: Lee-Fah Yap, Christopher W. Dawson, Rajadurai Pathmanathan, Hui-Min Lee, Paul Lim, Tracey Haigh, John R. Arrand, Graham S. Taylor, Ian C. Paterson, Paul G. Murray. Aberrant phospholipid signalling in EBV-associated nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4263. doi:10.1158/1538-7445.AM2013-4263

Snoring and Obstructive Sleep Apnoea Syndrome: Aspects of Diagnosis and Evaluation

Snoring is prevalent and closely related to the less common obstructive sleep apnoea syndrome. The effects of sleep apnoea on concentration leading to accidents are well documented. Untreated obstructive sleep apnoea is associated with an increased risk of morbidity and mortality. Proper assessment of a patient presenting with snoring is important to distinguish between ‘simple snoring’ and obstructive sleep apnoea. It is also important in determining the treatment most suited for alleviating the patient’s symptoms. There are many issues regarding the assessment and investigations of these two conditions. The roles of clinical history, physical examination, specially designed questionnaires including the Epworth Sleepiness Scale, investigative procedures including Müller’s manoeuvre, sleep nasoendoscopy and polysomnography, and available techniques for diagnosis and evaluation of snoring and obstructive sleep apnoea are discussed.