Identification of pathogens causing invasive fungal rhinosinusitis in surgical biopsies using polymerase chain reaction.

BACKGROUND: Invasive fungal rhinosinusitis is associated with high morbidity and mortality. Rapid pathogen identification is mandatory, but fresh tissue is not always available. A polymerase chain reaction method was designed in order to detect fungi in formalin-fixed paraffin-embedded samples. This was applied to a retrospective series of tissue biopsies from Thai patients with invasive fungal rhinosinusitis. METHODS: Tissue blocks from 64 cases yielded adequate DNA. Three sequential polymerase chain reaction were performed: ZP3 (housekeeping gene) and panfungal polymerase chain reactions, and a differentiating polymerase chain reaction based on the 5.8s ribosomal RNA and internal transcribed spacer 2 regions. The polymerase chain reaction products were then sequenced. RESULTS: Polymerase chain reaction identified a fungal pathogen in 20 of 64 cases (31 per cent). Aspergillus species was the most common cause of invasive fungal rhinosinusitis (nine cases). Other causes included candida (n = 4), cladosporium (n = 4), mucor (n = 1), alternaria (n = 1) and dendryphiella (n = 1) species. CONCLUSION: Polymerase chain reaction can provide rapid identification of fungal pathogens in paraffin-embedded tissue, enabling prompt treatment of invasive fungal rhinosinusitis.

Investigation of PTEN promoter methylation in ameloblastoma.

BACKGROUND: Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor gene. Inactivation of PTEN has been reported in various types of cancers. PTEN promoter methylation possibly underlies PTEN inactivation, which results in tumorigenesis. The aim of this study was to investigate whether PTEN promoter methylation contributes to PTEN inactivation in ameloblastoma and its associated protein expression. MATERIAL AND METHODS: In total, 20 fresh-frozen ameloblastoma samples were evaluated for PTEN promoter methylation using methylation-specific polymerase chain reaction (MS-PCR). A subset of 10 paraffin-embedded ameloblastoma samples was examined for PTEN expression through immunohistochemistry. Four primary cultured ameloblastoma cells were investigated for PTEN promoter methylation and PTEN transcriptional expression via reverse transcription PCR. RESULTS: PTEN promoter methylation was detected in 65% (13/20) of the ameloblastoma samples. Of 10 ameloblastoma samples, 4 exhibited reduced PTEN expression. Of 5 samples with methylated PTEN, 3 (60%) were associated with loss of PTEN expression. However, PTEN expression was detected in 4 (80%) of 5 samples with unmethylated PTEN. In addition, 3 (75%) of 4 primary ameloblastoma cell cultures exhibited an inverse correlation between PTEN promoter methylation and PTEN transcription level. CONCLUSIONS: PTEN promoter methylation is found in a number of ameloblastomas but not significantly correlated with loss of PTEN expression. Genetic or epigenetic mechanisms other than PTEN promoter methylation may contribute to PTEN inactivation in ameloblastoma tumor cells.

TRH site-specific methylation in oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) continues to increase each year. Clinical examination and biopsy usually detect OSCC at an advanced stage that is difficult to treat, leading to poor prognosis. DNA methylation pattern is tissue specific and has emerged as a biomarker for the detection of cancers of tissue origin. Herein, we aimed to discover a novel site-specific methylation marker for OSCC. METHODS: We selected OSCC datasets analyzed using the IlluminaHumanMethylation27 BeadChip from the Gene Expression Omnibus repository of the National Center for Biotechnology Information using a bioinformatics approach. From 27,578 CG dinucleotide (CpG) sites, the CpG site with the highest difference in methylation level between healthy and cancerous cells was selected for further validation. A total of 18 mucosal tissue samples were collected from nine healthy controls and nine from OSCC subjects and subjected to microdissection for cell purification, followed by DNA extraction, bisulfite conversion, and pyrosequencing. Additionally, epithelial cells were collected from 2 cohorts including oral rinse from healthy controls, oral rinse and oral swab from OSCC subjects and oral rinse from oropharyngeal squamous cell carcinoma (SCC) were examined for their methylation status using real-time polymerase chain reaction (PCR). RESULTS: Among the 27,578 differentially methylated CpG sites, cg01009664 of the thyrotropin-releasing hormone (TRH) gene showed the greatest difference in methylation level between healthy and cancerous cells. Validation of the TRH gene using pyrosequencing revealed a methylation percentage of 7% ± 3.43% in healthy cells in contrast to 63% ± 19.81% in cancerous cells. Screening of epithelial cells using real-time PCR showed that the DNA methylation level was significantly higher in oral swab and rinse samples collected from OSCC and oropharyngeal SCC subjects than those from healthy controls (p < 0.001). In addition, when using a cutoff at 3.31 ng/µL, the TRH methylation biomarker was able to distinguish OSCC and oropharyngeal SCC subjects from healthy controls with high level of area under the curve, sensitivity and specificity. CONCLUSION: We demonstrated cg01009664 of TRH as a potential biomarker for OSCC and oropharyngeal SCC screening using oral rinse and swab techniques.

Detection of global hypermethylation in well-differentiated thyroid neoplasms by immunohistochemical (5-methylcytidine) analysis.

PURPOSE: While global hypomethylation of DNA has been found in several malignancies, studies on thyroid tumours have shown controversial results using different techniques. To help resolve this issue, we assessed methylation status using two different techniques in papillary thyroid carcinomas (PTC) and follicular adenomas (FA) and carcinomas (FTC), comparing adjacent non-neoplastic thyroid tissue. METHODS: A series of 15 FA, 18 FTC and 17 PTC were assessed by: (1) measurement of methylation levels of long interspersed nuclear elements (LINE-1) using a combined bisulfite restriction analysis polymerase chain reaction protocol and (2) immunostaining with an anti-5-methylcytidine antibody that detects methylated DNA regardless of the DNA sequence. Immunostaining was scored by image analysis. RESULTS: Methylation levels of LINE-1 in FA, FTC and PTC were not significantly different from adjacent normal tissue. There was no significant difference in methylation levels of LINE-1 between FA, FTC and PTC (p = 0.44). By immunohistochemical staining for methylation, the 5-methylcytidine score was significantly higher in tumours than in normal tissue counterparts, for FA (p < 0.001), FTC (p = 0.04) and PTC (p = 0.02). PTC showed the highest 5-methylcytidine expression amongst all tumours which was significantly different from FTC (p = 0.015), but not FA (p = 0.09). There was no correlation in methylation level between LINE-1 and 5-methylcytidine scores for each group and overall. CONCLUSIONS: Well-differentiated thyroid neoplasms (FA, FTC and PTC) were not found by two independent methods to undergo global hypomethylation as part of an oncogenic sequence from normal tissue to carcinoma. Instead, hypermethylation was detected in all types of tumours, implying that this epigenetic event may contribute to oncogenic development of thyroid neoplasms (both benign and malignant).

Southeast Asian origins of five Hill Tribe populations and correlation of genetic to linguistic relationships inferred with genome-wide SNP data.

In Thailand, the term Hill Tribe is used to describe populations whose members traditionally practice slash and burn agriculture and reside in the mountains. These tribes are thought to have migrated throughout Asia for up to 5,000 years, including migrations through Southern China and/or Southeast Asia. There have been continuous migrations southward from China into Thailand for approximately the past thousand years and the present geographic range of any given tribe straddles multiple political borders. As none of these populations have autochthonous scripts, written histories have until recently, been externally produced. Northern Asian, Tibetan, and Siberian origins of Hill Tribes have been proposed. All purport endogamy and have nonmutually intelligible languages. To test hypotheses regarding the geographic origins of these populations, relatedness and migrations among them and neighboring populations, and whether their genetic relationships correspond with their linguistic relationships, we analyzed 2,445 genome-wide SNP markers in 118 individuals from five Thai Hill Tribe populations (Akha, Hmong, Karen, Lahu, and Lisu), 90 individuals from majority Thai populations, and 826 individuals from Asian and Oceanean HGDP and HapMap populations using a Bayesian clustering method. Considering these results within the context of results ofrecent large-scale studies of Asian geographic genetic variation allows us to infer a shared Southeast Asian origin of these five Hill Tribe populations as well ancestry components that distinguish among them seen in successive levels of clustering. In addition, the inferred level of shared ancestry among the Hill Tribes corresponds well to relationships among their languages.

Association of P53 codon 72 polymorphism and ameloblastoma.

OBJECTIVE: To test the hypothesis that P53 codon 72 polymorphism was associated with an increased risk of developing ameloblastoma in the Thai population. MATERIALS AND METHODS: Seventy-eight ameloblastomas and 94 healthy controls were genotyped for the P53 codon 72 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequencies of the Arg/Arg, Arg/Pro and Pro/Pro genotypes were 28.72%, 50.00% and 21.28%, respectively, in the controls; and 44.87%, 51.28% and 3.85%, respectively, in ameloblastomas. Therefore, P53 Arg is an ameloblastoma-susceptible allele [OR (95% CI) = 2.06 (1.28-3.31), P = 0.002]. Sex-adjusted OR (95% CI) is 2.08 (1.29-3.34), P = 0.002; and adjusted OR by clinical type (95% CI) is 2.04 (1.34-3.13), P < 10(-3). Therefore, the increased risk associated with P53 Arg may not be influenced by either the sex of patients or clinical characteristics of the tumours. Moreover, when compared with homozygous P53 Pro, people who carried the Arg allele had a remarkably high risk of developing ameloblastoma [adjusted OR (95% CI) = 7.26 (2.34-23.41), P < 10(-3)]. CONCLUSION: The Arg allele of P53 gene codon 72 may increase susceptibility, and P53 may be important in the aetiology of ameloblastoma.

LINE-1 methylation difference between ameloblastoma and keratocystic odontogenic tumor.

OBJECTIVE: Global hypomethylation is a common epigenetic event in cancer. Keratocystic odontogenic tumor (KCOT) and ameloblastoma are different tumors but posses the same tissue in origin. Here, we investigated long interspersed nuclear element-1 (LINE-1 or L1) methylation status between ameloblastoma and KCOT. MATERIALS AND METHODS: We studied the methylation levels of the long interspersed nucleotide element-1 (LINE-1) in ameloblastoma and KCOT. After collecting ameloblastoma cells and epithelium lining cells of KCOT by laser capture microdissection from paraffin embedded tissue, combined bisulfite restriction analysis of LINE-1 (COBRALINE-1) was performed to measure LINE-1 methylation levels. RESULTS: The LINE-1 methylation level in KCOT (53.16 +/- 12.03%) was higher than that in ameloblastoma (36.90 +/- 16.52%), with a statistical significance of P = 0.001. The ranges of LINE-1 methylation of both lesions were not associated with either age or sex. CONCLUSION: We found LINE-1 hypomethylation levels between ameloblastoma and KCOT are different. Therefore, global methylations between these tumors are processed differently.

LINE-1 hypomethylation level as a potential prognostic factor for epithelial ovarian cancer.

A genome-wide hypomethylation is a common and crucial event in cancer. This study was to evaluate common epithelial ovarian cancer (EOC) if long interspersed element-1 (LINE-1) repetitive sequences methylation levels are progressively decreased during multistage carcinogenesis and there are the correlation between LINE-1 methylation levels and clinicopathologic characteristics. A total of 59 pairs of microdissected EOC tissues obtained from patients with EOC were examined for the methylation levels of LINE-1 repetitive sequences by a COBRALINE-1 (combined bisulfite restriction analysis of LINE-1) PCR protocol. The methylation levels were correlated with clinicopathologic parameters to determine the potential role of global hypomethylation as a prognostic marker for EOC. The LINE-1 methylation levels of 59 EOCs, 34.87 +/- 7.39%, were lower than in representative normal ovarian tissues (46.89 +/- 8.31%; 95% CI: 9.42-14.62; P < 0.001, paired-two-tailed t test). A decrease in the LINE-1 level of methylation was correlated with histological subtypes, higher FIGO and advanced tumor grade. Patients with greater hypomethylation (i.e., a methylation level

Role of human papillomavirus DNA testing in management of women with atypical squamous cells of undetermined significance.

To find the sensitivity, specificity, and positive and negative predictive values of the high-risk group human papillomavirus (HPV) DNA testing as a triage tool to detect high-grade squamous intraepithelial lesions (HSILs, ie, cervical intraepithelial neoplasia [CIN] 2 or worse) in women with a cytologic smear showing atypical squamous cells of undetermined significance (ASC-US). All new cases with cytologic smears showing ASC-US that presented in King Chulalongkorn Memorial Hospital from January 2003 to November 2003, excluding known cases of HSILs and pregnancies, were enrolled. Cervical cell samplings were done by cervical cytobrush technique and tested for high-risk group HPV with the Hybrid Capture 2 (HC2) test. All participants were examined under a colposcope. Then cervicographs were taken before colposcopic-directed cervical biopsies were done. Of the 90 ASC-US cases enrolled, the pathologic results were normal in 30.0%, squamous metaplasia in 16.7%, CIN 1 in 37.8%, CIN 2 in 1.1%, CIN 3 in 11.1%, and microinvasive cervical carcinoma in 3.3%. The prevalence of HSILs and the prevalence of high-risk HPV detection were 15.6% and 38.9%, respectively. Using pathologic results from cervical biopsy as the gold standard, the HC2 has the sensitivity, specificity, and positive and negative predictive values of 85.7%, 69.7%, 34.3%, and 96.4%, respectively, to detect HSILs. High-risk group HPV detection can be used as an additional triage test to detect HSILs in women having ASC-US with high sensitivity and negative predictive value.

High frequency of mutation of epidermal growth factor receptor in lung adenocarcinoma in Thailand.

Recent reports have suggested influences of racial difference on the frequency of mutation of EGFR in lung cancer. We therefore sought to characterize the frequency and pattern of mutation of EGFR in lung adenocarcinoma in Thai patients. Overall, EGFR catalytic domain mutations were detected in 35/61 (57.4%). We found 29/60 (48.3%) of exon 19 deletions, 5/54 (9.3%) of exon 21 point mutations, and 1/54 (1.9%) of double-mutation of both exons. The presence of these mutations was significantly associated with non-smoking habit. In summary, we report a strikingly high prevalence of mutation of EGFR in Thai lung adenocarcinoma, which may explain the high response rate to the treatment with TKI among Asian populations.

HLA-E polymorphism in patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) has been known to be associated with HLA class I region. The aim of this study was to investigate the association between HLA-E and genetic susceptibility to nasopharyngeal carcinogenesis by comparing the frequencies of HLA-E alleles in 100 Thai NPC patients and 100 healthy controls. HLA-E typing was performed by means of polymerase chain reaction-sequence-specific oligonucleotide probe method. The frequency of the HLA-E*0103 allele and HLA-E*0103, 0103 genotype, but not others, was increased in NPC patients, compared to controls. This observation suggests a possible role for HLA-E in NPC development, possibly via natural killer cell or cytotoxic lymphocyte function.

Distribution of HLA-B alleles in nasopharyngeal carcinoma patients and normal controls in Thailand.

HLA-B frequencies in 54 unrelated nasopharyngeal carcinoma (NPC) patients and 49 healthy random controls in Thailand were investigated by direct DNA sequencing. Similar to previous reports in Chinese NPC patients, HLA-B*4601 was observed at a greater frequency in patients (21/54 (40%)) compared to controls (7/49 (14%). An increase in HLA-B*51012 was also demonstrated. B*51012 was present in 6/54 (11%) NPC patients but was not observed (0%) in controls. B*44032 was associated with a decreased risk. Five out of 54 (9%) NPC patients had B*44032 compared to 14/49 (29%) in the control group.

Stage specificity of Plasmodium falciparum telomerase and its inhibition by berberine.

Telomerase activity in synchronized Plasmodium falciparum during its erythrocytic cycle was examined using the TRAP assay. Telomerase activity was detected at all stages of the parasite intraerythrocyte development, with higher activity in trophozoite and schizont stages compared with ring form. Berberine, extracted from Arcangelisia flava (L.) Merr., inhibited telomerase activity in a dose-dependent manner over a range of 30-300 microM, indicating that P. falciparum telomerase might be a potential target for future malaria chemotherapy.

Serum/plasma viral DNA: mechanisms and diagnostic applications to nasopharyngeal and cervical carcinoma.

Following reports describing circulating tumor DNA, serum/plasma viral nucleic acid has shown its potential as a new diagnostic target in cancer. In the majority of examples of viral carcinogenesis, the viral genome is consistently present in certain tumors and serves as an effective marker. This article reviews recent findings, proposes possible mechanisms, and examines the potential clinical application of serum/plasma Epstein-Barr virus (EBV) DNA in nasopharyngeal cancer (NPC) and human papillomavirus (HPV) DNA in cervical carcinoma (CC). These tumors share a DNA viral etiology and present similar histopathological findings. However, plasma EBV and HPV DNA are distinct in several aspects, including incidence, mechanism of release from tumor, and clinical application. Both circulating cell-free EBV and HPV DNA reveal the same viral type as their matched tumors, indicating both are derived from the neoplastic tissue. Plasma viral DNA incidence and copy number are high in NPC, but low in HPV-associated cancers. Whereas much EBV DNA in NPC is episomal, the resistance to DNase treatment of serum EBV DNA and evidence confirming lytic EBV replication in NPC suggest that a reasonable proportion of plasma EBV DNA is virions. On the contrary, plasma HPV genomes, as in CC, integrate into host chromosome. Plasma EBV DNA copy number, by quantitative PCR, is related to tumor mass, predicts prognosis, measures immediate response to treatment, and is useful in early detection of recurrence. Plasma HPV DNA, on the other hand, is associated with and can be considered as an early tumor marker for distant metastasis.

Fibroblast growth factor receptor 3 S249C mutation in virus associated squamous cell carcinomas.

An S249C mutation in fibroblast growth factor receptor 3 (FGFR3) gene was recently identified in patients with cervical carcinomas (CC). However, its importance in cervical tumorigenesis is still inconclusive. Apart from CC, nasopharyngeal carcinoma (NPC) is the other major virus associated squamous cell carcinoma. We sought to clarify the frequency of the FGFR3 S249C mutation in 75 primary CC in the Thai population and to determine its prevalence in 69 primary NPC by PCR and restriction enzyme digestion. None of the patients but one NPC showed the enzyme digestion pattern consistent with the mutation. This is the first report demonstrating the role of FGFR3 in the development of human NPC. This study confirms the low frequency of the FGFR3 S249C mutation in CC. Nevertheless, the discovery of the mutation, not only in CC as reported by previous studies, but in NPC based on this report, suggests that FGFR3 may play a significant role in human CC and NPC development.

Clinical and molecular characteristics of Thai patients with achondroplasia.

Achondroplasia is an autosomal dominant disorder characterized by disproportionately short stature, frontal bossing, rhizomelia, and trident hands. Most patients appear sporadically resulting from a de novo mutation associated with advanced paternal age. A glycine to arginine mutation at codon 380 (G380R) of the fibroblast growth factor receptor 3 gene (FGFR3) was found to be the most common cause of achondroplasia in various populations. We identified and clinically characterized 3 Thai patients with achondroplasia. In all of them, we also successfully identified the G380R mutation supporting the observation that this is the most common mutation in achondroplasia across different ethnic groups including Thai.

Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare tumor in most parts of the world but occurs at relatively high frequency among people of Chinese descent. The cytochrome P450 2E1 enzyme (CYP2E1) is responsible for the metabolic activation of nitrosamines, and has been shown to be a susceptibility gene for NPC development in Taiwan [RR = 2.6; 95%CI = 1.2-5.7]. Since there has been only one report of this link, it was decided to investigate the susceptibility of CYP2E1 to NPC development in other populations. Therefore, the correlation between the RsaI polymorphism of this gene and NPC was studied in-patients including Thai and Chinese in Thailand. The present study comprised 217 cases diagnosed with NPC and 297 healthy controls. RESULTS: Similar to the result found in Taiwanese, a homozygous uncut genotype demonstrated a higher relative risk both when all cases were analyzed [RR = 2.19; 95%CI = 0.62-8.68] or individual racial groups, Thai [RR = 1.51; 95%CI = 0.08-90.06] or Chinese [RR = 1.99; 95%CI = 0.39-10.87]. The ethnicity-adjusted odds ratio is 2.39 with 95%CI, 0.72-7.89. CONCLUSIONS: Though our finding was not statistically significant due to the moderate sample size of the study, similarity to the study in Taiwan with only a slight loss in precision was demonstrated. The higher RR found for the same genotype in distinct populations confirmed that CYP2E1 is one of several NPC susceptibility genes and that the RsaI minus variant is one mutation that affects phenotype.