Youngho Moon

Diagnostic value of pyrosequencing for the BRAFV600E mutation in ultrasound-guided fine-needle aspiration biopsy samples of thyroid incidentalomas

Context  Dideoxy sequencing is the most commonly used method for detecting the BRAFV600E mutation in thyroid cancer and melanoma. However, this gold standard method often makes less definite results in detecting the BRAFV600E mutation when there are relatively low amounts of the mutant template in biopsy specimens, which are invariably contaminated with normal tissues. Pyrosequencing, which measures the incorporation of each of the four nucleotides at each template position and indicates the amounts of mutant template present, may be more useful in such situations.

Genome-wide identification and validation of a novel methylation biomarker, SDC2, for blood-based detection of colorectal cancer

Aberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.

Pyrosequencing cut‐off value identifying BRAFV600E mutation in fine needle aspiration samples of thyroid nodules

Context  Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAFV600E mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi‐quantitative methods, such as dual‐priming oligonucleotide (DPO)‐based multiplex polymerase chain reaction (PCR), have the potential to generate false‐positive (FP) results.

Hypermethylation of EBF3 and IRX1 genes in synovial fibroblasts of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease of unknown origin, which exhibits a complex heterogeneity in its pathophysiological background, resulting in differential responses to a range of therapies and poor long-term prognosis. RA synovial fibroblasts (RASFs) are key player cells in RA pathogenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the process of diagnosis and prognosis of various human diseases. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with a high resolution CpG microarray for discovery of novel hypermethylated genes in RASFs. Thirteen genes (APEX1, EBF3, EGR2, EN1, IRX1, IRX6, KIF12, LHX2, MIPOL1, SGTA, SIN3A, TOLLIP, and ZHX2) with three consecutive hypermethylated probes were isolated as candidate genes through two CpG microarrays. Pyrosequencing assay was performed to validate the methylation status of TGF-β signaling components, EBF3 and IRX1 genes in RASFs and osteoarthritis (OA) SFs. Hypermethylation at CpG sites in the EBF3 and IRX1 genes was observed with a high methylation index (MI) in RASFs (52.5% and 41.4%, respectively), while a lower MI was observ ed in OASFs and h ealthy SFs (13.2% for EBF3 and 4.3% for IRX1). In addition, RT-PCR analysis showed a remarkable decrease in their mRNA expression in the RA group, compared with the OA or healthy control, and their reduction levels correlated with MI. The current findings suggest that methylation-associated down-regulation of EBF3 and IRX1 genes may play an important role in a pathogenic effect of TGF-β on RASFs. However, further clinical validation with large numbers of patients is needed in order to confirm our findings.

Abstract LB-91: Identification and feasibility test of novel methylation biomarkers for stool DNA-based colorectal cancer screening

Aberrant DNA methylation event is known to be early and frequent process in tumorigenesis and could therefore serve as a convenient biomarker for early detection of cancers. For the identification of novel methylation markers we compared each methylation profiles in super normal tissues (n=2) of healthy individuals and primary tumors and their matched normal appearing adjacent tissues (non-tumor) from colorectal cancer patients (n=12) relative to that of common reference DNA through genome-wide CpG microarray analysis following methylated DNA enrichment with a minimum size of methyl binding domain. Unsupervised hierarchical clustering of with entire methylation pattern data successfully discriminated normal, match-paired normal and primary tumors. We then statistically selected novel methylation candidates showing high frequency of methylation in primary tumor samples and intermediate in non-tumor but no methylation in supernormal tissues for early detection and confirmed in colorectal cancer cell lines by quantitative pyrosequencing-based methylation assay. For final candidates with high specificity, we selected 4 genes, GABRA1, gt-SD, gt-SI, gt-SO which showed a low methylation level in 2 healthy normal tissues. All of 4 genes appeared to be much more frequently hypermethylated in primary tumors than in non-tumor tissues in pyrosequencing assay. For the applicability into non-invasive sample we analyzed methylation status of 4 genes in stool DNAs obtained from patients with colorectal cancer (n=106) and healthy volunteers (n=10) by modified two-round nested MSP for improving analytic sensitivity. The sensitivity and specificity of each of 4 genes were estimated as 50.5% to 60.2% and 85.7% to 100%, respectively. The sensitivity of the four-gene panel was 88.7% (94/106) and its specificity was 80.0% (2/10) in detecting colorectal cancer with stool DNA. Consequently, 4 novel genes have a great potential in noninvasive stool DNA testing for colorectal cancer screening. Further large-scale clinical validation will be needed for clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-91.