Sungwhan An

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.

Development and Clinical Evaluation of a Highly Sensitive DNA Microarray for Detection and Genotyping of Human Papillomaviruses

ABSTRACT Human papillomavirus (HPV) has been found in cervical cancer, tonsillar cancer, and certain types of head and neck cancers. We report on a DNA microarray-based method for the simultaneous detection and typing of HPVs. The genotype spectrum discriminated by this HPV DNA microarray includes 15 high-risk HPV genotypes and 12 low-risk HPV genotypes. The HPV DNA microarray showed high degrees of specificity and reproducibility. We evaluated the performance of the HPV DNA microarray by application to three HPV-positive cell lines (HeLa, Caski, and SiHa cells) and two HPV-negative cell lines (C33A and A549 cells). The HPV DNA microarray successfully identified the known types of HPV present in the cell lines. The detection limit of the HPV DNA microarray was at least 100-fold higher than that of PCR. To assess the clinical applicability of the HPV DNA microarray, we performed the HPV genotyping assay with 73 nonmalignant and malignant samples from 39 tonsillar cancer patients. Twenty-five of the 39 (64.1%) malignant samples were positive for HPV, whereas 3 of 34 (8.8%) nonmalignant samples were positive for HPV. This result shows a preferential association of HPV with tonsillar carcinomas. The correlations of the presence of HPV with the grade of differentiation and risk factors were not significant. Our data show that the HPV DNA microarray may be useful for the diagnosis and typing of HPV in large-scale epidemiological studies.

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.

The role of vimentin as a methylation biomarker for early diagnosis of cervical cancer

Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM promoter methylation and VIM gene expression were investigated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-formation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multistep carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is responsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cervical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.

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.

Epigenetic regulation of the potential tumor suppressor gene, hLHX6.1, in human cervical cancer.

It is well known that the Homo sapiens LIM homeobox domain 6 gene (hLHX6), a putative transcription regulator, controls the differentiation and development of neural and lymphoid cells, particularly in the central nervous system. In this study, we investigated hLHX6.1 (an isoform of hLHX6), which functions as a tumor suppressor gene in the cervix. Firstly, the methylation levels of the hLHX6 and hLHX6.1 promoters were investigated in 8 cervical cancer cell lines and human tissue samples with a distinctive degree of malignant transformation. In spite of the presence of multiple cytosine guanine dinucleotides (CpG islands) in 2 proximal promoters of the hLHX6 and hLHX6.1 genes, only the hLHX6.1 promoters were found to be mostly hypermethylated and associated with transcriptional silencing by promoter methylation, whereas the hLHX6 promoters were not. Methylation levels in the hLHX6.1 promoter were also found to be strongly related to cervical cancer development. The level of hLHX6.1 gene expression was found to be relatively high in normal cells, in which the hLHX6.1 promoter was mostly unmethylated. However, the hLHX6.1 gene expression was down-regulated or undetectable in cervical cancer cell lines and cancer tissues, in which the hLHX6.1 promoter was hypermethylated. This epigenetic alteration in the hLHX6.1 promoter begins at a relatively early stage, suggesting its potential as a biomarker for the early diagnosis and prevention of cervical cancer. Moreover, the overexpression of the hLHX6.1 gene in cervical cancer cells suppressed the tumorigenic phenotype, as shown by soft agar colony formation and migration assays, suggesting that hLHX6.1 could be a new tumor suppressor gene in the cervix.

PACAP inhibits tumor growth and interferes with clusterin in cervical carcinomas.

Secretory clusterin (sCLU), an anti‐apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo‐resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt–Raf–ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.

PINCH-2 presents functional copy number variation and suppresses migration of colon cancer cells by paracrine activity

In recent years, characterization of cancer and its environment has become necessary. However, studies of the cancer microenvironment remain insufficient. Copy number variations (CNVs) occur in 40% of cancer‐related genes, but few studies have reported the correlation between CNVs in morphologically normal tissues adjacent to cancer and cancer progression. In this study, we evaluated cancer cell migration and invasion according to the genetic differences between cancer tissues and their surrounding normal tissues. To study the field cancerization effect, we screened 89 systemic metastasis‐related CNVs from morphologically normal tissues adjacent to colon cancers. Among these CNVs, LIM and senescent cell antigen‐like domain 2 (PINCH‐2) showed copy number amplification and upregulation of mRNA in the nonrelapsed group compared to the systemic relapse group. PINCH‐2 expression in colon cancer cells was lower than that in normal epithelial colon cells at both the protein and mRNA levels. Suppression of PINCH‐2 resulted in decreased formation of the PINCH‐2‐IPP (PINCH‐2, integrin‐linked kinase and α‐parvin) complex and reciprocally increased formation of the PINCH‐1‐IPP complex. Although PINCH‐2 expression of survival pathway‐related proteins (Akt and phospho‐Akt) did not change upon suppression of PINCH‐2 expression, cell migration‐related proteins [matrix‐metalloproteinase (MMP)−9 and −11] were upregulated through autocrine and paracrine activation. Thus, PINCH‐2 participates in decreased systemic recurrence by competitively regulating IPP complex formation with PINCH‐1, thereby suppressing autocrine and paracrine effects on motility in colon cancer. This genetic change in morphologically normal tissue suggests a field cancerization effect of the tumor microenvironment in cancer progression.

Abstract 4517: Syndecan-2 methylation is an early detection biomarker for colorectal cancer with high sensitivity and specificity in small serum sample volumes

BACKGROUND: Colorectal cancer (CRC) is a major cause of morbidity and mortality in the world. Epigenetics has been a useful tool in the development of sensitive and specific screening tests for CRC. Targeting genes highly methylated in CRC has allowed us to develop an accurate method to select novel candidate genes and validate their sensitivity and selectivity through testing in clinical samples. METHODS: We performed CpG microarray analysis using methylation enriched DNA from tumors and matched normal appearing tissues to identify genes methylated in CRC. We then applied a step-wise filtering method to narrow our field of candidate genes. Six genes were verified for high methylation-positivity in CRC cell lines and underwent negative selection in healthy normal samples using bisulfate-pyrosequencing. Genes SORCS3, SIM1, SDC2 were selected for low levels of methylation in normal tissue followed by verification with MSP-pyrosequencing. RESULTS: SDC2 was then selected because of high methylation levels in tumor and very low levels in normal tissue. SDC2 methylation levels were measured in 133 tumors and were found to be significantly methylated at all stages. Next, 500αl sera samples from 42 CRC patients were tested using qMSP which demonstrated sensitivity at 71.4% and specificity 92.9%. CONCLUSIONS: This study utilized refined technologies to identify and validate a novel candidate SCD2 for CRC screening in serum with high sensitivity and selectivity using only 500αl. These findings demonstrate a new level of candidate selection, verification and application that we feel will meet the need for a new minimally-invasive screening test. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4517. doi:1538-7445.AM2012-4517