Yu-Wei Cheng

CpG Island Methylator Phenotype Associates with Low-Degree Chromosomal Abnormalities in Colorectal Cancer

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

Loss of imprinting and marked gene elevation are 2 forms of aberrant IGF2 expression in colorectal cancer.

Loss of imprinting (LOI) of insulin‐like growth factor 2 (IGF2) is a common event in many cancers and typically activates the maternally silenced allele. The resulting biallelic IGF2 expression correlates strongly with the hypomethylation of a differentially methylated region (DMR) near its promoter. It has also been shown that IGF2 undergoes overexpression in human malignancies; nevertheless, this phenomenon and its link to aberrant DMR methylation have not been reported in colorectal cancer (CRC). The aim of this study was to determine the relationship between IGF2 LOI, overexpression and DMR hypomethylation in CRC. By analyzing IGF2 and H19 methylation in 97 primary CRC and 64 matched normal colorectal tissues, we have shown a significant correlation between IGF2 LOI and DMR hypomethylation of IGF2 and H19. Additionally, when analyzing Affymetrix expression data of 167 primary CRC tumors and 32 normal tissues, 15% of tumors showed marked IGF2 elevation. We further investigated if substantially elevated IGF2 levels were linked to IGF2 or H19 hypomethylation, but found no significant correlation. However, we demonstrated that noticeable IGF2 overexpression, rather than LOI, negatively correlated with CRC microsatellite instability. These observations indicate that IGF2 expression, particularly when transcribed at significantly high levels, is a result of mechanisms unrelated to LOI. Our results suggest that IGF2 participates in CRC tumorigenesis through 2 different forms of aberrant gene expression.

Applications of the Universal DNA Microarray in Molecular Medicine

Integration of molecular medicine into standard clinical practice will require the availability of diagnostics that are sensitive, rapid, and robust. The backbone technology underlying the diagnostic will likely serve double duty during clinical trials in order to first validate the biomarkers that contribute to both drug response and disease stratification. PCR/LDR/Universal DNA microarray is a promising technology to help drive the transition from the current paradigms of clinical decision making to the new era of personalized medicine. By uncoupling the mutation detection step from array hybridization, this technology becomes fully programmable. It exploits full use of the sensitivity that the ligase detection reaction can provide, while maintaining a rapid read out on a universal microarray. Thus, PCR/LDR/Universal DNA microarray is 50-fold more sensitive and 10-fold more rapid than conventional hybridization-only arrays. The intent of this article is to provide investigators with a perspective on current uses of this approach, as well as to serve as a practical guide to implementation.

LRAT Promoter Hypermethylation as a Prognostic Marker for Colorectal Cancer Impairs Retinol Metabolism

Retinoids and its derivatives are known to regulate tumor progression. Our previous study in Colorectal Cancer (CRC) has shown that the expression of LRAT, a gene converts excess retinol into retinyl ester to balance retinoids homeostasis, may be regulated by its promoter methylation status to modulate the retinoids synthesis. In this report, we begin to explore the potential mechanism of LRAT mediated retinoid metabolism. Our data indicate CRC patient of LRAT hypermethylation associated with better prognosis. A consistent finding is shown in siRNA mediated LRAT silencing, which leads to slow growth of CRC cell lines. We have also observed favorable CRC prognosis occurred in patients of both LRAT and RAR-beta hypermethylation, suggesting the better CRC prognosis may be mediated through RAR beta independent pathway.

Detection of low abundant mutations in DNA using single molecule FRET and ligase detection reactions

New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.