TCGA-My: A Systematic Repository for Systems Biology of Malaysian Colorectal Cancer.

Colorectal cancer (CRC) ranks second among the most commonly occurring cancers in Malaysia, and unfortunately, its pathobiology remains unknown. CRC pathobiology can be understood in detail with the implementation of omics technology that is able to generate vast amounts of molecular data. The generation of omics data has introduced a new challenge for data organization. Therefore, a knowledge-based repository, namely TCGA-My, was developed to systematically store and organize CRC omics data for Malaysian patients. TCGA-My stores the genome and metabolome of Malaysian CRC patients. The genome and metabolome datasets were organized using a Python module, pandas. The variants and metabolites were first annotated with their biological information using gene ontologies (GOs) vocabulary. The TCGA-My relational database was then built using HeidiSQL PorTable 9.4.0.512, and Laravel was used to design the web interface. Currently, TCGA-My stores 1,517,841 variants, 23,695 genes, and 167,451 metabolites from the samples of 50 CRC patients. Data entries can be accessed via search and browse menus. TCGA-My aims to offer effective and systematic omics data management, allowing it to become the main resource for Malaysian CRC research, particularly in the context of biomarker identification for precision medicine.

Epigenome-Wide DNA Methylation Profiling in Colorectal Cancer and Normal Adjacent Colon Using Infinium Human Methylation 450K.

The aims were to profile the DNA methylation in colorectal cancer (CRC) and to explore cancer-specific methylation biomarkers. Fifty-four pairs of CRCs and the adjacent normal tissues were subjected to Infinium Human Methylation 450K assay and analysed using ChAMP R package. A total of 26,093 differentially methylated probes were identified, which represent 6156 genes; 650 probes were hypermethylated, and 25,443 were hypomethylated. Hypermethylated sites were common in CpG islands, while hypomethylated sites were in open sea. Most of the hypermethylated genes were associated with pathways in cancer, while the hypomethylated genes were involved in the PI3K-AKT signalling pathway. Among the identified differentially methylated probes, we found evidence of four potential probes in CRCs versus adjacent normal; HOXA2 cg06786372, OPLAH cg17301223, cg15638338, and TRIM31 cg02583465 that could serve as a new biomarker in CRC since these probes were aberrantly methylated in CRC as well as involved in the progression of CRC. Furthermore, we revealed the potential of promoter methylation ADHFE1 cg18065361 in differentiating the CRC from normal colonic tissue from the integrated analysis. In conclusion, aberrant DNA methylation is significantly involved in CRC pathogenesis and is associated with gene silencing. This study reports several potential important methylated genes in CRC and, therefore, merit further validation as novel candidate biomarker genes in CRC.

Whole genome sequencing of Malaysian colorectal cancer patients reveals specific druggable somatic mutations.

The incidences of colorectal cancer (CRC) are continuously increasing in some areas of the world, including Malaysia. In this study, we aimed to characterize the landscape of somatic mutations using the whole-genome sequencing approach and identify druggable somatic mutations specific to Malaysian patients. Whole-genome sequencing was performed on the genomic DNA obtained from 50 Malaysian CRC patients' tissues. We discovered the top significantly mutated genes were APC, TP53, KRAS, TCF7L2 and ACVR2A. Four novel, non-synonymous variants were identified in three genes, which were KDM4E, MUC16 and POTED. At least one druggable somatic alteration was identified in 88% of our patients. Among them were two frameshift mutations in RNF43 (G156fs and P192fs) predicted to have responsive effects against the Wnt pathway inhibitor. We found that the exogenous expression of this RNF43 mutation in CRC cells resulted in increased cell proliferation and sensitivity against LGK974 drug treatment and G1 cell cycle arrest. In conclusion, this study uncovered our local CRC patients' genomic landscape and druggable alterations. It also highlighted the role of specific RNF43 frameshift mutations, which unveil the potential of an alternative treatment targeting the Wnt/ß-Catenin signalling pathway and could be beneficial, especially to Malaysian CRC patients.

Validation of immunogenic PASD1 peptides against HLA-A*24:02 colorectal cancer.

Colorectal cancer is the third commonest malignancy in Asia including Malaysia. The immunogenic cancer-testis antigens, which are expressed in a variety of cancers but with limited expression in normal tissues except the testis, represent an attractive approach to improve treatment options for colorectal cancer. We aimed to validate four PASD1 peptides as the immunotherapeutic targets in colorectal cancer. First, PASD1 mRNA and protein expression were determined via real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. The PASD1 peptides specific to HLA-A*24:02 were investigated using IFN-y-ELISpot assay, followed by the cytolytic and granzyme-B-ELISpot assays to analyze the cytolytic effects of CD8+ T cells. Gene and protein expressions of PASD1 were detected in 20% and 17.3% of colorectal cancer samples, respectively. PASD1(4) peptide was shown to be immunogenic in colorectal cancer samples. CD8+ T cells raised against PASD1(4) peptide were able to lyze HLA-A*24:02+ PASD1+ cells. Our results reveal that PASD1(4) peptide represents a potential target for colorectal cancer.

Integrated Characterization of MicroRNA and mRNA Transcriptome in Papillary Thyroid Carcinoma.

The incidence rate of papillary thyroid carcinoma (PTC) has rapidly increased in the recent decades, and the microRNA (miRNA) is one of the potential biomarkers in this cancer. Despite good prognosis, certain features such as lymph node metastasis (LNM) and BRAF V600E mutation are associated with a poor outcome. More than 50% of PTC patients present with LNM and BRAF V600E is the most common mutation identified in this cancer. The molecular mechanisms underlying these features are yet to be elucidated. This study aims to elucidate miRNA-genes interaction networks in PTC with or without LNM and to determine the association of BRAF V600E mutation with miRNAs and genes expression profiles. Next generation sequencing was performed to characterize miRNA and gene expression profiles in 20 fresh frozen tumor and the normal adjacent tissues of PTC with LNM positive (PTC LNM-P) and PTC without LNM (PTC LNN). BRAF V600E was genotyped using Sanger sequencing. Bioinformatics integration and pathway analysis were performed to determine the regulatory networks involved. Based on network analysis, we then investigated the association between miRNA and gene biomarkers, and pathway enrichment analysis was performed to study the role of candidate biomarkers. We identified 138 and 43 significantly deregulated miRNAs (adjusted p value < 0.05; log2 fold change ≤ -1.0 or ≥1.0) in PTC LNM-P and PTC LNN compared to adjacent normal tissues, respectively. Ninety-six miRNAs had significant expression ratios of 3p-to-5p in PTC LNM-P as compared to PTC LNN. In addition, ribosomal RNA-reduced RNA sequencing analysis revealed 699 significantly deregulated genes in PTC LNM-P versus normal adjacent tissues, 1,362 genes in PTC LNN versus normal adjacent tissue, and 1,576 genes in PTC LNM-P versus PTC LNN. We provide the evidence of miRNA and gene interactions, which are involved in LNM of papillary thyroid cancer. These findings may lead to better understanding of carcinogenesis and metastasis processes. This study also complements the existing knowledge about deregulated miRNAs in papillary thyroid carcinoma development.

MicroRNA-200c and microRNA-31 regulate proliferation, colony formation, migration and invasion in serous ovarian cancer.

BACKGROUND: Serous epithelial ovarian cancer (SEOC) is a highly metastatic disease and its progression has been implicated with microRNAs. This study aimed to identify the differentially expressed microRNAs in Malaysian patients with SEOC and examine the microRNAs functional roles in SEOC cells. METHODS: Twenty-two SEOC and twenty-two normal samples were subjected to miRNA expression profiling using the locked nucleic acid (LNA) quantitative real-time PCR (qPCR). The localization of miR-200c was determined via LNA in situ hybridization (ISH). Functional analysis of miR-200c and miR-31 on cell proliferation, migration and invasion and clonogenic cell survival were assessed in vitro. The putative target genes of the two miRNAs were predicted by miRWalk program and expression of the target genes in SEOC cell lines was validated. RESULTS: The miRNA expression profiling revealed thirty-eight significantly dysregulated miRNAs in SEOC compared to normal ovarian tissues. Of these, eighteen were up-regulated whilst twenty miRNAs were down-regulated. We observed chromogenic miR-200c-ISH signal predominantly in the cytoplasmic compartment of both epithelial and inflammatory cancer cells. Re-expression of miR-200c significantly increased the cell proliferation and colony formation but reduced the migration and invasion of SEOC cells. In addition, miR-200c expression was inversely proportionate with the expression of deleted in liver cancer-1 (DLC-1) gene. Over-expression of miR-31 in SEOC cells resulted in decreased cell proliferation, clonogenic potential, cell migration and invasion. Meanwhile, miR-31 gain-of-function led to the down-regulation of AF4/FMR2 family member 1 (AFF1) gene. CONCLUSIONS: These data suggested that miR-200c and miR-31 may play roles in the SEOC metastasis biology and could be considered as promising targets for therapeutic purposes.

Molecular characterization of serous ovarian carcinoma using a multigene next generation sequencing cancer panel approach.

BACKGROUND: High grade serous ovarian cancer is one of the poorly characterized malignancies. This study aimed to elucidate the mutational events in Malaysian patients with high grade serous ovarian cancer by performing targeted sequencing on 50 cancer hotspot genes. RESULTS: Nine high grade serous ovarian carcinoma samples and ten normal ovarian tissues were obtained from Universiti Kebangsaan Malaysia Medical Center (UKMMC) and the Kajang Hospital. The Ion AmpliSeq™ Cancer Hotspot Panel v2 targeting "mutation-hotspot region" in 50 most common cancer-associated genes was utilized. A total of 20 variants were identified in 12 genes. Eleven (55%) were silent alterations and nine (45%) were missense mutations. Six of the nine missense mutations were predicted to be deleterious while the other three have low or neutral protein impact. Eight genes were altered in both the tumor and normal groups (APC, EGFR, FGFR3, KDR, MET, PDGFRA, RET and SMO) while four genes (TP53, PIK3CA, STK11 and KIT) were exclusively altered in the tumor group. TP53 alterations were present in all the tumors but not in the normal group. Six deleterious mutations in TP53 (p.R175H, p.H193R, p.Y220C, p.Y163C, p.R282G and p.Y234H) were identified in eight serous ovarian carcinoma samples and none in the normal group. CONCLUSION: TP53 remains as the most frequently altered gene in high grade serous ovarian cancer and Ion Torrent Personal Genome Machine (PGM) in combination with Ion Ampliseq™ Cancer Hotspot Panel v2 were proven to be instrumental in identifying a wide range of genetic alterations simultaneously from a minute amount of DNA. However, larger series of validation targeting more genes are necessary in order to shed a light on the molecular events underlying pathogenesis of this cancer.