Rohaizak Muhammad

Gene expression patterns distinguish breast carcinomas from normal breast tissues: The Malaysian context

Genomic and transcriptomic alterations that affect cellular processes, such as cell proliferation, differentiation, apoptosis and invasion, commonly occur in breast oncogenesis. Epidemiological evidence has proven that the risk of breast cancer predisposition varies among different ethnicities. This study aims to identify the transcriptome changes that commonly occur during the transition of normal breast epithelium to carcinoma in three local ethnic groups (Malays, Chinese and Indians). The gene expression patterns of 43 breast carcinomas with 43 patient-matched normal breast tissues were investigated using Affymetrix U133A GeneChip (containing 22,283 probe sets targeting approximately 18,400 different transcripts) and analyzed with GeneSpring GX10. Our findings revealed a total of 33 significantly differentially expressed genes, which showed>2-fold change at a 99.9% confidence interval level (p<0.001). The significantly differentially expressed genes included CD24, CD36, CD9, TACSTD1, TACSTD2, HBB, LEP, LPL, AKR1C1, AKR1C2 and AKR1C3. Our results indicate that the vast majority of gene expression changes, from normal breast epithelial to carcinoma, found in our three major ethnic populations are similar to those in the Caucasian population. Further study of the differentially expressed genes identified in our present study is needed to search for potential breast tumor biomarkers. This will eventually help to improve the therapeutic and treatment strategies for breast cancer patients in the future.

Integrated microRNA, gene expression and transcription factors signature in papillary thyroid cancer with lymph node metastasis

Background. Papillary thyroid carcinoma (PTC) is the commonest thyroid malignancy originating from the follicle cells in the thyroid. Despite a good overall prognosis, certain high-risk cases as in those with lymph node metastasis (LNM) have progressive disease and poorer prognosis. MicroRNAs are a class of non-protein-coding, 19–24 nucleotides single-stranded RNAs which regulate gene expression and these molecules have been shown to play a role in LNM. The integrated analysis of miRNAs and gene expression profiles together with transcription factors (TFs) has been shown to improve the identification of functional miRNA-target gene-TF relationships, providing a more complete view of molecular events underlying metastasis process. Objectives. We reanalyzed The Cancer Genome Atlas (TCGA) datasets on PTC to identify differentially expressed miRNAs/genes in PTC patients with LNM-positive (LNM-P) versus lymph node negative (LNN) PTC patients and to investigate the miRNA-gene-TF regulatory circuit that regulate LNM in PTC. Results. PTC patients with LNM (PTC LNM-P) have a significantly shorter disease-free survival rate compared to PTC patients without LNM (PTC LNN) (Log-rank Mantel Cox test, p = 0.0049). We identified 181 significantly differentially expressed miRNAs in PTC LNM-P versus PTC LNN; 110 were upregulated and 71 were downregulated. The five topmost deregulated miRNAs were hsa-miR-146b, hsa-miR-375, hsa-miR-31, hsa-miR-7-2 and hsa-miR-204. In addition, 395 miRNAs were differentially expressed between PTC LNM-P and normal thyroid while 400 miRNAs were differentially expressed between PTC LNN and normal thyroid. We found four significant enrichment pathways potentially involved in metastasis to the lymph nodes, namely oxidative phosphorylation (OxPhos), cell adhesion molecules (CAMs), leukocyte transendothelial migration and cytokine–cytokine receptor interaction. OxPhos was the most significantly perturbed pathway (p = 4.70E−06) involving downregulation of 90 OxPhos-related genes. Significant interaction of hsa-miR-301b with HLF, HIF and REL/NFkB transcription factors were identified exclusively in PTC LNM-P versus PTC LNN. Conclusion. We found evidence of five miRNAs differentially expressed in PTC LNM-P. Alteration in OxPhos pathway could be the central event in metastasis to the lymph node in PTC. We postulate that hsa-miR-301b might be involved in regulating LNM in PTC via interactions with HLF, HIF and REL/NFkB. To the best of our knowledge, the roles of these TFs have been studied in PTC but the precise role of this miRNA with these TFs in LNM in PTC has not been investigated.

MicroRNAs and Lymph Node Metastasis in Papillary Thyroid Cancers.

Lymph node metastasis (LNM) in papillary thyroid cancer (PTC) has been shown to be associated with increased risk of locoregional recurrence, poor prognosis and decreased survival, especially in older patients. Hence, there is a need for a reliable biomarker for the prediction of LNM in this cancer. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene translation or degradation and play key roles in numerous cellular functions including cell-cycle regulation, differentiation, apoptosis, invasion and migration. Various studies have demonstrated deregulation of miRNA levels in many diseases including cancers. While a large number of miRNAs have been identified from PTCs using various means, association of miRNAs with LNM in such cases is still controversial. Furthermore, studies linking most of the identified miRNAs to the mechanism of LNM have not been well documented. The aim of this review is to update readers on the current knowledge of miRNAs in relation to LNM in PTC.

DURAL METASTASIS MIMICKING MENINGIOMA: AN INTERESTING CASE

Dural metastasis is a rare entity in clinical practice. We report a case of dural metastasis secondary to thyroid carcinoma, which on both preoperative CT and MRI and at surgery had the typical appearance of a meningioma. Histopathological findings confirmed metastatic follicular thyroid carcinoma as a primary site. Although rare, dural metastases can mimic a meningioma. Our experience in this case has led us to consider metastasis as a differential diagnosis even when a meningioma is suspected. We believe that reporting of the case of dural metastasis mimicking a meningioma may help clinicians in future.

Quantification of Her-2/Neu Gene in Breast Cancer Patients using Real Time-Polymerase Chain Reaction (Q-PCR) and Correlation with Immunohistochemistry Findings

BACKGROUND HER-2/neu is a proto-oncogene that encodes a transmembrane tyrosine kinase growth factor which is crucial for stimulating growth and cellular motility. Overexpression of HER-2/neu is observed in 10-35% of human breast cancers and is associated with pathogenesis, prognosis as well as response to therapy. Given the imperative role of HER-2/neu overexpression in breast cancer, it is important to determine the magnitude of amplification which may facilitate a better prognosis as well as personalized therapy in affected patients. In this study, we determined HER-2/neu protein expression by immunohistochemistry (IHC) concurrently with HER-2/neu DNA amplification by quantitative real time-polymerase chain reaction (Q-PCR). MATERIALS AND METHODS A total of 53 paired tissue samples from breast cancer patients were frozen-sectioned to characterize the tumour and normal tissues. Only tissues with 80% tumour cells were used in this study. For confirmation, Q-PCR was used to determine the HER-2/neu DNA amplification. RESULTS We found 20/53 (37.7%) of the tumour tissues to be positive for HER-2/neu protein overexpression using IHC. Out of these twenty, only 9/53 (17%) cases were in agreement with the Q-PCR results. The concordance rate between IHC and Q-PCR was 79.3%. Approximately 20.7% of positive IHC cases showed no HER-2/neu gene amplification using Q-PCR. CONCLUSION In conclusion, IHC can be used as an initial screening method for detection of the HER-2/neu protein overexpression. Techniques such as Q-PCR should be employed to verify the IHC results for uncertain cases as well as determination of HER-2/neu gene amplification.

Low Prevalence of CHEK2 Gene Mutations in Multiethnic Cohorts of Breast Cancer Patients in Malaysia

CHEK2 is a protein kinase that is involved in cell-cycle checkpoint control after DNA damage. Germline mutations in CHEK2 gene have been associated with increase in breast cancer risk. The aim of this study is to identify the CHEK2 gene germline mutations among high-risk breast cancer patients and its contribution to the multiethnic population in Malaysia. We screened the entire coding region of CHEK2 gene on 59 high-risk breast cancer patients who tested negative for BRCA1/2 germline mutations from UKM Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Sequence variants identified were screened further in case-control cohorts consisting of 878 unselected invasive breast cancer patients (180 Malays, 526 Chinese and 172 Indian) and 270 healthy individuals (90 Malays, 90 Chinese and 90 Indian). By screening the entire coding region of the CHEK2 gene, two missense mutations, c.480A>G (p.I160M) and c.538C>T (p.R180C) were identified in two unrelated patients (3.4%). Further screening of these missense mutations on the case-control cohorts unveiled the variant p.I160M in 2/172 (1.1%) Indian cases and 1/90 (1.1%) Indian control, variant p.R180C in 2/526 (0.38%) Chinese cases and 0/90 Chinese control, and in 2/180 (1.1%) of Malay cases and 1/90 (1.1%) of Malay control. The results of this study suggest that CHEK2 mutations are rare among high-risk breast cancer patients and may play a minor contributing role in breast carcinogenesis among Malaysian population.

Exploring breast carcinogenesis through integrative genomics and epigenomics analyses

There have been many DNA methylation studies on breast cancer which showed various methylation patterns involving tumour suppressor genes and oncogenes but only a few of those studies link the methylation data with gene expression. More data are required especially from the Asian region and to analyse how the epigenome data correlate with the transcriptome. DNA methylation profiling was carried out on 76 fresh frozen primary breast tumour tissues and 25 adjacent non-cancerous breast tissues using the Illumina Infinium(®) HumanMethylation27 BeadChip. Validation of methylation results was performed on 7 genes using either MS-MLPA or MS-qPCR. Gene expression profiling was done on 15 breast tumours and 5 adjacent non-cancerous breast tissues using the Affymetrix GeneChip(®) Human Gene 1.0 ST array. The overlapping genes between DNA methylation and gene expression datasets were further mapped to the KEGG database to identify the molecular pathways that linked these genes together. Supervised hierarchical cluster analysis revealed 1,389 hypermethylated CpG sites and 22 hypomethylated CpG sites in cancer compared to the normal samples. Gene expression microarray analysis using a fold-change of at least 1.5 and a false discovery rate (FDR) at p>0.05 identified 404 upregulated and 463 downregulated genes in cancer samples. Integration of both datasets identified 51 genes with hypermethylation with low expression (negative association) and 13 genes with hypermethylation with high expression (positive association). Most of the overlapping genes belong to the focal adhesion and extracellular matrix-receptor interaction that play important roles in breast carcinogenesis. The present study displayed the value of using multiple datasets in the same set of tissues and how the integrative analysis can create a list of well-focused genes as well as to show the correlation between epigenetic changes and gene expression. These gene signatures can help us understand the epigenetic regulation of gene expression and could be potential targets for therapeutic intervention in the future.

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.

Estrogen Receptor-Negative Breast Ductal Carcinoma: Clinicopathological Features and Mib-1 (Ki-67) Proliferative Index Association

Breast cancer estrogen receptor (ER) status is one of the strong additional factors in predicting response of patients towards hormonal treatment. The main aim of this study was to assess the morphological characteristics and proliferative activity using MIB-1(Ki-67) of estrogen receptor negative invasive breast ductal carcinoma (NOS type) as well as to correlate these features with clinicopathological data. We also aim to study the expression of c-erbB2 in ER negative breast tumors. High proliferative rate (MIB-1 above 20%) was observed in 63 (63.6%) of 99 ER negative tumors and that these tumors were associated with high expression of c-erbB2 (57.6%). We observed that MIB-1 is a reliable independent prognostic indicator for ER negative infiltrating ductal carcinoma in this study.

Metastasis within a metastasis to the thyroid: A rare phenomenon

Metastatic disease involving the thyroid gland is uncommon. Solitary thyroid metastases from various primary sites particularly kidney, lung, and breast had been previously described. To the best of our knowledge, metastases from two topographically separate primary malignancies to the thyroid have never been documented hitherto. This is the first reported case of cancer-to-cancer metastasis involving an invasive breast carcinoma metastasized within a metastatic renal cell carcinoma in the nonneoplastic thyroid in a 58-year-old woman. Distinguishing a secondary thyroid metastases from a primary thyroid malignancy is utmost crucial as treatment differs. The possibility of tumor metastases from two separated primaries should always be considered in a tumor exhibiting malignant cell populations with two distinctive histomorphological appearances. The role of immunohistochemistry stains in equivocal cases cannot be overemphasized.