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Federal Way, NC, United States

Ahmed F.E.,Institute for Research in Biotechnology
Expert Review of Anticancer Therapy | Year: 2014

Early screening for colon cancer (CC) allows for early stage diagnosis of the malignancy and potentially reduces disease mortality as the cancer is most likely curable at its earliest stages. Early detection would be desirable if accurate, practical and cost-effective diagnostic measures for this cancer were available. Mortality and morbidity from CC represent a major health problem involving a malignant disease that is theoretically preventable through screening. Current screening methods (e.g., the convenient and inexpensive immunological fecal occult blood test, FOBTi, obtained from patients' medical records) either lack sensitivity and require dietary restriction, which impedes compliance and use; are costly (e.g., colonoscopy), which decreases compliance; or could result in mortality. In comparison with the FOBT test, a non-invasive sensitive screen for which there is no requirement for dietary restriction would be a more convenient test. Colorectal cancer is the only cancer for which colonoscopy is recommended as a screening method. Although colonoscopy is a reliable screening tool, the invasive nature, abdominal pain, potential complications and high cost have hampered the application of this procedure worldwide. A screening approach using the stable miRNA molecules, which are relatively non-degradable when extracted from non-invasive stool and semi-invasive blood samples by commercially available kits and manipulated thereafter, would be preferable to a transcriptomic mRNA-, a mutation DNA-, an epigenetic- or a proteomic-based test. The approach uses reverse transcriptase, modified real-time quantitative PCR. Although exosomal RNA would be missed, using a restricted extraction of total RNA from stool or blood, a parallel test could also be carried out on RNA obtained from stool or plasma samples, and appropriate corrections for exsosomal loss can be made for accurate and quantitative test result. Eventually, a chip can be developed to facilitate diagnosis, as has been done for the quantification of genetically modified organisms in foods. The gold standard to which the molecular miRNA test is compared is colonoscopy, which can be obtained from patients' medical records. If performance criteria are met, as detailed herein, a miRNA test in human stool or blood samples based on high-throughput automated technologies and quantitative expression measurements commonly used in the diagnostic clinical laboratory should be advanced to the clinical setting, which will make a significant impact on CC prevention. © 2014 Informa UK, Ltd. Source


Ahmed F.E.,East Carolina University | Ahmed F.E.,Institute for Research in Biotechnology | Amed N.C.,Institute for Research in Biotechnology | Atkins J.N.,Southestern Medical Oncology Center | And 5 more authors.
Cancer Genomics and Proteomics | Year: 2012

We carried out this study to present proof-of-principal application, showing that by using a global microarray expression analysis, followed by quantitative stem-loop reverse transcriptase in conjunction with TaqMan® polymerase chain reaction (PCR) analysis of micro(mi)RNA genes, on limited number of plasma and tissue samples obtained from 20 individuals (five healthy, five TNM stage 0-1 colon cancer, five stage 2 and five stage 3), we were able to quantitatively monitor miRNA changes at the various TNM stages of colon cancer progression, particularly at the early, pre-malignant adenoma stage (e.g. polyps ≥1 cm with high grade dysplasia). The expression of some of the tested miRNAs showed less variability in tissue than in plasma. Nevertheless, our limited preliminary data on the plasma by itself show that plasma is well-suited for screening, and that the quantitative changes in the expression of a few cell-free circulatory mature miRNA molecules in plasma, that are associated with colon cancer progression, would provide for more sensitive and specific markers than those tests currently available on the market. In addition, analysis of miRNA molecules offers a quantitative and cost-effective non-invasive diagnostic approach for screening, than currently employed methods in a prevalent cancer that can be cured if it is detected at the early TNM stages, and that becomes deadly if not diagnosed before metastasis. Thus, a larger prospective and properly randomized clinical study using plasma derived from many control individuals and at various stages of colon cancer (TNM stages 0-IV) from patients, in order to corroborate the initial results, is now urgently needed in order to allow for a statistically valid analysis, standardizing test conditions which will provide a means for determining the true sensitivity and specificity of a miRNA-screening approach. This approach, when combined with bioinformatics analysis to correlate miRNA seed data with mRNA target data, would allow for a mechanistic understanding of how miRNAs regulate mRNA gene expression, and would offer a better comprehensive diagnostic screening test for early-detection of colon cancer non-invasively. Source


Ahmed F.E.,East Carolina University | Ahmed F.E.,Institute for Research in Biotechnology | Ahmed N.C.,Institute for Research in Biotechnology | Atkins J.N.,Southestern Medical Oncology Center | And 6 more authors.
Cancer Genomics and Proteomics | Year: 2013

To present proof-of-principle application for employing micro(mi)RNAs as diagnostic markers for colon cancer, we carried out global microarray expression studies on stool samples obtained from fifteen individuals (three controls, and three each with TNM stage 0-1, stage 2, stage 3, and stage 4 colon cancer), using Affymetrix GeneChip miRNA 3.0 Array, to select for a panel of miRNA genes for subsequent focused semiquantitative polymerase chain reaction (PCR) analysis studies. Microarray results showed 202 preferentially expressed miRNA genes that were either increased (141 miRNAs), or reduced (61 miRNAs) in expression. We then conducted a stem-loop reverse transcriptase (RT)-TaqMan® minor groove binding (MGB) probes, followed by a modified qPCR expression study on 20 selected miRNAs. Twelve of the miRNAs exhibited increased and 8 decreased expression in stool from 60 individuals (20 controls, 20 with tumor-lymph node-metastatic (TNM) stage 0-1, 10 with stage 2, five with stage 3, and 5 with stage 4 colon cancer) to quantitatively monitor miRNA changes at various TNM stages of colon cancer progression. We also used laser-capture microdissection (LCM) of colon mucosal epithelial tissue samples (three control samples, and three samples from each of the four stages of colon cancer, for a total of 15 samples) to find concordance or lack thereof with stool findings. The reference housekeeping pseudogene-free ribosomal gene (18S rRNA), which shows little variation in expression, was employed as a normalization standard for relative PCR quantification. Results of the PCR analyses confirmed that twelve miRNAs (miR-7, miR- 17, miR-20a, miR-21, miR-92a, miR-96, miR-106a, miR-134, miR-183, miR-196a, miR-199a-3p and miR214) had an increased expression in the stool of patients with colon cancer, and that later TNM carcinoma stages exhibited a more pronounced expression than did adenomas. On the other hand, eight miRNAs (miR-9, miR-29b, miR-127-5p, miR-138, miR-143, miR-146a, miR-222 and miR-938) had decreased expression in the stool of patients with colon cancer, which was also more pronounced from early to later TNM stages. Results from colon mucosal tissues were similar to those from stool samples, although with more apparent changes in expression. Cytological studies on purified stool colonocytes that employed Giemsa staining showed 80% sensitivity for detecting tumor cells in stool smears. The performance characteristics of the test confirmed that stool is a medium well-suited for colon cancer screening, and that the quantitative changes in the expression of few mature miRNA molecules in stool associated with colon cancer progression provided for more sensitive and specific non-invasive diagnostic markers than tests currently available on the market. Thus, a larger prospective and properly randomized validation study of control individuals and patients exhibiting various stages of colon cancer progression (TNM stages 0-IV) is now needed in order to standardize test conditions, and provide a means for determining the true sensitivity and specificity of a miRNA screening approach in stool for the non-invasive detection of colon cancer, particularly at an early stage (0-I). Eventually, we will develop a chip to enhance molecular screening for colon cancer, as has been accomplished for the detection of genetically-modified organisms (GMOs) in foods. Source

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