Plasma Samples Of Colorectal Cancer Patients Research Articles

Oncogenic microRNA-1290 and SCAI Gene as Potential Biomarker for Colorectal Carcinoma.

Colorectal cancer (CRC) is the world's third most frequent cancer, with a significant mortality rate due to late detection. There is a need to search for biomarkers that can detect colorectal cancer at an early stage. MicroRNAs (miRNAs) regulate several targets that function as oncogenes and/or tumor suppressor genes, so any change in microRNA expression level can predict abnormality. The objective of the study was to evaluate the expression of miR-1290, and Suppressor of cancer cell invasion (SCAI) gene that may be used as biomarkers for early diagnosis of colorectal carcinoma. This study included 50 subjects consisting of newly diagnosed colorectal carcinoma patients (n = 25), and healthy controls (n = 25). After RNA isolation and reverse transcription, the expression level of miR-1290 and SCAI gene in the tissues and plasma samples of CRC patients were analyzed using real time PCR and compared with healthy individuals as normal controls. The 2-ΔΔCt formula was used to compute the fold-change, while using miR-16 and GAPDH as reference genes for normalization. We found that miR-1290 is upregulated, whereas SCAI gene is downregulated in both plasma and tissue samples of CRC patients. For miR-1290, the sensitivity was 96% and specificity was 100%, and for SCAI, 100% sensitivity and 88% specificity was calculated by ROC analysis. The expression of miR-1290 and SCAI gene may be utilized as biomarkers for diagnosis of colorectal carcinoma.

A novel liquid biopsy methylation-based technology and early detection of colorectal cancer in patients.

e15527 Background: Since colorectal cancer (CRC) remains as the third most common and second most deadly malignant neoplasm, accurate and non-invasive detection technologies became a promising strategy to enhance early diagnosis. Our goal is to develop a liquid biopsy test able to detect methylation-based biomarkers in circulating tumor DNA (ct-DNA) collected from CRC patients' plasma. We are currently working on a digital PCR (dPCR)-based technology focused on detecting with high sensitivity and specificity our proprietary CRC specific epigenetic biomarker panel. Methods: We developed a bioinformatic platform that identifies methylation-based biomarkers from genome-wide Illumina 450K methylation data available in GDC-TCGA public databases. The platform uses multidimensional analysis algorithms and, through a predictive model, establishes a ranking with the most appropriate diagnostic epigenetic biomarkers. First, we tested the selected biomarkers in tumor tissue and in distant colonic mucosa from average risk CRC patients using qPCR. Then, ct-DNA from plasma samples of CRC patients and circulating DNA (cf-DNA) from risk matched negative colonoscopy-confirmed healthy individuals was analyzed by a novel dPCR-based technology in order to detect our panel of epigenetic biomarkers. Lastly, we applied a ROC analysis to the Area Under the Curve (AUC) values and obtained the corresponding sensitivity and specificity results. Results: Illumina 450K methylation data from primary tumor and normal tissue was incorporated into our bioinformatic platform that subsequently ranked 275 candidate genes. Based on several classification criteria we selected a panel composed of the three best ranked candidates. We validated the bioinformatic platform performance in identifying sensitive and specific CRC epigenetic biomarkers by detecting the presence of these biomarkers in tumor mucosa from paired CRC patients and its absence in distant colonic mucosa (n = 20) using qPCR. Furthermore, we applied our dPCR-based technology to simultaneously detect the three-gene panel of epigenetic biomarkers in ctDNA from 34 CRC patients and cfDNA from 33 healthy individuals. We obtained an overall AUC = 0.92, with 76% sensitivity (95% CI: 60-87%) and 90.9% specificity (95% CI: 76-97%). Conclusions: We validated a technology capable of detecting a novel and proprietary methylation-based biomarkers panel. This accurate liquid biopsy technology based on epigenetic biomarker detection applies dPCR and might be implemented in screening or early colorectal cancer detection. Although our technology needs further clinical validations and optimizations, we consider that the obtained results are strong enough to evolve in a complete epigenetic biomarker's blood-based test.

Evaluation of plasma circ_0006282 as a novel diagnostic biomarker in colorectal cancer.

BackgroundNowadays, non‐invasive and rapid detection of cancers through molecular biomarkers has received much attention. Therefore, this study investigated the non‐invasive and rapid diagnosis of colorectal cancer through one of the newest biomarkers (circular RNA).MethodsFor this purpose, we collected tumoral, adjacent normal tissue, and plasma samples from 100 colorectal cancer (CRC) patients, 25 postoperative CRC patients, 28 colitis patients, and 108 healthy donors. First Illumina high‐throughput (Hi Seq 2000) sequencing was performed to identify known and novel differentially expressed circRNAs in the cancerous and adjacent normal tissues (n = 3). We used quantitative real‐time fluorescent polymerase chain reaction (qRT‐PCR) to detect the expression level of hsa_circ_0006282 among the different samples. Moreover, inter‐ and intra‐assays were performed to evaluate the potential of hsa_circ_0006282 as being a biomarker. The receiver operating characteristic curve (ROC) was drawn to appraise its diagnostic efficacy, and the sensitivity of this circ RNA was evaluated.ResultsBased on RNA‐sequencing results circ_0006282, cirs7, circ‐0001313, circ_0055625, circ_000984, circ_0055625, circ_0001178, circ_0071589, circ‐001569 were upregulated, and circ‐ITGA7, circ‐CDYL, circITCH, circ_0026344, circ_0000038, circ_0002220, circ_0067480, circIGHV3‐20‐1, circ_104916, circ_0009361 were downregulated circRNA. The hsa_circ_0006282 was the highest upregulated differentially expressed circRNA. Expression evaluation of this circRNA on different samples showed upregulation in CRC tissues (p < 0.0001) and plasma samples of CRC patients in comparison to healthy controls (p < 0.0001), while the area under the curve (AUC) was 0.831 (95% CI: 0.779–0.883). Expression of hsa_circ_0006282 in CRC patients decreased to normal after surgery (p < 0.0001). Our results showed high specificity and sensitivity of CRC detection when hsa_circ_0006282, carcinoembryonic antigen (CEA), and carbohydrate antigen199 (CA199) are combined.ConclusionPlasma hsa_circ_0006282 can be used as a novel diagnostic and dynamic monitoring biomarker in CRC patients.

Plasma microRNA Levels Combined with CEA and CA19-9 in the Follow-Up of Colorectal Cancer Patients.

Colorectal cancer (CRC) ranks among the most common cancers worldwide. Surgical removal remains the best strategy for treatment of resectable tumors. An important part of caring for patients after surgery is monitoring for early detection of a possible relapse of the disease. Efforts are being made to improve the sensitivity and specificity of routinely used carcinoembryonic antigen (CEA) with the use of additional biomarkers such as microRNAs. The aim of our study was to evaluate the prognostic potential of microRNAs and their use as markers of disease recurrence. The quantitative estimation of CEA, CA19-9, and 22 selected microRNAs (TaqMan Advanced miRNA Assays) was performed in 85 paired (preoperative and postoperative) blood plasma samples of CRC patients and in samples taken during the follow-up period. We have revealed a statistically significant decrease in plasma levels for miR-20a, miR-23a, miR-210, and miR-223a (p = 0.0093, p = 0.0013, p = 0.0392, and p = 0.0214, respectively) after surgical removal of the tumor tissue. A statistically significant relation to prognosis (overall survival; OS) was recorded for preoperative plasma levels of miR-20a, miR-21, and miR-23a (p = 0.0236, p = 0.0316, and p =0.0271, respectively) in a subgroup of patients who underwent palliative surgery. The best discrimination between patients with favorable and unfavorable outcomes was achieved by a combination of CEA, CA19-9 with miR-21, miR-20a, and miR-23a (p < 0.0001). The use of these microRNAs for early disease recurrence detection was affected by a low specificity in comparison with CEA and CA19-9. CEA and CA19-9 had high specificity but low sensitivity. Our results show the benefit of combining currently used standard biomarkers and microRNAs for precise prognosis estimation.

A Novel Method to Detect Early Colorectal Cancer Based on Chromosome Copy Number Variation in Plasma

Background/Aims: Colonoscopy screening has been accepted broadly to evaluate the risk and incidence of colorectal cancer (CRC) during health examination in outpatients. However, the intrusiveness, complexity and discomfort of colonoscopy may limit its application and the compliance of patients. Thus, more reliable and convenient diagnostic methods are necessary for CRC screening. Genome instability, especially copy-number variation (CNV), is a hallmark of cancer and has been proved to have potential in clinical application. Methods: We determined the diagnostic potential of chromosomal CNV at the arm level by whole-genome sequencing of CRC plasma samples (n = 32) and healthy controls (n = 38). Arm level CNV was determined and the consistence of arm-level CNV between plasma and tissue was further analyzed. Two methods including regular z score and trained Support Vector Machine (SVM) classifier were applied for detection of colorectal cancer. Results: In plasma samples of CRC patients, the most frequent deletions were detected on chromosomes 6, 8p, 14q and 1p, and the most frequent amplifications occurred on chromosome 19, 5, 2, 9p and 20p. These arm-level alterations detected in plasma were also observed in tumor tissues. We showed that the specificity of regular z score analysis for the detection of colorectal cancer was 86.8% (33/38), whereas its sensitivity was only 56.3% (18/32). Applying a trained SVM classifier (n = 40 in trained group) as the standard to detect colorectal cancer relevance ratio in the test samples (n = 30), a sensitivity of 91.7% (11/12) and a specificity 88.9% (16/18) were finally reached. Furthermore, all five early CRC patients in stages I and II were successfully detected. Conclusion: Trained SVM classifier based on arm-level CNVs can be used as a promising method to screen early-stage CRC.

Expression Analysis of Previously Verified Fecal and Plasma Dow-regulated MicroRNAs (miR-4478, 1295-3p, 142-3p and 26a-5p), in FFPE Tissue Samples of CRC Patients.

Colorectal cancer (CRC) is one of the most common causes of cancer-related mortality worldwide. Early diagnosis of this neoplasm is critical and may reduce patients' mortality. MicroRNAs are small non-coding RNA molecules whose expression pattern can be altered in various diseases such as CRC. In this study, we evaluated the expression levels of miR-142-3p, miR-26a-5p (their reduced expression in plasma samples of CRC patients was previously confirmed), miR-4478 and miR-1295-3p (their reduced expression in stool samples of CRC patients was previously confirmed) in tissue samples of CRC patients in comparison to healthy subjects. To achieve this purpose, total RNA including small RNA was extracted from 53 CRC and 35 normal subjects' Formalin-fixed, Paraffin-embedded (FFPE) tissue samples using the miRNeasy FFPE Mini Kit. The expression levels of these four selected miRNAs were measured using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). We found that the expression levels of miR-4478 and miR-1295b-3p (two previously down-regulated fecal miRNAs) were significantly decreased in FFPE samples of CRC patients compared to healthy controls. On the other hand, no significant differences were seen in expression levels of miR-142-3p and miR-26a-5p (two previously down-regulated circulating miRNAs) in FFPE samples between these two groups. Regarding current findings, it may be concluded that to diagnose CRC patients based on the miRNAs approach, stool samples are more likely preferable to plasma samples; nevertheless, additional studies with more samples are needed to confirm the results.

Combined detection of plasma GATA5 and SFRP2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas.

To investigate GATA5, SFRP2, and ITGA4 methylation in plasma DNA as noninvasive biomarkers for colorectal cancer (CRC) or adenomas. There were 57 CRC patients, 30 adenomas patients, and 47 control patients enrolled in this study. Methylation-specific polymerase chain reaction was used to determine the promoter methylation status of GATA5, SFRP2, and ITGA4 genes in plasma DNA, and their association with clinical outcome in CRC. The predictive ability of GATA5, SFRP2, and ITGA4 methylation, individually or in combination, to detect CRC or adenomas was further analyzed. Hypermethylated GATA5 was detected in plasma in 61.4% (35/57) of CRC cases, 43.33% (13/30) of adenoma cases, and 21.28% (10/47) of control cases. The hypermethylation of SFRP2 was detected in 54.39% (31/57), 40.00% (12/30), and 27.66% (13/47) in plasma samples from CRC, adenomas, and controls, respectively. ITGA4 methylation was detected in 36.84% (21/57) of plasma samples of CRC patients and in 30.00% (9/30) of plasma samples from patients with colorectal adenomas, and the specificity of this individual biomarker was 80.85% (9/47). Moreover, GATA5 methylation in the plasma was significantly correlated with larger tumor size (P = 0.019), differentiation status (P = 0.038), TNM stage (P = 0.008), and lymph node metastasis (P = 0.008). SFRP2 and ITGA4 methylation in plasma significantly correlated with differentiation status (SFRP2, P = 0.012; ITGA4, P = 0.007), TNM stage (SFRP2, P = 0.034; ITGA4, P = 0.021), and lymph node metastasis (SFRP2, P = 0.034; ITGA4, P = 0.021). From the perspective of predictive power and cost-performance, using GATA5 and SFRP2 together as methylation markers seemed the most favorable predictor for CRC (OR = 8.06; 95%CI: 2.54-25.5; P < 0.01) and adenomas (OR = 3.35; 95%CI: 1.29-8.71; P = 0.012). A combination of GATA5 and SFRP2 methylation could be promising as a marker for the detection and diagnosis of CRC and adenomas.

The expression of microRNA-375 in plasma and tissue is matched in human colorectal cancer.

BackgroundMicroRNAs (miRNAs) offer great potential as cancer biomarkers. The importance of miRNAs profiling in tissue and body fluids in colorectal cancer (CRC) have been addressed respectively in many studies. The purpose of our study is to systematically assess the expression of miRNAs in cancer tissue and matched plasma samples and to evaluate their usefulness as minimally invasive diagnostic biomarkers for the detection of CRC.MethodsThe study was divided into two phases: firstly, qRT-PCR based TaqMan Low Density MiRNA Arrays (TLDAs) was used to screen the differentially expressed miRNAs in 6 plasma samples of CRC patients and 6 healthy controls. Secondly, marker validation by stem-loop reverse transcription real-time PCR using an independent set of paired cancer tissues (n = 88) and matched plasma samples (CRC, n = 88; control, n = 40). Correlation analysis was determined by Pearson’s test. Receiver operating characteristic curve analyses were applied to obtain diagnostic utility of the differentially expressed miRNAs. Target gene prediction and signal pathway analyses were used to predict the function of miRNAs.ResultsTLDAs identified 42 miRNAs, which were differentially expressed in patients and healthy individuals. Five of them (miR-375, miR-150, miR-206, miR-125b and miR-126*) were chosen to be validated in plasma and tissue samples. The results indicated that for plasma sample, miR-375 (p < 0.0001) and miR-206 (p = 0.0002) were dysregulated and could discriminate CRC patients from healthy controls. For tissue samples, miR-375 (p < 0.0001), miR-150 (p < 0.0001), miR-125b (p = 0.0065) and miR-126*(p = 0.0009) were down-regulated. miR-375 was significantly down-regulated and positively correlated in both tissue and plasma samples (r = 0.4663, p = 0.0007). Gene ontology and signal pathway analyses showed that most of the target genes that were regulated by miR-375 were involved in some critical pathways in the development and progression of cancer.ConclusionsOur results indicate that the down-regulation of miR-375 in plasma and tissue is matched in CRC. Moreover, bioinformatics prediction revealed miR-375 association with some critical signal pathways in the development and progression of CRC. Therefore, plasma miR-375 holds great promise to be an alternative tissue biomarker for CRC detection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-714) contains supplementary material, which is available to authorized users.

Identification and Evaluation of Plasma MicroRNAs for Early Detection of Colorectal Cancer

BackgroundColorectal cancer (CRC) is one of the most commonly diagnosed cancers. Circulating microRNAs (miRNAs) have been suggested as potentially promising markers for early detection of CRC. We aimed to identify and evaluate a panel of miRNAs that might be suitable for CRC early detection.MethodsMiRNAs were profiled by TaqMan MicroRNA Array and screened for differential expression in 5 pools of plasma samples of CRC patients (N = 50) and 5 pools of neoplasm-free controls (N = 50). Additional miRNAs were selected from a literature review. Identified candidates were evaluated in independent validation samples with respect to discrimination of CRC patients (N = 80) or advanced adenoma patients (N = 50) and neoplasm-free controls (N = 194). Diagnostic performance of the panel of miRNAs was assessed by multiple logistic regression, using bootstrap analysis to correct for over-optimism.ResultsFive miRNAs identified to be differentially expressed from TaqMan MicroRNA Array (miR-29a, -106b, -133a, -342-3p, -532-3p), and seven miRNAs reported to be differentially expressed in the literature (miR-18a, -20a, -21, -92a, -143, -145, -181b) were selected for validation. Nine of the twelve miRNAs (miR-18a, -20a, -21, -29a, -92a, -106b, -133a, -143, -145) were found to be differentially expressed in CRC patients and controls in the validation samples. The optimism-corrected area under the curve was 0.745 (95% confidence interval: 0.708–0.846). None of the selected miRNAs showed significant differential expression between advanced adenoma patients and neoplasm-free controls.ConclusionThe identified panel of miRNAs could be of potential use in the development of a multi-marker blood based test for early detection of CRC. Impact: The study underscores the high potential of plasma miRNAs for the improvement of current offers of non-invasive CRC screening.

Circulating MicroRNA in Digestive Tract Cancers

d t For many decades, cell-free nucleic acids have been known to be present in peripheral blood. Several tudies have identified tumor-specific and/or tumor-assoiated alterations in the circulating nucleic acids of paients with various cancers. In recent years, cell-free miroRNA (miRNA) have been stably detected in the plasma nd serum, like other molecules; their presence in the lood has attracted the attention of researchers due to heir potential use as valuable blood biomarkers.1 MiRNAs are short, noncoding RNAs that play important roles in various physiologic and developmental processes. The mature miRNAs are produced from long primary transcripts through 2 sequential cleavage steps. The long primary miRNA transcript is cleaved by the Drosha complex in the nucleus, generating intermediate precursor miRNA. Precursor miRNA is transported by exportin-5 from the nucleus into the cytoplasm, and then subjected to further cleavage by a Dicer RNAase III enzyme, generating a short double-strand miRNA. One strand (guided strand) of mature miRNA is then incorporated into the RNA-induced silencing complex and subsequently hybridize to the 3=-untranslated region of their target mRNAs to repress translation or degrade these mRNAs. Thus, a single miRNA can influence the expression of hundreds of genes and allow them to function in a coordinated manner. Therefore, miRNAs have been implicated as key molecules in all cellular processes. Numerous studies have shown that alterations in miRNA expression correlate with various diseases, including the development and progression of cancer, and some miRNAs can function as oncogenes or tumor suppressors. These findings have opened up a new and interesting field in the diagnosis of cancer and the treatments of cancer patients. Mitchell et al2 first demonstrated that circulating miRNAs had the potential to be new biomarkers in patients with solid cancers. In recent years, several papers have demonstrated that circulating miRNAs can also be detected in the peripheral blood of patients with digestive tract cancers. Although the origins and physiologic functions of cell-free miRNAs in the blood remain to be fully elucidated, a noninvasive assay for miRNAs should be developed to exploit these molecules as potential diagnostic and prognostic biomarkers. This assay undoubtedly contributes to an improvement in the clinical outcomes of cancer patients. In this article, we review the current state of biological and clinical research regarding circulating miRNAs of digestive tract cancer patients and discuss the future perspectives.