Noninvasive Diagnosis Of Colorectal Cancer Research Articles

Plasma Circulating mRNA Profile for the Non-Invasive Diagnosis of Colorectal Cancer Using NanoString Technologies.

Colorectal cancer (CRC) is one of the most prevalent cancers and the second leading cause of cancer deaths in developed countries. Early CRC may have no symptoms and symptoms usually appear with more advanced diseases. Regular screening can identify people who are at increased risk of CRC in order to offer earlier treatment. A cost-effective non-invasive platform for the screening and monitoring of CRC patients allows early detection and appropriate treatment of the disease, and the timely application of adjuvant therapy after surgical operation is needed. In this study, a cohort of 71 plasma samples that include 48 colonoscopy- and histopathology-confirmed CRC patients with TNM stages I to IV were recruited between 2017 and 2019. Plasma mRNA profiling was performed in CRC patients using NanoString nCounter. Normalized data were analyzed using a Mann-Whitney U test to determine statistically significant differences between samples from CRC patients and healthy subjects. A multiple-group comparison of clinical phenotypes was performed using the Kruskal-Wallis H test for statistically significant differences between multiple groups. Among the 27 selected circulating mRNA markers, all of them were found to be overexpressed (gene expression fold change > 2) in the plasma of patients from two or more CRC stages. In conclusion, NanoString-based targeted plasma CRC-associated mRNAs circulating the marker panel that can significantly distinguish CRC patients from a healthy population were developed for the non-invasive diagnosis of CRC using peripheral blood samples.

The non-invasive diagnosis of colorectal cancer via a SOX9-based gene panel.

Colorectal cancer (CRC) threatens human health seriously. Early diagnosis of CRC is critical to improving patient survival. Meanwhile, non-invasive detection through tumor-circulating markers can be an important auxiliary diagnosis. In this study, we performed targeted RNA sequencing in paired tumor and adjacent normal fresh frozen tissues from 68 patients, and we also measured circulating mRNA levels in 4 time-point plasma samples collected before and after operation or chemotherapy. Our results showed that SOX9 (6.73-fold with adjusted p value < 1 × 10-45), MYC (20.59-fold with adjusted p value < 1 × 10-57), and MMP7 (131.94-fold with adjusted p value < 1 × 10-78) highly expressed in tumor compared with adjacent normal tissues. Besides, the circulating mRNA of SOX9 (41.14-fold with adjusted p value < 1 × 10-13) in CRC was significantly higher than in the normal control as well. Moreover, a SOX9-based 9-gene panel (SOX9, GSK3A, FZD4, LEF1, DVL1, FZD7, NFATC1, KRT19, and RUVBL1) showed the non-invasive diagnostic value of CRC (AUC: 0.863 (0.766-0.960), TPR: 0.92, TNR: 0.87). In summary, SOX9 expression consistently increases in tumor and plasma samples from CRC patients, which indicates the important role of SOX9 in CRC progression and its potential in non-invasive diagnosis of CRC.

TUSC3 Methylation in Peripheral Blood Cells as a Biomarker for Diagnosis of Colorectal Cancer.

Several case-control studies have suggested that global and loci-specific deoxyribonucleic acid (DNA) methylation in peripheral blood mononuclear cells (PBMCs) of DNA might be potential biomarkers of cancer diagnosis and prognosis. In this study, for the first time, we intended to assess the diagnostic power of the methylation level of tumor suppressor candidate 3 (TUSC3) gene promoter in patients with colorectal cancer (CRC). In the current study, we quantitatively assessed the promoter methylation level of TUSC3 in PBMCs of 70 CRC cases and 75 non-cancerous subjects via methylation quantification of endonuclease-resistant DNA (MethyQESD) method. The methylation level of the TUSC3 was meaningfully higher in CRC cases than in non-CRC subjects (43.55 ± 21.80% vs. 16.07 ± 13.63%, respectively; P < 0.001). The sensitivity and specificity of this gene for the detection of CRC were 88.6% and 76.0%, respectively. The receiver operating characteristic (ROC) curve examination discovered an area under the curve (AUC) of 0.880, representing a very high accuracy of the TUSC3 methylation marker in distinguishing CRC subjects from healthy individuals. However, there was no substantial diversity in methylation level between various CRC stages (P: 0.088). For CRC screening, PBMCs are a reliable source for DNA methylation analysis and TUSC3 promoter methylation can be utilized as a hopeful biomarker for early and non-invasive diagnosis of CRC.

Microbiome analysis reveals universal diagnostic biomarkers for colorectal cancer across populations and technologies.

The gut microbial dysbiosis is a risk of colorectal cancer (CRC) and some bacteria have been reported as potential markers for CRC diagnosis. However, heterogeneity among studies with different populations and technologies lead to inconsistent results. Here, we investigated six metagenomic profiles of stool samples from healthy controls (HC), colorectal adenoma (CA) and CRC, and six and four genera were consistently altered between CRC and HC or CA across populations, respectively. In FengQ cohort, which composed with 61 HC, 47 CA, and 46 CRC samples, a random forest (RF) model composed of the six genera, denoted as signature-HC, distinguished CRC from HC with an area under the curve (AUC) of 0.84. Similarly, another RF model composed of the four universal genera, denoted as signature-CA, discriminated CRC from CA with an AUC of 0.73. These signatures were further validated in five metagenomic sequencing cohorts and six independent 16S rRNA gene sequencing cohorts. Interestingly, three genera overlapped in the two models (Porphyromonas, Parvimonas and Peptostreptococcus) were with very low abundance in HC and CA, but sharply increased in CRC. A concise RF model on the three genera distinguished CRC from HC or CA with AUC of 0.87 and 0.67, respectively. Functional gene family analysis revealed that Kyoto Encyclopedia of Genes and Genomes Orthogroups categories which were significantly correlated with markers in signature-HC and signature-CA were mapped into pathways related to lipopolysaccharide and sulfur metabolism, which might be vital risk factors of CRC development. Conclusively, our study identified universal bacterial markers across populations and technologies as potential aids in non-invasive diagnosis of CRC.

An electrochemical genosensing platform for the relative quantification of the circulating long noncoding RNA CCAT1 to aid in the diagnosis of colorectal cancer

Long noncoding RNAs (lncRNAs) are promising biomarkers for the noninvasive diagnosis of colorectal cancer, but detection of cancer-associated changes in their expression levels is now restricted to the use of RT-qPCR. To extend the clinical applicability of these biomarkers, we developed an electrochemical platform, integrated by two different sandwich genosensors, for the relative quantification of a lncRNA called colon cancer associated transcript-1 (CCAT1) with respect to the mRNA GAPDH as an endogenous control. To achieve highly sensitive detection, and in contrast to previous sensors of this class, we use multiple fluorescein-tagged hybridization assistant probes allowing the incorporation of multiple redox enzymes per target molecule, in turn favoring the selective capture of these highly-structured targets onto the sensing platform. The resultant biosensors show high sensitivity (9.7 ± 0.3 µA nM−1 for CCAT1 and 7.3 ± 0.1 µA nM−1 for GAPDH) and low limits of detection (990 fM and 1830 fM for CCAT1 and GAPDH, respectively). Coupled to a PCR assay, we detect the lncRNA at clinically relevant concentrations. Preliminary results using total RNA extracted from the colorectal cancer cell line HT29 and spiked plasma samples from healthy individuals suggest that this new sensing architecture is sensitive and selective enough to obtain the expression level of CCAT1 simply from the ratio of outputs from the two platform sensors. We envision that the same design could be applied to detect other lncRNAs associated to various malignancies, facilitating early cancer diagnosis.

Fecal biomarkers: Non-invasive diagnosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer in the world in terms of morbidity and mortality, which brings great health hazards and economic burdens to patients and society. A fecal examination is an effective method for clinical examination and the most commonly used method for the census. It is simple, non-invasive, and suitable for large-scale population screening. With the development of molecular biology, lots of efforts have been made to discover new fecal biomarkers for the early screening of colorectal cancer. In this review, we summarize and discuss the recent advances of fecal biomarkers for CRC screening or diagnosis, including DNA biomarkers, RNA biomarkers, protein biomarkers, gut microbes and volatile organic compounds focusing on their diagnostic evaluation for CRC, which can provide a basis for the further development of new and effective CRC fecal screening and early diagnosis techniques.

A New Biomarker of Fecal Bacteria for Non-Invasive Diagnosis of Colorectal Cancer.

BackgroundThe intestinal flora is correlated with the occurrence of colorectal cancer. We evaluate a new predictive model for the non-invasive diagnosis of colorectal cancer based on intestinal flora to verify the clinical application prospects of the intestinal flora as a new biomarker in non-invasive screening of colorectal cancer.MethodsSubjects from two independent Asian cohorts (cohort I, consisting of 206 colorectal cancer and 112 healthy subjects; cohort II, consisting of 67 colorectal cancer and 54 healthy subjects) were included. A probe-based duplex quantitative PCR (qPCR) determination was established for the quantitative determination of candidate bacterial markers.ResultsWe screened through the gutMEGA database to identify potential non-invasive biomarkers for colorectal cancer, including Prevotella copri (Pc), Gemella morbillorum (Gm), Parvimonas micra (Pm), Cetobacterium somerae (Cs), and Pasteurella stomatis (Ps). A predictive model with good sensitivity and specificity was established as a new diagnostic tool for colorectal cancer. Under the best cutoff value that maximizes the sum of sensitivity and specificity, Gm and Pm had better specificity and sensitivity than other target bacteria. The combined detection model of five kinds of bacteria showed better diagnostic ability than Gm or Pm alone (AUC = 0.861, P < 0.001). These findings were further confirmed in the independent cohort II. Particularly, the combination of bacterial markers and fecal immunochemical test (FIT) improved the diagnostic ability of the five bacteria (sensitivity 67.96%, specificity 89.29%) for patients with colorectal cancer.ConclusionFecal-based colorectal cancer-related bacteria can be used as new non-invasive diagnostic biomarkers of colorectal cancer. Simultaneously, the molecular biomarkers in fecal samples are similar to FIT, have the applicability in combination with other detection methods, which is expected to improve the sensitivity of diagnosis for colorectal cancer, and have a promising prospect of clinical application.

Non-invasive diagnosis of colorectal cancer by Raman spectroscopy: Recent developments in liquid biopsy and endoscopy approaches

Colorectal cancer (CRC) is the third most common cancer diagnosed globally and is also one of the leading causes of cancer deaths in both men and women. The progression of CRC is slow and is often contained in colon but the risk increases with age. Based on the high certainty that the net benefit of screening in an age group is substantial, screening for CRC is recommended beginning at the age of 50. Currently, most of the incidence is concentrated in developed countries but the rate is increasing rapidly in developing geographies. Detecting CRC at an early stage is critical to reduce morbidity and mortality. Colonoscopy is the most preferred screening method but not very widely implemented due to practical considerations such as cost involved, lack of personnel and facility. To address these concerns, Raman spectroscopy (RS) has been suggested as a viable alternative due to its potential as a rapid non-invasive diagnostic tool. Recently, several studies have been reported but many variations of RS applications in CRC exists and are not well understood by non-specialists. This review focuses particularly on developments of Raman based liquid biopsy and endoscopic studies in order to throw light on each of their significance and limitations. Necessary developments in the future to translate RS into a clinical tool for screening and diagnosis of CRC are also briefly presented.

Quantitative detection of SRY-Box 21 (SOX21) gene promoter methylation as a stool-based noninvasive biomarker for early diagnosis of colorectal cancer by MethyLight method.

In recent years, the study of potential epigenetic biomarkers in feces has been an attractive research approach for the noninvasive diagnosis of colorectal cancer (CRC). The aim of this study was to evaluate the stool-based DNA methylation potential of SRY-Box 21 (SOX21) gene promoter as an appropriate candidate biomarker for differentiating CRC patients and healthy individuals for the first time. The MethyLight method was performed to analyze the methylation status of SOX21 gene promoter in fecal samples from 40 patients with CRC and 40 healthy controls. In addition, the diagnostic efficiency of measuring the hypermethylated SOX21 gene in the feces to the fecal occult blood test (FOBT) was compared. The percentage of methylated reference (PMR) values in the stool of CRC patients (median 1.44) was higher than those of healthy individuals (median 0.00) (P < 0.001). A sensitivity of 72.5% and specificity of 100% were obtained for SOX21 gene promoter methylation status and 29 of the patients were considered as positive in methylation status. There was no significant association between PMR values and demographic/clinicopathological features (P > 0.05). The results of the present study demonstrated that the stool-based assay of SOX21 gene promoter methylation has a relatively high sensitivity and specificity and it may serve as a noninvasive biomarker for early detection of CRC. However, more studies with a wide range of samples are required to further confirm the role of hypermethylation of SOX21 in the early CRC diagnosis.

Fecal microbial DNA markers serve for screening colorectal neoplasm in asymptomatic subjects.

Background and AimWe have previously shown that fecal microbial markers might be useful for non‐invasive diagnosis of colorectal cancer (CRC) and adenoma. Here, we assessed the application of microbial DNA markers, as compared with and in combination with fecal immunochemical test (FIT), in detecting CRC and adenoma in symptomatic patients and asymptomatic subjects.MethodsWe recruited 676 subjects [210 CRC, 115 advanced adenoma (AA), 86 non‐advanced adenoma, and 265 non‐neoplastic controls], including 241 symptomatic and 435 asymptomatic subjects. Fecal abundances of Fusobacterium nucleatum, a Lachnoclostridium sp. m3, Bacteroides clarus, and Clostridium hathewayi were quantified by quantitative PCR. Combining score of the four microbial markers (4Bac) and diagnostic prediction were determined using our previously established scoring model and cutoff values and FIT with a cutoff of 100 ng Hb/mL.Results4Bac detected similar percentages of CRC [85.3% (95%CI: 79.2–90.2%) vs 84.9% (68.1–94.9%)] and AA [35.7% (12.8–64.9%) vs 38.6% (29.1–48.8%)], while FIT detected more CRC [72.1% (63.7–79.4%) vs 66.7% (48.2–82.0%)] and AA [28.6% (8.4–58.1%) vs 16.8% (10.1–25.6%)], in symptomatic vs asymptomatic subjects, respectively. Focusing on the asymptomatic cohort, 4Bac was more sensitive for diagnosing CRC and AA than FIT (P < 0.001), with lower specificity [83.3% (77.6–88.0%) vs 98.6% (96.0–99.7%)]. FIT failed to detect any non‐advanced adenoma [0% (0.0–4.2%)] compared with 4Bac [41.9% (31.3–53.0%), P < 0.0001]. Combining 4Bac with FIT improved sensitivities for CRC [90.9% (75.7–98.1%)] and AA [48.5% (38.4–58.7%)] detection.ConclusionQuantitation of fecal microbial DNA markers may serve as a new test, stand alone, or in combination with FIT for screening colorectal neoplasm in asymptomatic subjects.

Identification and Validation of Stage-Associated Serum Biomarkers in Colorectal Cancer Using MS-Based Procedures.

Successful prevention of colorectal cancer (CRC) would benefit from a rapid serum screening for early detection. Here, a novel strategy for CRC biomarker discovery and validation exclusively based on MS procedures is reported. Identification of CRC serum biomarkers is initially made using label-free quantification on pooled serum samples from different CRC stages followed by two consecutive steps of targeted parallel reaction monitoring assays in different serum cohorts. Relevance of different protein depletion and peptide fractionation extent is investigated. Absolute quantification of a selected peptide is performed as a proof-of-concept. A total of 945 proteins showed differential abundance in the discovery phase. Based on their statistical significance and relative expression in disease stages, 123 potential biomarkers are selected for a training step. In the final validation step, five peptides belonging to four proteins are consistently quantified in individual CRC serum samples and controls. Different statistical analyses indicate that peptides GWVTDGFSSLK (APOC3) and LCNNPTPQFGGK (THBS1) are candidate biomarkers. Absolute quantification of LCNNPTPQFGGK shows statistical significance for the diagnosis of early respect to late CRC stages. Two peptides from APOC3 and THBS1 are validated by PRM as potential biomarkers for non-invasive diagnosis of colorectal cancer.

Abstract 3310: Non-invasive diagnosis of colorectal cancer via targeted high-throughput DNA methylation sequencing of circulating tumor DNA (ctDNA)

Abstract Colorectal cancer (CRC) commonly develops from precancerous polyps. Most polyps are benign. The current screening method involves stool tests and colonoscopy, which is an unpleasant procedure. Even though stool DNA-based approach has been developed, it is still a clinical challenge to differentiate benign complications and malignant CRC. We took the approach of high-throughput DNA bisulfide sequencing of tissue samples to identify cancer-specific methylation signatures. We learned methylation patterns by in-depth data mining and then applied pattern recognition approach to classify plasma samples. From a cohort of 70 pairs of tumor-normal matched tissue samples, we identified 1062 differential methylation markers. Next, we applied a greedy optimization algorithm to build a malignancy classification model using a training set of 118 plasma samples (malignant CRC: 56, benign complications: 23, healthy subjects: 39). In the training set, we achieved an AUC of 91.9% (95%CI: 87.3-96.5%). In an independent test set of 150 plasma samples (malignant CRC: 56, benign complications: 23, healthy subjects: 71), we achieved an AUC of 92.9% (95%CI: 88.3%~97.5%). Specifically, the model had a sensitivity of 91.1% for the identification of malignant CRC, and a specificity of 84.5% and 78.3% for healthy subjects and benign complications respectively. Our assay was demonstrated to be very sensitive towards early-stage CRC detection, with a sensitivity of 76.5% and 95% for stage I and II CRC respectively. The performance of this assay still awaits further validation in a large-scale clinical trial. In summary, we have developed a highly sensitive blood-based non-invasive diagnostic assay for the identification of malignant CRC, which can aid clinical decisions for doctors. This approach can also be extended to non-invasive early screening for various other cancer types. Citation Format: Xianrui Wu, Jiacheng Chuan, Tuo Hu, Tingting Peng, Meng Yang, Hui Shen, Peter Laird, Yangbin Gao, Xuyu Cai, Weihong Xu, Jian-bing Fan, Ping Lan. Non-invasive diagnosis of colorectal cancer via targeted high-throughput DNA methylation sequencing of circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3310.

Binary-blend fibber-based capture assay of circulating tumor cells for clinical diagnosis of colorectal cancer

BackgroundIn addition to conventional approaches, detecting and characterizing CTCs in patient blood allows for early diagnosis of cancer metastasis.MethodsWe blended poly(ethylene oxide) (PEO) into nylon-6 through electrospinning to generate a fibrous matbased circulating tumour cells (CTCs) assay. The contents of nylon-6 and PEO in the electrospun blend fibrous mats (EBFMs) were optimized to facilitate high cell-substrate affinity and low leukocyte adsorption.ResultsCompared with the IsoFlux System, a commercial instrument for CTC detection, the CTC assay of EBFMs exhibited lower false positive readings and high sensitivity and selectivity with preclinical specimens. Furthermore, we examined the clinical diagnosis accuracy of colorectal cancer, using the CTC assay and compared the results with those identified through pathological analyses of biopsies from colonoscopies. Our positive expressions of colorectal cancer through CTC detection completely matched those recognized through the pathological analyses for the individuals having stage II, III, and IV colorectal cancer. Nevertheless, two in four individuals having stage I colorectal cancer, recognized through pathological analysis of biopsies from colonoscopies, exhibited positive expression of CTCs. Ten individuals were identified through pathological analysis as having no colorectal tumours. Nevertheless, two of these ten individuals exhibited positive expression of CTCs.ConclusionsThus, in this population, the low cost EBFMs exhibited considerable capture efficiency for the non-invasive diagnosis of colorectal cancer.

Fecal Bacteria Act as Novel Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

Purpose: Gut microbiota have been implicated in the development of colorectal cancer. We evaluated the utility of fecal bacterial marker candidates identified by our metagenome sequencing analysis for colorectal cancer diagnosis.Experimental Design: Subjects (total 439; 203 colorectal cancer and 236 healthy subjects) from two independent Asian cohorts were included. Probe-based duplex quantitative PCR (qPCR) assays were established for the quantification of bacterial marker candidates.Results: Candidates identified by metagenome sequencing, including Fusobacterium nucleatum (Fn), Bacteroides clarus (Bc), Roseburia intestinalis (Ri), Clostridium hathewayi (Ch), and one undefined species (labeled as m7), were examined in fecal samples of 203 colorectal cancer patients and 236 healthy controls by duplex-qPCR. Strong positive correlations were demonstrated between the quantification of each candidate by our qPCR assays and metagenomics approach (r = 0.801-0.934, all P < 0.0001). Fn was significantly more abundant in colorectal cancer than controls (P < 0.0001), with AUROC of 0.868 (P < 0.0001). At the best cut-off value maximizing sum of sensitivity and specificity, Fn discriminated colorectal cancer from controls with a sensitivity of 77.7%, and specificity of 79.5% in cohort I. A simple linear combination of four bacteria (Fn + Ch + m7-Bc) showed an improved diagnostic ability compared with Fn alone (AUROC = 0.886, P < 0.0001) in cohort I. These findings were further confirmed in an independent cohort II. In particular, improved diagnostic performances of Fn alone (sensitivity 92.8%, specificity 79.8%) and four bacteria (sensitivity 92.8%, specificity 81.5%) were achieved in combination with fecal immunochemical testing for the detection of colorectal cancer.Conclusions: Stool-based colorectal cancer-associated bacteria can serve as novel noninvasive diagnostic biomarkers for colorectal cancer. Clin Cancer Res; 23(8); 2061-70. ©2016 AACR.

Faecal bacteria act as novel biomarkers for non-invasive diagnosis of colorectal cancer

The global incidence of colorectal cancer is increasing, and especially so in many Asian societies such as China, which has seen a two to four-fold upsurge in occurrence of the disease over the past decade.

303 Fecal Bacteria Act as Novel Biomarkers for Non-Invasive Diagnosis of Colorectal Cancer

303 Fecal Bacteria Act as Novel Biomarkers for Non-Invasive Diagnosis of Colorectal Cancer

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

Detection of miR-34a and miR-34b/c in stool sample as potential screening biomarkers for noninvasive diagnosis of colorectal cancer

Explore potential screening biomarkers for noninvasive diagnosis of colorectal cancer (CRC) by testing methylation of the miR-34a and miR-34b/c promoter in CRC patients' tissue and stool samples. Methylation-specific PCR analyses were performed on sample DNAs: 82 pairs of normal/cancer samples, 82 CRC patients' stool samples, 40 healthy volunteer stool samples, and 20 healthy volunteer blood samples were recruited. miR-34a has been found methylated in 65 of 82 (79.3%) the CRC tissue samples, but only 36 of 82 (43.9%) in corresponding normal samples. And when testing miR-34a in stool, 63 of 82 (76.8 %) CRC stool samples were observed methylated, and 2 of 40 (5%) healthy samples were observed methylated. The methylation for miR-34b/c has been found in 80 of 82 (97.5%) CRC tissue samples, 49 of 82 (59.8%) corresponding CRC normal samples, and 74 of 79 (93.6%) CRC stool samples. Yet we did not detect any methylation from healthy volunteers stool samples or healthy adult blood samples. Results indicated 76.8 % sensitivity and 93.6% specificity of the miR-34a methylation test for detecting CRC using stool samples. Meanwhile, the sensitivity and specificity of miR-34b/c were 95 and 100%, respectively. Moreover, our results revealed that abnormal DNA methylation of miR-34a was correlated with lymph metastasis (P = 0.010). Abnormal methylation of miR-34a and miR-34b/c genes might be regarded as potential biomarkers for noninvasive screening and diagnosis of colorectal cancer.

DNA Methylation Biomarkers for Noninvasive Diagnosis of Colorectal Cancer

DNA methylation biomarkers for noninvasive diagnosis of colorectal cancer (CRC) and precursor lesions have been extensively studied. Different panels have been reported attempting to improve current protocols in clinical practice, although no definite biomarkers have been established. In the present study, we have examined patient biopsies starting from a comprehensive analysis of DNA methylation differences between paired normal and tumor samples in known cancer-related genes aiming to select the best performing candidates informative for CRC diagnosis in stool samples. Five selected markers were considered for subsequent analyses in independent biologic cohorts and in silico data sets. Among the five selected genes, three of them (AGTR1, WNT2 and SLIT2) were validated in stool DNA of affected patients with a detection sensitivity of 78% [95% confidence interval (CI), 56%-89%]. As a reference, DNA methylation of VIM and SEPT9 was evaluated in a subset of stool samples yielding sensitivities of 55% and 20%, respectively. Moreover, our panel may complement histologic and endoscopic diagnosis of inflammatory bowel disease (IBD)-associated neoplasia, as it was also efficient detecting aberrant DNA methylation in non-neoplastic tissue samples from affected patients. This novel panel of specific methylation markers can be useful for early diagnosis of CRC using stool DNA and may help in the follow-up of high-risk patients with IBD.

Fecal DNA for Noninvasive Diagnosis of Colorectal Cancer in Immunochemical Fecal Occult Blood Test–Positive Individuals

We aimed to define the potential of the fecal DNA assay as an alternative or in addition to the currently used immunochemical fecal occult blood test (iFOBT) for the early diagnosis of colorectal cancer. A total of 560 individuals aged 50 to 69 years with a positive iFOBT were recruited from an Italian FOBT regional screening program. Twenty-six were diagnosed with adenocarcinoma, 264 with high-risk adenoma, and 54 with low-risk adenoma, whereas 216 subjects did not have premalignant or malignant lesions. Fecal DNA integrity was analyzed blindly by the fluorescence long DNA (FL-DNA) test. iFOBT and FL-DNA were largely independent variables (rs = 0.036, P = 0.42), with values ranging from 101 to 5,826 ng/mL and from 0 to 515 ng, respectively. Median values of both variables were significantly higher in cancer patients than in patients with noncancerous lesions or in healthy individuals. Moreover, iFOBT and FL-DNA values were individually associated with a number of pathologic parameters. Sequential use of the diagnostic iFOBT and FL-DNA methods showed that fecal DNA provided more accurate diagnostic information and was able to identify subgroups at different risk of cancer in iFOBT-positive individuals. A combined approach based on FL-DNA and iFOBT evaluation could help to better identify colorectal cancers and to determine a patient's risk of harboring a preneoplastic or neoplastic lesion. Further evaluation in a screening setting is needed to confirm this hypothesis. Fecal DNA could be a useful tool to better predict cancer risk in FOBT-positive individuals.