SnoRNA profiling in colorectal cancer and assessment of non-invasive biomarker capacity by ddPCR in fecal samples.

Small nucleolar RNAs (snoRNAs) have been identified dysregulated in several pathologies, and these alterations can be detected in tissues and in circulation. The main aim of this study was to analyze the whole snoRNome in advanced colorectal neoplasms and to identify new potential non-invasive snoRNA-based biomarkers in fecal samples by different analytical approaches. SNORA51, SNORD15B, SNORA54, SNORD12B, SNORD12C, SNORD72, SNORD89, and several members of SNORD115 and SNORD116 clusters were consistently deregulated in both tissue sets. After technical validation, SNORA51 and SNORD15B were detected in FIT+ samples. SNORA51 was significantly upregulated in FIT+ samples from CRC patients compared to healthy controls. This upregulation, together with the fecal hemoglobin concentration, was sufficient to identify, among FIT+ individuals, patients with CRC (AUC = 0.86) and individuals with advanced adenomas (AUC = 0.68). These findings portray snoRNAs as an alternative source of candidates for further studies and SNORA51 appears as a potential non-invasive biomarker for CRC detection.

Fecal MicroRNA-Based Algorithm Increases Effectiveness of Fecal Immunochemical Test-Based Screening for Colorectal Cancer.

BACKGROUND & AIMS: An algorithm based on fecal levels of 2 microRNAs (miR-421 and miR-27a-3p), fecal hemoglobin concentration, and patient age and sex can identify patients with advanced colorectal neoplasia. We investigated whether this algorithm, called miRFec, could increase effectiveness and efficiency of fecal immunochemical test (FIT)-based colorectal cancer (CRC) screening programs. METHODS: We obtained data and fecal samples from 767 persons with a positive result from the FIT who then underwent colonoscopy examination while participating a population-based CRC screening program, from March 2011 through May 2017 in Barcelona, Spain. Fecal miRNAs were isolated from the buffer contained in the original FIT collection device and analyzed by quantitative reverse transcription PCR. Aims were to evaluate the usefulness of the miRFec algorithm in identifying persons at greatest risk for CRC who should be prioritized for colonoscopy examination and individuals at low risk for whom colonoscopy could be avoided. RESULTS: Of the 767 study subjects, 414 (54.0%) were found by colonoscopy to have advanced colorectal neoplasia (67 with CRC and 347 with advanced adenomas) and 353 (46.0%) were found to have either non-advanced adenomas (n = 136) or a normal examination (n = 217). MiRFec algorithm scores (1-4) were independently associated with the presence of advanced colorectal neoplasia (P < .001). The miRFec algorithm differentiated patients with CRC from those with non-advanced adenomas or normal colonoscopy with an area under the receiver operating characteristic curve of 90% (95% CI, 86-94). Subjects with miRFec scores in the 4th quartile (above 3.09, high-risk group) were 8-fold more likely to have advanced colorectal neoplasia than subjects with miRFec scores in the 1st quartile (below 2.14, low-risk group). Subjects in the low-risk group had a positive predictive value below 30% for detection of advanced colorectal neoplasia. When we used a 50% specificity cut-off value, the miRFec algorithm identified 97% of patients with CRC and would allow 264 subjects (34.4%) to avoid colonoscopy examination. CONCLUSIONS: An algorithm based on fecal levels of 2 miRNAs and hemoglobin, patient age and sex (miRFec) differentiated patients with CRC from those with non-advanced adenomas or normal colonoscopy with an area under the receiver operating characteristic curve value of 90% and avoided 34% of colonoscopies. Inclusion of this algorithm in FIT-based CRC screening programs could increase their effectiveness and efficiency.

Nuevos biomarcadores no invasivos para el cribado del cáncer colorrectal / No disponible

El cáncer colorrectal (CCR) representa una de las neoplasias más comunes en los países desarrollados y la segunda causa de muerte por cáncer en España. Se desarrolla mediante la acumulación de alteraciones genéticas y epigenéticas en la mucosa colónica, dando lugar a adenomas colorrectales y carcinomas invasivos. Este proceso multietapa hace que el CCR sea susceptible a la implementación de medidas preventivas como los programas de cribado. Para ello, hoy en día existen diferentes estrategias, aunque ninguna cumple las condiciones ideales de una prueba de cribado. En este contexto, se ha visto que diversas alteraciones moleculares implicadas en la carcinogénesis del CCR podrían contribuir a la generación de nuevos métodos no invasivos, sensibles y específicos. En la siguiente revisión se comentarán brevemente algunos de los biomarcadores no invasivos más relevantes para el diagnóstico del CCR

No disponible

New non-invasive biomarkers for colorectal cancer screening.

Colorectal cancer (CRC) is one of the most frequent neoplasia in developed countries and the second leading cause of cancer death in Spain. It develops through the accumulation of genetic and epigenetic alterations in the normal mucosa, producing advanced adenomas and invasive cancers. This multistep process makes CRC highly traceable, making it a good candidate for the implementation of preventive measures such as screening programs. Although there are different strategies, none of them meet all the criteria for CRC screening purposes. In this context, many molecular alterations involved in CRC carcinogenesis could contribute to the generation of new non-invasive methods with a high sensitivity and specificity. In this review, some of the most promising diagnostic non-invasive biomarkers for CRC will be briefly discussed.

Identification and Validation of MicroRNA Profiles in Fecal Samples for Detection of Colorectal Cancer.

BACKGROUND & AIMS: Screening for colorectal cancer (CRC) is effective in the population at average risk. The most extended strategy in organized programs involves the fecal immunochemical test, which is limited by low sensitivity for the detection of advanced adenomas (AAs). We aimed to identify microRNA (miRNA) signatures in fecal samples that identify patients with AAs or CRC and might be used in noninvasive screening. METHODS: Our study comprised 4 stages. In the discovery phase, we performed genome-wide miRNA expression profiling of 124 fresh, paired colorectal tumor and nontumor samples (30 CRC; 32 AAs) from patients in Spain. In the technical validation stage, miRNAs with altered expression levels in tumor vs nontumor tissues were quantified by reverse-transcription polymerase chain reaction in fecal samples from a subset of patients included in the discovery phase (n = 39) and individuals without colorectal neoplasms (controls, n = 39). In the clinical validation stage, the miRNAs found to be most significantly up-regulated by quantitative reverse transcription polymerase chain reaction analysis were measured in an independent set of fecal samples (n = 767) from patients with positive results from fecal immunochemical tests in a CRC screening program. Finally, we developed a model to identify patients with advanced neoplasms (CRCs or AAs) based on their miRNA profiles, using findings from colonoscopy as the reference standard. RESULTS: Among 200 and 324 miRNAs significantly deregulated in CRC and AA tissues, respectively, 7 and 5 of these miRNAs were also found to be deregulated in feces (technical validation). Of them, MIR421, MIR130b-3p, and MIR27a-3p were confirmed to be upregulated in fecal samples from patients with advanced neoplasms. In our model, the combination of fecal level of MIR421, MIR27a-3p, and hemoglobin identified patients with CRC with an area under the curve (AUC) of 0.93, compared with an AUC of 0.67 for fecal hemoglobin concentration alone. CONCLUSIONS: We found that increased levels of 2 miRNAs and hemoglobin in feces can identify patients with AAs or CRC more accurately than fecal hemoglobin concentration alone. Assays for these miRNAs might be added to fecal tests for the detection of CRC or AAs.

Validation of miR-1228-3p as Housekeeping for MicroRNA Analysis in Liquid Biopsies from Colorectal Cancer Patients.

BACKGROUND: Circulating microRNA (miRNA) analysis is a growing research field. However, it usually requires an endogenous control or housekeeping (HK) in order to normalize expression of specific miRNAs throughout different samples. Unfortunately, no adequate HK for circulating miRNA analysis is still known in the colorectal cancer (CRC) context whereas several have been suggested. Hence, our aims were to validate the previously suggested miR-1228-3p as HK for CRC studies, to compare its suitability with the widely used miR-16-5p, and to evaluate the influence of hemolysis on both miRNAs. METHODS: We analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) the expression of miR-1228-3p, miR-16-5p and the spike-in cel-miR-39 in a set of 297 plasmas (92 CRC, 101 advanced adenomas -AA-, and 100 controls) and 213 serum samples (59 CRC, 74 AA and 80 controls). We also analyzed both miRNAs depending on the hemolysis degree in 7 plasmas and 31 serums. RESULTS: Levels of miR-1228-3p and miR-16-5p did not show significant differences between groups although miR-16-5p exhibited more variability in plasma and serum samples. Importantly, the combination of cel-miR-39 and miR-1228-3p was the most stable one. Moreover, we observed that miR-16-5p was significantly influenced by hemolysis in contrast with miR-1228-3p that exhibited no correlation with this confounding factor in both biofluids. CONCLUSION: MiR-1228-3p has been validated as an adequate endogenous control for circulating miRNA analysis in CRC and AA liquid biopsies.

Analysis of A 6-Mirna Signature in Serum from Colorectal Cancer Screening Participants as Non-Invasive Biomarkers for Advanced Adenoma and Colorectal Cancer Detection.

Early detection of colorectal cancer (CRC) and its precancerous lesion, advanced adenomas (AA), is critical to improve CRC incidence and prognosis. Circulating microRNAs (miRNAs or miR) are promising non-invasive biomarkers for cancer detection. Our previous results showed that a plasma 6-miRNA signature (miR-15b-5p, miR-18a-5p, miR-29a-3p, miR-335-5p, miR-19a-3p and miR-19b-3p) could distinguish between CRC or AA and healthy individuals (controls). However, its diagnostic performance in serum is unknown. In this exploratory study we aim to evaluate the diagnostic performance of the 6-miRNA signature in serum samples in a cohort of individuals participating in Barcelona's CRC Screening Programme. We prospectively collected serums from 264 faecal immunochemical test (FIT)-positive participants and total RNA was extracted. Finally, 213 individuals (CRC, 59, AA, 74, controls, 80) were included. MiRNA expression was quantified by real-time RT-qPCR and data analysis was performed by logistic regression. Faecal hemoglobin concentration (f(Hb)) from FIT of the same individuals was also considered. As previously described in plasma, serum from patients with AA or CRC presented significant differences in the 6-miRNA signature compared to controls. Moreover, when combined with f(Hb), the final signature showed high discriminative capacity to distinguish CRC from controls (area under the curve (AUC) = 0.88), and even AA (AUC = 0.81) that otherwise are poorly detected if we only consider f(Hb) (AUC = 0.64). Addition of the serum 6-miRNA signature to quantitative f(Hb) show high accuracy to detect patients with advanced colorectal neoplasia in average-risk individuals. A combination of these two non-invasive methods could be a good strategy to improve diagnostic performances of current CRC screening programmes.

Circulating biomarkers for early detection and clinical management of colorectal cancer.

New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.

Novel Circulating miRNA Signatures for Early Detection of Pancreatic Neoplasia.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) presents the lowest survival rate of all cancers because only 6% of patients reach five-year survival. Alterations in the expression of several microRNAs (miRNAs) occur in the tumor of PDAC and in preneoplastic lesions as the called intraductal papillary mucinous neoplasm (IPMN). Here, we aimed at identifying which miRNAs are significantly altered in liquid biopsies from patients with PDAC and IPMN to find new noninvasive biomarkers for early detection of PDAC. METHODS: We analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) the expression of 17 circulating miRNAs, previously found to be significantly overexpressed in tissue pancreatic neoplasms, in a set of 182 plasma samples (94 PDAC, 19 IPMN, 18 chronic pancreatitis, and 51 disease-free controls). Then, we analyzed CA19.9 levels in the same plasma set, and we assessed the diagnostic values of differentially expressed miRNAs, CA19.9, and all possible combinations. RESULTS: Of note, 16, 14, and 9 miRNAs were significantly increased in PDAC, IPMN, and chronic pancreatitis, respectively, compared with control plasmas. miR-21-5p, miR-33a-3p, miR-320a, and miR-93-5p showed the highest discriminating capacity for pancreatic neoplasia (PDAC or IPMN) with an area under the receiver operating characteristic curve (AUC) of 0.86, 0.85, 0.85, and 0.80, respectively. 2-miRNA combinations improved these performances reaching AUC = 0.90 for "miR-33a-3p+miR-320a." Addition of CA19.9 increased the diagnostic potential of miRNA signatures even further achieving an AUC of 0.95 (93% sensitivity and 85% specificity) for the combination of "miR-33a-3p+miR-320a+CA19.9." CONCLUSIONS: Novel signatures combining miRNAs and CA19.9 could be used as noninvasive biomarkers for early detection of PDAC.

Plasma MicroRNA Signature Validation for Early Detection of Colorectal Cancer.

OBJECTIVES: Specific microRNA (miRNA) signatures in biological fluids can facilitate earlier detection of the tumors being then minimally invasive diagnostic biomarkers. Circulating miRNAs have also emerged as promising diagnostic biomarkers for colorectal cancer (CRC) screening. In this study, we investigated the performance of a specific signature of miRNA in plasma samples to design a robust predictive model that can distinguish healthy individuals from those with CRC or advanced adenomas (AA) diseases. METHODS: Case control study of 297 patients from 8 Spanish centers including 100 healthy individuals, 101 diagnosed with AA, and 96 CRC cases. Quantitative real-time reverse transcription was used to quantify a signature of miRNA (miRNA19a, miRNA19b, miRNA15b, miRNA29a, miRNA335, and miRNA18a) in plasma samples. Binary classifiers (Support Vector Machine [SVM] linear, SVM radial, and SVM polynomial) were built for the best predictive model. RESULTS: Area under receiving operating characteristic curve of 0.92 (95% confidence interval 0.871-0.962) was obtained retrieving a model with a sensitivity of 0.85 and specificity of 0.90, positive predictive value of 0.94, and negative predictive value of 0.76 when advanced neoplasms (CRC and AA) were compared with healthy individuals. CONCLUSIONS: We identified and validated a signature of 6 miRNAs (miRNA19a, miRNA19b, miRNA15b, miRNA29a, miRNA335, and miRNA18a) as predictors that can differentiate significantly patients with CRC and AA from those who are healthy. However, large-scale validation studies in asymptomatic screening participants should be conducted.

MicroRNAs for Detection of Pancreatic Neoplasia: Biomarker Discovery by Next-generation Sequencing and Validation in 2 Independent Cohorts.

OBJECTIVE: The aim of our study was to analyze the miRNome of pancreatic ductal adenocarcinoma (PDAC) and its preneoplastic lesion intraductal papillary mucinous neoplasm (IPMN), to find new microRNA (miRNA)-based biomarkers for early detection of pancreatic neoplasia. OBJECTIVE: Effective early detection methods for PDAC are needed. miRNAs are good biomarker candidates. METHODS: Pancreatic tissues (n = 165) were obtained from patients with PDAC, IPMN, or from control individuals (C), from Hospital Clínic of Barcelona. Biomarker discovery was done using next-generation sequencing in a discovery set of 18 surgical samples (11 PDAC, 4 IPMN, 3 C). MiRNA validation was carried out by quantitative reverse transcriptase PCR in 2 different set of samples. Set 1-52 surgical samples (24 PDAC, 7 IPMN, 6 chronic pancreatitis, 15 C), and set 2-95 endoscopic ultrasound-guided fine-needle aspirations (60 PDAC, 9 IPMN, 26 C). RESULTS: In all, 607 and 396 miRNAs were significantly deregulated in PDAC and IPMN versus C. Of them, 40 miRNAs commonly overexpressed in both PDAC and IPMN were selected for further validation. Among them, significant up-regulation of 31 and 30 miRNAs was confirmed by quantitative reverse transcriptase PCR in samples from set 1 and set 2, respectively. CONCLUSIONS: miRNome analysis shows that PDAC and IPMN have differential miRNA profiles with respect to C, with a large number of deregulated miRNAs shared by both neoplastic lesions. Indeed, we have identified and validated 30 miRNAs whose expression is significantly increased in PDAC and IPMN lesions. The feasibility of detecting these miRNAs in endoscopic ultrasound-guided fine-needle aspiration samples makes them good biomarker candidates for early detection of pancreatic cancer.