miRNA Classifier Research Articles

Potentiality of a triple microRNA classifier: miR-193a-3p, miR-23a and miR-338-5p for early detection of colorectal cancer

BackgroundMicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).MethodsThe study was divided into two phases: (I) Marker discovery by miRNA microarray using paired cancer tissues (n?=?30) and blood samples (CRC, n?=?42; control, n?=?18). (II) Marker validation by stem-loop reverse transcription real time PCR using an independent set of paired cancer tissues (n?=?30) and blood samples (CRC, n?=?70; control, n?=?32). Correlation analysis was determined by Pearson’s test. Logistic regression and receiver operating characteristics curve analyses were applied to obtain diagnostic utility of the miRNAs.ResultsSeven miRNAs (miR-150, miR-193a-3p, miR-23a, miR-23b, miR-338-5p, miR-342-3p and miR-483-3p) have been found to be differentially expressed in both tissue and blood samples. Significant positive correlations were observed in the tissue and blood levels of miR-193a-3p, miR-23a and miR-338-5p. Moreover, increased expressions of these miRNAs were detected in the more advanced stages. MiR-193a-3p, miR-23a and miR-338-5p were demonstrated as a classifier for CRC detection, yielding a receiver operating characteristic curve area of 0.887 (80.0% sensitivity, 84.4% specificity and 83.3% accuracy).ConclusionDysregulations in circulating blood miRNAs are reflective of those in colorectal tissues. The triple miRNA classifier of miR-193a-3p, miR-23a and miR-338-5p appears to be a potential blood biomarker for early detection of CRC.

Abstract 1963: Prognosis-related miRNA expression profiles of neuroblastoma.

Abstract PURPOSE: Neuroblastoma (NB) is known to exhibit clinical heterogeneity and its accurate risk classification is one of the major challenges to improve the personalized therapy. We have been constructing the genomic/genetic profile-based risk classification system for NB. Recent studies indicated that miRNA expression levels are strongly correlated with patient prognosis of NB. To further improve our tumor stratification system by adding the miRNA classifier, we conducted miRNA expression profiling of favorable (F) and unfavorable (UF) NBs and searched for the prognosis-related miRNAs in our sample set. METHODS: 48 primary samples (19 stage 1 or 2; 16 stage 3 or 4 without MYCN amplification; 13 stage 3 or 4 with MYCN amplification) were analyzed by miRNA microarray (Agilent G4470A). Statistical analysis was conducted by using the information of patient outcome at 3 years after diagnosis. Quantitative RT-PCR (qRT-PCR) was also conducted to validate the results in 101 primary tumors by using Taqman microRNA assays. RESULTS: Among the 470 miRNAs analyzed, 79 miRNAs showed strong correlation to the prognosis (p<0.05): 17 and 62 miRNAs showed higher expressions in UF and F phenotypes, respectively. In the former miRNA group, 5 were located in 1q whose genomic copy number is frequently increased in UF type tumors. On the other hand, the latter includes 5, 6 and 9 miRNAs in 1p, 19q13 and 14q32, respectively, which are frequently decreased in copy numbers in UF type. In addition, miR-542-5p, whose expression has been reported as a prognostic marker by other groups, was also in the latter group in our dataset (p<0.001, 3.3-fold lower in UF). We further conducted qRT-PCR of these picked-up miRNAs with 101 independent NB samples for validation and found that higher expression of miR-181b and miR-149 showed significant correlation with poor and good prognosis of patients (both logrank p<0.001), respectively. In addition, intermediate-risk type of tumors (stage 3 or 4, no MYCN amplification, n=40) were also significantly classified into favorable or unfavorable by these two miRNA classifiers (p=0.021 and p=0.005, respectively). Interestingly, miR-149 has been reported that it has pro-apoptotic function by repressing Akt1 and E2F1 expressions in a neuroblastoma cell line. CONCLUSION: The result suggested that miRNA expression profile exhibited strong correlation with NB prognosis, and integration of miRNA classifier with other markers could improve the prediction system for NB prognosis. Citation Format: Miki Ohira, Eriko Isogai, Yohko Nakamura, Takehiko Kamijo, Akira Nakagawara. Prognosis-related miRNA expression profiles of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1963. doi:10.1158/1538-7445.AM2013-1963

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis

AbstractmiRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1–positive MCL (n = 30) and cyclin D1–negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1–negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

Evaluation of the prognostic utility of miRNA and multiple genetic markers in stage IIA colon cancer.

3630 Background: The variable prognoses of stage II colon cancer subjects have provoked a therapeutic dilemma of who should receive adjuvant therapy. Presently, prognosis is evaluated on often subjective histopathological features. Reliable, objective markers that can accurately predict recurrence risk are needed. Using retrospectively collected tumor samples with known clinical outcome, we evaluated the prognostic utility of miRNA together with clinicopathological features and molecular markers with suggestive prognostic value. Methods: We evaluated the expression of miRNA in 118 stage IIA FFPE tumors (73 non-recurrent, NR; 45 recurrent, R) using the Affymetrix Gene Chip V1.0 microarray, and performed RT-qPCR to verify select miRNAs. We also sequenced KRAS codons 12/13, and BRAF codon 600, and assessed MSI status using a fluorescent PCR-based assay. MMR protein expression was determined by IHC. An empirical approach was applied to screen miRNA using combinatorial criteria of microarray 2-way ANOVA analysis, Kaplan-Meier curve logrank, and single and multivariate Cox-regression. miRNA was the most significant predictor among all histopathological and molecular features (FDR<0.05). A miRNA classifier was built with nested two-layer cross-validation. Results: The top miRNA model was selected based on AUC estimates and Cox-regression p-value. This model included 30 miRNAs; 25 of these were common with the empirical approach, confirming the robustness of candidate selection. The miRNA classifier distinguished R from NR cases with an AUC of ~0.74. Among all histopathological and molecular features evaluated, grade was significantly associated with disease-free survival (logrank p<0.05). Both single variate and multivariate Cox analysis revealed that grade and PMS2 status were strongly associated with recurrence (p<0.05). Lastly, lack of expression of PMS2 and MLH1, BRAF V600E and MSI was highly correlated (Φ coefficient > 0.6), and associated with non-recurrent disease. Conclusions: These data provide strong support for miRNAs as independent prognostic biomarkers for stage IIA colon cancer.Validation work is ongoing to demonstrate the potential utility of these miRNAs in patient stratification.

Abstract 4148: MicroRNA biomarkers prognostic for disease-free and overall survival in stage I and II lung adenocarcinoma

Abstract Background: Approximately 50% of all non-small cell lung carcinoma (NSCLC) cases in the United States are adenocarcinoma histology. While definitive lung cancer resection offers a chance for cure in stage I and II NSCLC, up to 57% of patients will have disease relapse or metastatic disease within 5 years. A majority of the relapsed cases will take place within 2 years of definitive surgical resection. Thus, pathologic staging alone does not stratify patients for prognosis sufficiently. Identifying patients with aggressive versus indolent tumors would have critical implications for directing treatment and designing future clinical trials. We propose using microRNA (miRNA) for discovery and validation of prognostic biomarkers for stage I and II lung adenocarcinoma. Methods: For the Discovery Cohort, we identified treatment-naïve AJCC 7th edition stage I and II lung adenocarcinoma patient samples from 26 disease-free survivors (DFS) and overall survival (OS) at least 47 months and 14 patients with DFS and overall survival less than 23 months. Total RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) samples. Differential miRNA array expression profiling between these two groups was assessed in the Discovery cohort on an 8x15 Agilent microarray printed with 886 miRNAs. Target miRNAs were selected based on minimum of 2-fold change in expression and Welch t-test p-value cut-off of <0.05. For our Validation Cohort, qPCR assay of these individual miRNAs were measured from the 40 Discovery Cohort samples, as well as additional stage I and II lung adenocarcinoma patient samples (N=37 with DFS and OS>47 months and N=12 with DFS and OS<23 months), to confirm differential expression of target miRNAs. Fold-change values were generated using the ΔΔCt method. A PCR-based miRNA classifier was developed. Results: Microarray analysis of the Discovery Cohort revealed 7 miRNAs that were significantly under-expressed in lung adenocarcinoma tumor from patients with DFS and OS <23 months. They included let-7a/b/c, miR-130a, -451, -29c, and -99a. PCR results in the Validation Cohort (total N=89) remained statistically significant for differential expression for all target miRNAs (p<0.05) except miR-99a. A two miRNA PCR-based classifier using let-7c and miR-451 had the best performance with an accuracy of 77.5%. Application of this classifier to an Independent Cohort of additional stage I and II lung adenocarcinoma samples from another institution blinded for DFS/OS comparisons (N=21 DFS/OS >47 months and N=4 DFS/OS <23 months) is ongoing. Conclusion: We have validated miRNAs prognostic for DFS and OS in stage I and II lung adenocarcinoma in at least one cohort of 89 patients so far. A two miRNA classifier is currently being evaluated and we anticipate results for accuracy prediction on the Independent cohort at the time of the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4148. doi:1538-7445.AM2012-4148

MiRNA Expression Profiling Enables Risk Stratification in Archived and Fresh Neuroblastoma Tumor Samples

More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples. Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples. The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples. In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material.

New miRNA classifier distinguishes CTCL from benign skin disorders

Cutaneous T-cell lymphomas (CTCL) are the most frequently occurring primary skin lymphoma. These skin lesions contain a high number of inflammatory cells but early diagnosis of CTCL is challenging because of its clinical, pathological and histological similarity to benign inflammatory skin lesions such as psoriasis and eczema. Now, a study has shown that a microRNA (miRNA) classifier can distinguish CTCL from benign skin disorders. Senior researcher Niels Odum explains the background to the study “we decided to address if a novel miRNA technology could help solve a 100-year-old problem—namely, how to distinguish cutaneous CTCL from benign dermatosis”.

A miRNA assay for the classification of benign and neoplastic lesions in pancreatic fine needle aspirates.

10614 Background: Improved diagnosis of pancreatic ductal adenocarcinoma (PDAC) versus benign pancreatic lesions is an urgent clinical need. We previously reported a laboratory-developed test based on the differential expression of 2 miRNAs, miR-196a and miR-217, with a sensitivity and specificity of 95% in formalin-fixed, paraffin-embedded (FFPE) specimens with ≥60% tumor content. Here, we report the development and validation of a miRNA-based research test optimized for low % tumor specimens and un-enriched fine needle aspirates (FNA). Methods: RNA was extracted using methods developed and validated by the Asuragen Services Laboratory from frozen, FFPE or FNA pancreatic specimens. One to 3 endoscopic ultrasonography-guided FNAs were collected, stored, and shipped in RNARetain, an RNA stabilization solution formulated in 1 mL single-use vials. Expression levels of up to 12 miRNAs were interrogated using TaqMan qRT-PCR assays. Results: A set of 211 specimens, including 94 PDAC, 96 chronic pancreatitis, and 21 normal, was used to develop a miRNA classifier to discriminate benign and low tumor content PDAC specimens. Training on 95 FFPE specimens showed that expression analysis of miR-196a, -210, and -375 improved by 2- to 3-fold the detection rate of PDAC in specimens with 5-88% tumor. The classifier performance was further verified on 4 independent sets of specimens (n=116): frozen with high tumor content (80-100%), FFPE with high tumor content (60-95%), FFPE with low tumor content (10-60%), and FNA. The signature alongside the remaining 9 miRNAs, is now being evaluated in a multi-site prospective study using FNAs collected in RNARetain; 133 specimens have been analyzed to date and we will report the relative performance of the top miRNA classifiers. Conclusions: Benign lesions and PDAC can be accurately classified based on the expression level of a few miRNAs in specimens with 5-100% tumor and in FNA. Testing of pre-operative FNAs collected in RNARetain as part of routine clinical evaluation could in the future improve the differential diagnosis of PDAC and potentially reduce the rate of indeterminate or false negative calls.

Abstract 127: MicroRNA-328 is associated with non-small cell lung cancer (NSCLC) brain metastasis and mediates NSCLC migration

Abstract Background: Lung cancer is the leading cause of cancer deaths and ∼88% of patients with primary lung malignancy have non-small cell lung cancer (NSCLC). Brain metastasis (BM) can affect up to 25% of these patients during their lifetime. There is surely a need for improvement in patient stratification to identify lung cancer patients at higher risk of developing BM. Recently, microRNAs (miRNAs) have been studied to characterize tumors. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. Experimental Design: A miRNA brain metastasis classifier described in Nasser et al was validated on a set of 15 other patients(ref1). Following these experiments, miR-328 (one of the two markers that could successfully classify BM+ cases) was over-expressed in A549, a NSCLC cell line, using a lentiviral system. These miR-328 over-expressing cells were then used for gene expression studies using Agilent microarrays. These cells were also used for cell migration studies. Transient transfection of gene specific siRNA was used to demonstrate the role of PRKCA (gene upregulated in miR-328 over-expressing cells) in migration of NSCLC cells. Results: A miRNA classifier was built based on 12 samples from clinically matched NSCLC from patients with BM (BM+) and without BM (BM-)(ref1). This classifier consisting of miR-328 and miR-330-3p expression was able to correctly classify BM+ vs. BM- patients. It was then used on a validation cohort and correctly classified 12/15 patients. Comparison of gene expression profiles of A549 parental and A549 cells stably transfected to over-express miR-328 (A549-328) identified several significantly differentially-expressed genes. PRKCA was one of the genes upregulated in A549-328 cells. Its upregulation in A549-328 cells was confirmed at the protein level by western blotting. A549-328 cells have significantly increased cell migration compared to A549 cells, and knockdown of PRKCA by transient siRNA transfection leads to significant reduction in this migration potential. Conclusion: miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA. With further corroboration, these miRNAs may be incorporated into clinical treatment decision-making to stratify NSCLC patients at higher risk for developing brain metastasis. Reference 1. Nasser S, Ranade A, Sridhar S, Haney L, Korn RL, Gotway MB, Weiss GJ, Kim S. Identifying MiRNA and Imaging Features Associated with Metastasis of Lung Cancer to the Brain. Proceedings of the IEEE International Conference on Bioinformatics &amp; Biomedicine 2009:246-51. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2011-127

An miRNA assay for the classification of benign and neoplastic lesions in pancreatic fine-needle aspirates.

163 Background: Improved diagnosis of pancreatic ductal adenocarcinoma (PDAC) versus benign pancreatic lesions is an urgent clinical need. We previously reported a laboratory-developed test based on the differential expression of two miRNAs, miR-196a and miR-217, with a sensitivity and specificity of 95% in formalin-fixed, paraffin-embedded (FFPE) specimens with ≥60% tumor content. Here, we report the development and validation of an miRNA-based research test optimized for low % tumor specimens and fine needle aspirates (FNA). Methods: RNA was extracted using methods developed and validated by the Asuragen Services Laboratory from frozen, FFPE or FNA pancreatic specimens. One to three endoscopic ultrasonography-guided FNAs were collected, stored, and shipped in RNARetain, an RNA stabilization solution formulated in 1 mL single-use vials. Expression levels of up to 12 miRNAs were interrogated using TaqMan qRT-PCR assays. Results: A set of 211 specimens, including 94 PDAC, 96 chronic pancreatitis, and 21 normal, was used to develop a miRNA classifier to discriminate benign and low tumor content PDAC. Training on 95 FFPE specimens showed that expression analysis of miR-196a, -210, and -375 improved by 2- to 3-fold the detection rate of PDAC in specimens with 5-88% tumor. The classifier performance was further verified on 4 independent sets of specimens (n=116): frozen with high tumor content (80-100%), FFPE with high tumor content (60-95%), FFPE with low tumor content (10-60%), and FNA. The signature is now being evaluated in a multisite prospective study using FNAs collected in RNARetain; 110 specimens have been analyzed to date and we will report the relative performance of the 2- and 3-miRNA classifiers. Conclusions: Benign lesions and PDAC can be accurately classified based on the expression level of a few miRNAs in specimens with 5-100% tumor and in FNA. Testing of preoperative FNAs collected in RNARetain as part of routine clinical evaluation could in the future improve the differential diagnosis of PDAC and potentially reduce the rate of indeterminate or false-negative calls. [Table: see text]

MicroRNA Expression Profiles Associated with Mutational Status and Survival in Malignant Melanoma

Malignant cutaneous melanoma is a highly aggressive form of skin cancer. Despite improvements in early melanoma diagnosis, the 5-year survival rate remains low in advanced disease. Therefore, novel biomarkers are urgently needed to devise new means of detection and treatment. In this study, we aimed to improve our understanding of microRNA (miRNA) deregulation in melanoma development and their impact on patient survival. Global miRNA expression profiles of a set of melanoma lymph node metastases, melanoma cell lines, and melanocyte cultures were determined using Agilent array. Deregulated miRNAs were evaluated in relation with clinical characteristics, patient survival, and mutational status for BRAF and NRAS. Several miRNAs were differentially expressed between melanocytes and melanomas as well as melanoma cell lines. In melanomas, miR-193a, miR-338, and miR-565 were underexpressed in cases with a BRAF mutation. Furthermore, low expression of miR-191 and high expression of miR-193b were associated with poor melanoma-specific survival. In conclusion, our findings show miRNA dysregulation in malignant melanoma and its relation to established molecular backgrounds of BRAF and NRAS oncogenic mutations. The identification of an miRNA classifier for poor survival may lead to the development of miRNA detection as a complementary prognostic tool in clinical practice.

Abstract A31: Prognostic microRNA biomarkers for recurrence of stage II colorectal cancer

Abstract Reliable prediction of recurrence in stage II colorectal cancer (CRC) patients is a major unresolved clinical need as recurrence is often the ultimate cause of death in these individuals. Several mRNA and DNA prognostic markers have been evaluated and, to date, none have been recommended by oncology groups due to poor specificity and sensitivity. MicroRNA (miRNA) have been shown to regulate fundamental cellular processes and have been implicated in several cancer types, including CRC. We used a microarray platform to examine the expression of more than 600 miRNAs in tumors from stage II patients with or without recurrence. Using an exploratory sample set of 12 tumors (6 nonrecurrent and 6 recurrent), we identified several miRNAs that were significantly deregulated in tumors from recurrent patients compared to non-recurrent patients. We used RT-qPCR to interrogate the differentially expressed miRNAs on the original 12 samples plus an additional set of 39 stage II CRC tumors that were not used in the initial miRNA discovery process. Using cross-validation, we derived a miRNA-based classifier that was strongly associated with recurrence (hazard ratio (HR), 3.83; p=0.005). We further evaluated associations of MSI status and mutations in KRAS, BRAF and PIK3CA genes with recurrence (n=39). Multivariate Cox analysis revealed that only the miRNA classifier was significantly associated with recurrence (HR 3.08; p=0.03). Interestingly, the combination of the miRNA classifier and mutant KRAS status was highly predictive of recurrence (p&amp;lt;0.0001). These data provide strong support for miRNAs as prognostic biomarkers for stage II CRC and warrant further validation. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A31.