Iben Daugaard

Increased sensitivity of KRAS mutation detection by high‐resolution melting analysis of COLD‐PCR products

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low‐level tumor specific KRAS mutations. Coamplification at lower denaturation temperature‐PCR (COLD‐PCR) is a new form of PCR that selectively amplifies mutation‐containing templates based on the lower melting temperature of mutant homoduplexes versus wild‐type homoduplexes. We have developed a fast COLD‐PCR and high‐resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD‐PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen® KRAS mutation kit. The sensitivity was increased by 5‐ to 100‐fold for melting temperature decreasing mutations when using COLD‐PCR compared to standard PCR. Mutations, undetectable by the TheraScreen® kit in clinical samples, were detected by COLD‐PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD‐PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Hum Mutat 31:1–8, 2010.

Biogenesis and Function of Ago-Associated RNAs

Numerous sophisticated high-throughput sequencing technologies have been developed over the past decade, and these have enabled the discovery of a diverse catalog of small non-coding (nc)RNA molecules that function as regulatory entities by associating with Argonaute (Ago) proteins. MicroRNAs (miRNAs) are currently the best-described class of post-transcriptional regulators that follow a specific biogenesis pathway characterized by Drosha/DGCR8 and Dicer processing. However, more exotic miRNA-like species that bypass particular steps of the canonical miRNA biogenesis pathway continue to emerge, with one of the most recent additions being the agotrons, which escape both Drosha/DGCR8- and Dicer-processing. We review here the current knowledge and most recent discoveries relating to alternative functions and biogenesis strategies for Ago-associated RNAs in mammals.

Identification and validation of candidate epigenetic biomarkers in lung adenocarcinoma

Lung cancer is the number one cause of cancer-related deaths worldwide. DNA methylation is an epigenetic mechanism that regulates gene expression, and disease-specific methylation changes can be targeted as biomarkers. We have compared the genome-wide methylation pattern in tumor and tumor-adjacent normal lung tissue from four lung adenocarcinoma (LAC) patients using DNA methylation microarrays and identified 74 differentially methylated regions (DMRs). Eighteen DMRs were selected for validation in a cohort comprising primary tumors from 52 LAC patients and tumor-adjacent normal lung tissue from 32 patients by methylation-sensitive high resolution melting (MS-HRM) analysis. Significant increases in methylation were confirmed for 15 DMRs associated with the genes and genomic regions: OSR1, SIM1, GHSR, OTX2, LOC648987, HIST1H3E, HIST1H3G/HIST1H2BI, HIST1H2AJ/HIST1H2BM, HOXD10, HOXD3, HOXB3/HOXB4, HOXA3, HOXA5, Chr1(q21.1).A, and Chr6(p22.1). In particular the OSR1, SIM1 and HOXB3/HOXB4 regions demonstrated high potential as biomarkers in LAC. For OSR1, hypermethylation was detected in 47/48 LAC cases compared to 1/31 tumor-adjacent normal lung samples. Similarly, 45/49 and 36/48 LAC cases compared to 3/31 and 0/31 tumor-adjacent normal lung samples showed hypermethylation of the SIM1 and HOXB3/HOXB4 regions, respectively. In conclusion, this study has identified and validated 15 DMRs that can be targeted as biomarkers in LAC.

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

miR-151a induces partial EMT by regulating E-cadherin in NSCLC cells

miR-151a and its host gene, focal adhesion kinase, FAK, are located in a region of chromosome 8q that is frequently amplified in solid tumors, including lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and metastasis remains the major challenge in battling lung cancer mortality. Here, we demonstrate that miR-151a is overexpressed in non-small cell lung cancer (NSCLC) patient specimens, as compared to healthy lung. In addition, miR-151a overexpression promotes proliferation, epithelial-to-mesenchymal transition (EMT) and induces tumor cell migration and invasion of NSCLC cells. Blocking miR-151a expression using anti-miR-151a approaches significantly reduced NCSLC cell proliferative and motility potential. Furthermore, we determined that miR-151a significantly regulates E-cadherin expression. Finally, functional rescue experiments determined that overexpression of E-cadherin in miR-151a NSCLC cell lines potently repressed miR-151a-induced partial EMT and cell migration of NSCLC cells. In conclusion, our findings suggest that miR-151a functions as an oncomiR in NSCLC by targeting E-cadherin mRNA and inducing proliferation, migration and partial EMT.

Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.

The association between miR-34 dysregulation and distant metastases formation in lung adenocarcinoma

Lung cancer has the highest mortality rate amongst human cancers and the majority of deaths can be attributed to metastatic spread. The miR-34 family includes three tumor suppressive miRs: miR-34a, miR-34b and miR-34c. miR-34 downregulation is a frequent observation in human malignancies and is often attributed to hypermethylation of the miR-34a and miR-34b/c promoters. Here, the potential association between aberrant miR-34 expression and promoter methylation and distant metastases formation in lung adenocarcinoma (LAC) is investigated. The expression levels of miR-34a, miR-34b and miR-34c, as well as the methylation status of the miR-34a and miR-34b/c promoters were determined in a LAC patient cohort comprising 26 non-metastasizing and 26 metastasizing primary LACs, as well as 24 paired distant metastases and 25 tumor-adjacent normal lung samples using RT-qPCR and Methylation-Sensitive High Resolution Melting (MS-HRM) analysis. No difference in expression was observed for miR-34a when comparing metastasizing and non-metastasizing LACs (p=0.793). For both miR-34b and miR-34c, a significantly lower expression level was determined in metastasizing LACs compared to non-metastasizing LACs (p=0.0005 and p=0.002) with similarly decreased expression levels observed in the paired distant metastases. Hypermethylation was detected in 35/51 LACs compared to 0/25 tumor-adjacent normal lungs for the miR-34a promoter (p<0.0001). Similarly, 18/51 LACs compared to 1/25 tumor-adjacent normal lungs showed hypermethylation of the miR-34b/c promoter (p=0.003). No difference in methylation was observed between metastasizing and non-metastasizing LACs for neither the miR-34a (p=0.832) nor the miR-34b/c (p=0.900) promoter. In conclusion, miR-34a and miR-34b/c promoter hypermethylation is a frequent event in LAC occurring in 68.7% and 35.3% of tested cases (n=51), respectively. Low miR-34b and miR-34c expression was associated with distant metastases formation in LAC. These changes can be targeted as novel biomarkers in LAC.

Non-coding AUG circRNAs constitute an abundant and conserved subclass of circles

Circular RNAs (circRNAs) are a subset of noncoding RNAs (ncRNAs) previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated, and therefore the vast majority of circRNAs are without known relevance. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, defined by spanning the canonical translational start site in the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide ORF, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling analyses and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the analyses suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements. Moreover, AUG circRNAs exhibit high cross-species conservation and are therefore likely to be functionally relevant.

Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies

Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3′-end of an exon to the 5′-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.Endogenous circular RNA (circRNA) expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations. Here, the authors provide a map of circRNA expression in B-cell malignancies based on high-throughput RNA sequencing and present an enzyme-free digital counting methodology, which has the potential to become the gold standard for circRNA quantification.

The agotrons: Gene regulators or Argonaute protectors?

Over the last decades, it has become evident that highly complex networks of regulators govern post-transcriptional regulation of gene expression. A novel class of Argonaute (Ago)-associated RNA molecules, the agotrons, was recently shown to function in a Drosha- and Dicer-independent manner, hence bypassing the maturation steps required for canonical microRNA (miRNA) biogenesis. Agotrons are found in most mammals and associate with Ago as ∼100 nucleotide (nt) long RNA species. Here, we speculate on the functional and biological relevance of agotrons: (i) agotrons could serve as non-promiscuous miRNA-like regulators with reduced off-targeting or (ii) agotrons could encompass other putative functions, such as protecting Ago proteins from taking up aberrant short RNAs or by rescuing and stabilizing otherwise unloaded Ago-proteins from degradation. Collectively, agotrons have emerged as a novel class of interesting non-coding RNA molecules, but their full functional potential and biological impact still remain to be disclosed.