Lorenzo F. Sempere

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that less than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database--MirGeneDB ( http://mirgenedb.org )--to catalog this set of miRNAs, which complements the efforts of miRBase but differs from it by annotating the mature versus star products and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

Critical analysis of the potential for microRNA biomarkers in breast cancer management

Breast cancer is a complex and heterogeneous disease. Signaling by estrogen receptor (ER), progesterone receptor (PR), and/or human EGF-like receptor 2 (HER2) is a main driver in the development and progression of a large majority of breast tumors. Molecular characterization of primary tumors has identified major subtypes that correlate with ER/PR/HER2 status, and also subgroup divisions that indicate other molecular and cellular features of the tumors. While some of these research findings have been incorporated into clinical practice, several challenges remain to improve breast cancer management and patient survival, for which the integration of novel biomarkers into current practice should be beneficial. microRNAs (miRNAs) are a class of short non-coding regulatory RNAs with an etiological contribution to breast carcinogenesis. miRNA-based diagnostic and therapeutic applications are rapidly emerging as novel potential approaches to manage and treat breast cancer. Rapid technological development enables specific and sensitive detection of individual miRNAs or the entire miRNome in tissues, blood, and other biological specimens from breast cancer patients. This review focuses on recent miRNA research and its potential to address unmet clinical needs and challenges. The four sections presented discuss miRNA findings in the context of the following clinical challenges: biomarkers for early detection; prognostic and predictive biomarkers for treatment decisions using targeted therapies against ER and HER2; diagnostic and prognostic biomarkers for subgrouping of triple-negative breast cancer, for which there are currently no targeted therapies; and biomarkers for monitoring and characterization of metastatic breast cancer. The review concludes with a critical analysis of the current state of miRNA breast cancer research and the need for further studies using large patient cohorts under well-controlled conditions before considering the clinical implementation of miRNA biomarkers.

Stromal Expression of miR-21 Identifies High-Risk Group in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. Expression of miR-21, an oncomiR, is frequently altered and may be distinctly expressed in the tumor stroma. Because tumor lesions are a complex mixture of cell types, we hypothesized that analysis of miR-21 expression at single-cell resolution could provide more accurate information to assess disease recurrence risk and BC-related death. We implemented a fully automated, tissue slide-based assay to detect miR-21 expression in 988 patients with BC. The miR-21(High) group exhibited shorter recurrence-free survival [hazard ratio (HR), 1.71; P < 0.001] and BC-specific survival (HR, 1.96; P < 0.001) in multivariate regression analyses. When tumor compartment and levels of miR-21 expression were considered, significant associations with poor clinical outcome were detected exclusively in tumor epithelia from estrogen receptor- and/or progesterone receptor-positive human epidermal growth factor receptor 2-negative cases [recurrence-free survival: HR, 3.67 (P = 0.006); BC-specific survival: HR, 5.13 (P = 0.002)] and in tumor stroma from TNBC cases [recurrence-free survival: HR, 2.59 (P = 0.013); BC-specific survival: HR, 3.37 (P = 0.003)]. These findings suggest that the context of altered miR-21 expression provides clinically relevant information. Importantly, miR-21 expression was predominantly up-regulated and potentially prognostic in the tumor stroma of TNBC.

Expression of tumor suppressive microRNA-34a is associated with a reduced risk of bladder cancer recurrence.

Bladder cancer is the fourth most common cancer among men in the United States and more than half of patients experience recurrences within 5 years after initial diagnosis. Additional clinically informative and actionable biomarkers of the recurrent bladder cancer phenotypes are needed to improve screening and molecular therapeutic approaches for recurrence prevention. MicroRNA‐34a (miR‐34a) is a short noncoding regulatory RNA with tumor suppressive attributes. We leveraged our unique, large, population‐based prognostic study of bladder cancer in New Hampshire, United States to evaluate miR‐34a expression levels in individual tumor cells to assess prognostic value. We collected detailed exposure and medical history data, as well as tumor tissue specimens from bladder patients and followed them long‐term for recurrence, progression and survival. Fluorescence‐based in situ hybridization assays were performed on urothelial carcinoma tissue specimens (n = 229). A larger proportion of the nonmuscle invasive tumors had high levels of miR‐34a within the carcinoma cells compared to those tumors that were muscle invasive. Patients with high miR‐34a levels in their baseline nonmuscle invasive tumors experienced lower risks of recurrence (adjusted hazard ratio 0.57, 95% confidence interval 0.34–0.93). Consistent with these observations, we demonstrated a functional tumor suppressive role for miR‐34a in cultured urothelial cells, including reduced matrigel invasion and growth in soft agar. Our results highlight the need for further clinical studies of miR‐34a as a guide for recurrence screening and as a possible candidate therapeutic target in the bladder.

Tissue slide-based microRNA characterization of tumors: how detailed could diagnosis become for cancer medicine?

miRNAs are short, non-coding, regulatory RNAs that exert cell type-dependent, context-dependent, transcriptome-wide gene expression control under physiological and pathological conditions. Tissue slide-based assays provide qualitative (tumor compartment) and semi-quantitative (expression levels) information about altered miRNA expression at single-cell resolution in clinical tumor specimens. Reviewed here are key technological advances in the last 5 years that have led to implementation of fully automated, robust and reproducible tissue slide-based assays for in situ miRNA detection on US FDA-approved instruments; recent tissue slide-based discovery studies that suggest potential clinical applications of specific miRNAs in cancer medicine are highlighted; and the challenges in bringing tissue slide-based miRNA assays into the clinic are discussed, including clinical validation, biomarker performance, biomarker space and integration with other biomarkers.

Fully Automated Fluorescence-Based Four-Color Multiplex Assay for Co-detection of MicroRNA and Protein Biomarkers in Clinical Tissue Specimens

The application of locked nucleic acid chemistry for microRNA detection by in situ hybridization, and thereby visualization of microRNA expression at single-cell resolution, has contributed to our understanding of the roles that these short noncoding regulatory RNAs play during development, physiology, and disease. Several groups have implemented chromogenic-based and fluorescence-based protocols to detect microRNA expression in formalin-fixed paraffin-embedded clinical tissue specimens. These emerging robust and reproducible tissue slide-based assays are valid tools to bring about the clinical application of in situ microRNA detection for routine diagnostics. Here, I describe a fully automated fluorescence-based four-color multiplex assay for co-detection of a microRNA (e.g., let-7a, miR-10b, miR-21, miR-34a, miR-126, miR-145, miR-155, miR-205, miR-210), reference RNA (e.g., U6 snRNA, 18S rRNA), and protein markers (e.g., CD11b, CD20, CD45, collagen I, cytokeratin 7, cytokeratin 19, smooth muscle actin, tubulin, vimentin) in FDA-approved Leica Bond-MAX staining station.

Segment and Fit Thresholding: A New Method for Image Analysis Applied to Microarray and Immunofluorescence Data

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsus method for selected images. SFT promises to advance the goal of full automation in image analysis.

Individual Noncoding RNA Variations: Their Role in Shaping and Maintaining the Epigenetic Landscape

While a small portion of the human genome is composed of protein-coding genes, a far greater portion is now known to be transcribed into noncoding RNAs (ncRNAs). Noncoding RNAs have many regulatory functions, including those of shaping and maintaining the epigenetic landscape. MicroRNAs, trinucleotide repeats, and long ncRNAs are three classes of ncRNAs involved in the regulation of epigenetic processes whose expression can also be regulated by epigenetic changes. Disruption of these regulatory interactions contributes to the etiology of human diseases. This chapter focuses on the interactions and regulatory networks involving these ncRNA classes and the epigenetic machinery that shape and remodel the epigenetic landscape, which in turn dictates transcriptional programs that can affect ncRNA expression. We provide examples of their close interplay and reciprocal regulatory feedback loops with local and global effects on transcriptional programs. We discuss the clinical implications of genetic, epigenetic, and expression variations of these ncRNAs for inherited, congenital, and sporadic neurodegenerative conditions as well as for complex somatic diseases including cancers of the breast, bladder, and lung. Variations of these ncRNA classes can be exploited as biomarkers for risk assessment and prognosis. Thus, ncRNA biomarkers could inform personalized therapies throughout the course of a variety of human diseases.

MIR21 is differentially expressed in the lymphoid tissue and modulated by stromal signalling in chronic lymphocytic leukaemia

MicroRNAs contribute to the initiation, progression, and dissemination of malignant clone and serve as important prognostic indicators in chronic lymphocytic leukaemia (CLL) (Calin & Croce, 2009). MIR21 is an oncogenic microRNA; its overexpression in peripheral blood (PB) CLL cells was linked to fludarabine resistance and unfavourable prognosis (Ferracin et al, 2010; Rossi et al, 2010; Ferrajoli et al, 2013). MIR21 promotes survival by blocking expression of apoptosis-related genes, for example via targeting phosphatase and tensin homolog (PTEN) and activation of phosphoinositide-3 kinase (Calin & Croce, 2009). We and others have shown that B-cell receptor (BCR) engagement may upregulate MIR21 in CLL (Humphries et al, 2013; Pede et al, 2013). While recent literature addresses the impact of MIR21 in the malignant B cells circulating in the periphery, few studies have focused on its expression in CLL lymph nodes, where the BCR is most active (Herishanu et al, 2011). The lymphoid tissue constitutes a vital niche where CLL cells receive pro-survival stromal signals and express additional anti-apoptotic BCL2 family members absent in the peripheral cells. In those tissues, some microRNAs demonstrate distinct expression patterns. For example, high MIR155HG (BIC) expression (the primary transcript of MIR155) was found in CLL proliferation centres using in situ hybridization (ISH). However, this technique failed to detect microRNAs expressed at lower levels, including MIR21 (Wang et al, 2008). Here we investigated MIR21 expression in the lymphoid tissue of patients with CLL. MIR21 expression was characterized at single cell resolution in 30 formalin-fixed paraffin-embedded (FFPE) lymph nodes from patients with CLL and four control (tonsil) tissues. We employed a modified combined fluorescence-based ISH (FISH)/immunohistochemistry (IHC) assay as previously reported (Sempere et al, 2010). This assay enabled us to co-detect expression of MIR21 and RNU6-1 RNAs as well as CD20 protein (also termed MS4A1) in FFPE tissues. Briefly, 50 and 30-terminally tagged locked nucleic acid-modified DNA probes against MIR21 (2x-fluorescein tag) and RNU6-1 (2x-biotin tag), both at 50 nmol/l, were hybridized to tissue slides for 75 min at 45°C. Sequential rounds of horseradish peroxidase (HRP)-mediated deposition for 20 min with fluoresceinand rodhamine-conjugated substrates were performed to detect MIR21 and RNU6-1 probes, respectively. A final round of HRP-mediated deposition for 20 min with Dylight594-conjugated substrate was performed to detect CD20. MIR21 was differentially expressed in the lymphoid tissues of patients with CLL (Fig 1A). Of the 30 lymph nodes, MIR21 was detected in 13 (43%); of these, three lymph nodes demonstrated strong staining and 10 showed weak staining. The remaining 17 tissue specimens (56%) stained negative for MIR21 despite RNU6-1 staining. Where present, strong MIR21 expression followed a focal staining pattern. Co-staining for CD20 confirmed MIR21 detection in the neoplastic CLL cells (Fig 1B). In this small cohort, MIR21 positivity did not predict time to first treatment or correlate with CD38 or ZAP70 expression. Interestingly, two of the four control tissues demonstrated strong staining for MIR21, indicating that its expression in the lymphoid organs is not restricted to clonal neoplastic B-cells. Stromal signalling may modulate microRNA expression in CLL. Lymph nodes and bone marrow are the sites of CLL cell proliferation, with expression of gene signatures indicating activation of the BCR and nuclear factor-jB (NF-jB) pathways (Herishanu et al, 2011). We have previously shown that BCR signalling induces MIR21 expression in a subset of CLL, with 5/10 tested samples demonstrating an increase in this microRNA (Humphries et al, 2013). Given the variability of MIR21 expression in primary lymphoid tissues, we then studied if MIR21 was further modulated by stromal signalling in vitro. First, MIR21 expression levels were evaluated in PB. PB CLL cells (n = 33; 90 9% of patients previously untreated) and normal B-cells (from healthy volunteers, n = 7) were processed as described earlier (Humphries et al, 2013). While it was previously reported that RNU6-1, a commonly used normalizer, is differentially expressed between the normal and neoplastic B-cells (Ferrajoli et al, 2013), we did not detect a significant difference in the cycle thresholds between these two groups. Consistent with previous reports, we found increased levels of MIR21 in PB CLL cells compared with normal B-cells, although both groups demonstrated extensive variability in expression of this microRNA (Fig 2A). Similar results were obtained using GAPDH as a normalizer. Subsequently, we modelled lymph node microenvironment in vitro using CD40LG-expressing L cells or control L cells. Stromal co-cultures protect CLL cells from spontaneous apoptosis, while CD40LG-expressing stroma further Correspondence