A comprehensive framework for analysis of microRNA sequencing data in metastatic colorectal cancer.

Although microRNAs (miRNAs) contribute to all hallmarks of cancer, miRNA dysregulation in metastasis remains poorly understood. The aim of this work was to reliably identify miRNAs associated with metastatic progression of colorectal cancer (CRC) using novel and previously published next-generation sequencing (NGS) datasets generated from 268 samples of primary (pCRC) and metastatic CRC (mCRC; liver, lung and peritoneal metastases) and tumor adjacent tissues. Differential expression analysis was performed using a meticulous bioinformatics pipeline, including only bona fide miRNAs, and utilizing miRNA-tailored quality control and processing. Five miRNAs were identified as up-regulated at multiple metastatic sites Mir-210_3p, Mir-191_5p, Mir-8-P1b_3p [mir-141-3p], Mir-1307_5p and Mir-155_5p. Several have previously been implicated in metastasis through involvement in epithelial-to-mesenchymal transition and hypoxia, while other identified miRNAs represent novel findings. The use of a publicly available pipeline facilitates reproducibility and allows new datasets to be added as they become available. The set of miRNAs identified here provides a reliable starting-point for further research into the role of miRNAs in metastatic progression.

MirGeneDB 2.1: toward a complete sampling of all major animal phyla.

We describe an update of MirGeneDB, the manually curated microRNA gene database. Adhering to uniform and consistent criteria for microRNA annotation and nomenclature, we substantially expanded MirGeneDB with 30 additional species representing previously missing metazoan phyla such as sponges, jellyfish, rotifers and flatworms. MirGeneDB 2.1 now consists of 75 species spanning over ∼800 million years of animal evolution, and contains a total number of 16 670 microRNAs from 1549 families. Over 6000 microRNAs were added in this update using ∼550 datasets with ∼7.5 billion sequencing reads. By adding new phylogenetically important species, especially those relevant for the study of whole genome duplication events, and through updating evolutionary nodes of origin for many families and genes, we were able to substantially refine our nomenclature system. All changes are traceable in the specifically developed MirGeneDB version tracker. The performance of read-pages is improved and microRNA expression matrices for all tissues and species are now also downloadable. Altogether, this update represents a significant step toward a complete sampling of all major metazoan phyla, and a widely needed foundation for comparative microRNA genomics and transcriptomics studies. MirGeneDB 2.1 is part of RNAcentral and Elixir Norway, publicly and freely available at http://www.mirgenedb.org/.

T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases.

BACKGROUND: Colorectal liver metastasis (CLM) is a leading cause of colorectal cancer mortality, and the response to immune checkpoint inhibition (ICI) in microsatellite-stable CRC has been disappointing. Administration of cytotoxic chemotherapy may cause increased density of tumor-infiltrating T cells, which has been associated with improved response to ICI. This study aimed to quantify and characterize T-cell infiltration in CLM using T-cell receptor (TCR) repertoire sequencing. Eighty-five resected CLMs from patients included in the Oslo CoMet study were subjected to TCR repertoire sequencing. Thirty-five and 15 patients had received neoadjuvant chemotherapy (NACT) within a short or long interval, respectively, prior to resection, while 35 patients had not been exposed to NACT. T-cell fractions were calculated, repertoire clonality was analyzed based on Hill evenness curves, and TCR sequence convergence was assessed using network analysis. RESULTS: Increased T-cell fractions (10.6% vs. 6.3%) were detected in CLMs exposed to NACT within a short interval prior to resection, while modestly increased clonality was observed in NACT-exposed tumors independently of the timing of NACT administration and surgery. While private clones made up >90% of detected clones, network connectivity analysis revealed that public clones contributed the majority of TCR sequence convergence. CONCLUSIONS: TCR repertoire sequencing can be used to quantify T-cell infiltration and clonality in clinical samples. This study provides evidence to support chemotherapy-driven T-cell clonal expansion in CLM in a clinical context.

MapToCleave: High-throughput profiling of microRNA biogenesis in living cells.

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

Neoadjuvant chemotherapy is associated with a transient increase of intratumoral T-cell density in microsatellite stable colorectal liver metastases.

Patients with colorectal liver metastases (CLM) commonly receive neoadjuvant chemotherapy (NACT) prior to surgical resection. NACT may induce immunogenic cell death with subsequent recruitment of T-cells to the tumor microenvironment, which could be exploited by immune checkpoint inhibition (ICI). In theory, this could expand the use of ICI to obtain responses also in microsatellite stable colorectal cancer, but evidence to suggest optimal treatment schedules are lacking. In this study, densities of total-, cytotoxic-, helper- and regulatory T-cells were quantified by immunohistochemistry in resected CLM from 92 patients included in the OSLO-COMET trial (NCT01516710). All but one patient had microsatellite stable tumors (91/92). Associations between T-cell densities and clinicopathological parameters were analyzed. Fluoropyrimidine-based NACT (in most cases with addition of oxaliplatin or irinotecan) was administered to 45 patients completed median 8 weeks prior to surgical resection. No overall association was found between NACT administration and intratumoral T-cell densities. However, within the NACT group, a short time interval (<9.5 weeks) between NACT completion and CLM resection was strongly associated with high intratumoral T-cell densities compared to the long-interval and no NACT groups (medians 491, 236, and 292 cells/mm2, respectively; P < .0001). The results from this study suggest that the observed increase in intratumoral T-cells after NACT administration may be transient. The significance of this finding should be further explored to ensure that optimal treatment schedules are chosen for studies combining cytotoxic chemotherapy and ICI.

MirGeneDB 2.0: the metazoan microRNA complement.

Small non-coding RNAs have gained substantial attention due to their roles in animal development and human disorders. Among them, microRNAs are special because individual gene sequences are conserved across the animal kingdom. In addition, unique and mechanistically well understood features can clearly distinguish bona fide miRNAs from the myriad other small RNAs generated by cells. However, making this distinction is not a common practice and, thus, not surprisingly, the heterogeneous quality of available miRNA complements has become a major concern in microRNA research. We addressed this by extensively expanding our curated microRNA gene database - MirGeneDB - to 45 organisms, encompassing a wide phylogenetic swath of animal evolution. By consistently annotating and naming 10,899 microRNA genes in these organisms, we show that previous microRNA annotations contained not only many false positives, but surprisingly lacked >2000 bona fide microRNAs. Indeed, curated microRNA complements of closely related organisms are very similar and can be used to reconstruct ancestral miRNA repertoires. MirGeneDB represents a robust platform for microRNA-based research, providing deeper and more significant insights into the biology and evolution of miRNAs as well as biomedical and biomarker research. MirGeneDB is publicly and freely available at http://mirgenedb.org/.

microRNAs as cancer biomarkers.

Since their discovery in 1993, microRNAs (miRNAs) have been identified as important gene regulators in many biological processes and as key molecular players in human disease, including cancer where they show specific pathogenic deregulation. Their remarkable chemical stability, the availability of very sensitive miRNA detection methods and the fact that miRNAs can be extracted from and detected in various kinds of clinically relevant samples, such as solid tissues, body fluids and secretions make them excellent candidate biomarkers. However, no miRNA has yet entered the level of practical clinical relevance. We present a brief background and some key aspects and challenges of miRNAs as cancer biomarkers, we discuss shortfalls and identify possible routes towards the use of miRNAs as reliable biomarkers for cancer.