Integrated circulating tumour DNA and cytokine analysis for therapy monitoring of ALK-rearranged lung adenocarcinoma.

BACKGROUND: Detection of circulating tumour DNA (ctDNA) in biological fluids is a minimally invasive alternative to tissue biopsy for therapy monitoring. Cytokines are released in the tumour microenvironment to influence inflammation and tumorigenic mechanisms. Here, we investigated the potential biomarker utility of circulating cytokines vis-à-vis ctDNA in ALK-rearranged+ lung adenocarcinoma (ALK + NSCLC) and explored the optimal combination of molecular parameters that could indicate disease progression. METHODS: Longitudinal serum samples (n = 296) were collected from ALK + NSCLC patients (n = 38) under tyrosine kinase inhibitor (TKI) therapy and assayed to quantify eight cytokines: IFN-γ, IL-1ß, IL-6, IL-8, IL-10, IL-12p70, MCP1 and TNF-α. Generalised linear mixed-effect modelling was performed to test the performance of different combinations of cytokines and previously determined ctDNA parameters in identifying progressive disease. RESULTS: Serum IL-6, IL-8 and IL-10 were elevated at progressive disease, with IL-8 having the most significant impact as a biomarker. Integrating changes in IL-8 with ctDNA parameters maximised the performance of the classifiers in identifying disease progression, but this did not significantly outperform the model based on ctDNA alone. CONCLUSIONS: Serum cytokine levels are potential disease progression markers in ALK + NSCLC. Further validation in a larger and prospective cohort is necessary to determine whether the addition of cytokine evaluation could improve current tumour monitoring modalities in the clinical setting.

DNA Promoter Methylation and ERG Regulate the Expression of CD24 in Prostate Cancer.

CD24 is overexpressed in many human cancers and is a driver of tumor progression. Herein, molecular mechanisms leading to up-regulation of CD24 in prostate cancer were studied. DNA methylation of the CD24 gene promoter at four loci using quantitative methylation-specific PCR was evaluated. Expression of CD24 in tumor tissues was studied by immunohistochemistry. To corroborate the results in vitro, ERG-inducible LNCaP TMPRSS2:ERG (T2E) cells and luciferase promoter assays were used. DNA methylation of the CD24 promoter was significantly higher in tumors than in benign tissue and was associated with biochemical recurrence-free survival, tumor grade, and stage. CD24 mRNA and protein expression were significantly higher in T2E-positive, ERG-overexpressing, and/or PTEN-deficient cases. Higher levels of CD24 protein expression conferred shorter biochemical recurrence-free survival, and these observations were confirmed using The Cancer Genome Atlas prostate adenocarcinoma data. In silico analysis of the CD24 promoter revealed an ERG binding site in between the DNA methylation sites. ERG overexpression led to a strong induction of CD24 mRNA and protein expression. Luciferase promoter assays using the wild-type and mutated ERG binding site within the CD24 promoter showed ERG-dependent activation. Collectively, our results suggest that promoter DNA methylation of the CD24 gene and T2E fusion status are factors involved in the up-regulation of CD24 in patients with prostate cancer.

KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

Experimental design and data analysis of Ago-RIP-Seq experiments for the identification of microRNA targets.

The identification of microRNA (miRNA) target genes is crucial for understanding miRNA function. Many methods for the genome-wide miRNA target identification have been developed in recent years; however, they have several limitations including the dependence on low-confident prediction programs and artificial miRNA manipulations. Ago-RNA immunoprecipitation combined with high-throughput sequencing (Ago-RIP-Seq) is a promising alternative. However, appropriate statistical data analysis algorithms taking into account the experimental design and the inherent noise of such experiments are largely lacking.Here, we investigate the experimental design for Ago-RIP-Seq and examine biostatistical methods to identify de novo miRNA target genes. Statistical approaches considered are either based on a negative binomial model fit to the read count data or applied to transformed data using a normal distribution-based generalized linear model. We compare them by a real data simulation study using plasmode data sets and evaluate the suitability of the approaches to detect true miRNA targets by sensitivity and false discovery rates. Our results suggest that simple approaches like linear regression models on (appropriately) transformed read count data are preferable.

Circulating MicroRNAs as Potential Biomarkers for Lung Cancer.

Lung cancer is the number one cause of cancer-related mortality worldwide. To improve disease outcome, it is crucial to implement biomarkers into the clinics which assist physicians in their decisions regarding diagnosis, prognosis, as well as prediction of treatment response. Liquid biopsy offers an opportunity to obtain such biomarkers in a minimal invasive manner by retrieving tumor-derived material from body fluids of the patient. The abundance of circulating microRNAs is known to be altered in disease and has therefore been studied extensively as a cancer biomarker. Circulating microRNAs present a variety of favorable characteristics for application as liquid biopsy-based biomarkers, including their high stability, relatively high abundance, and presence is nearly all body fluids. Although the application of circulating microRNAs for the management of lung cancer has not entered the clinics yet, several studies showed their utility for diagnosis, prognosis, and efficacy prediction of various treatment strategies, including surgery, radio-/chemotherapy, as well as targeted therapy. To compensate for their limited tumor specificity, several microRNAs are frequently combined into microRNA panels. Moreover, the possibility to combine single microRNAs or microRNA panels with tumor imaging or other cancer-specific biomarkers has the potential to increase specificity and sensitivity and could lead to the clinical application of novel multi-marker combinations.

Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study.

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN.

Global DNA methylation reflects spatial heterogeneity and molecular evolution of lung adenocarcinomas.

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases.

The BRCA2 mutation status shapes the immune phenotype of prostate cancer.

Defects in DNA damage repair caused by mutations in BRCA1/2, ATM or other genes have been shown to play an important role in the development and progression of prostate cancer. The influence of such mutations on anti-tumor immunity in prostate cancer, however, is largely unknown. To better understand the correlation between BRCA1/2 mutations and the immune phenotype in prostate cancer, we characterized the immune infiltrate of eight BRCA2-mutated tumors in comparison with eight BRCA1/2 wild-type patients by T-cell receptor sequencing and immunohistochemistry for CD45, CD4, CD8, FOXP3, and CD163. In addition, we analyzed seven prostate cancer biopsies that were either BRCA2 or ATM-mutated in comparison with wild-type tumors. Whereas in BRCA1/2 wild-type tumors, immune cells were found predominantly extratumorally, most BRCA2-mutated tumors including one biopsy showed a significantly increased intratumoral immune cell infiltration. The ratio of intratumoral to extratumoral immune cells was considerably higher in BRCA2-mutated tumors for all markers and reached statistical significance for CD4 (p = 0.007), CD8 (p = 0.006), and FOXP3 (p = 0.001). However, the intratumoral CD8 to FOXP3 ratio showed a trend to be lower in BRCA2-mutated tumors suggesting a more suppressed tumor immune microenvironment. Our findings provide a rationale for the future use of immune oncological approaches in BRCA2-mutated prostate cancer and may encourage efforts to target immunosuppressive T-cell populations to prime tumors for immunotherapy.

QSEA-modelling of genome-wide DNA methylation from sequencing enrichment experiments.

Genome-wide enrichment of methylated DNA followed by sequencing (MeDIP-seq) offers a reasonable compromise between experimental costs and genomic coverage. However, the computational analysis of these experiments is complex, and quantification of the enrichment signals in terms of absolute levels of methylation requires specific transformation. In this work, we present QSEA, Quantitative Sequence Enrichment Analysis, a comprehensive workflow for the modelling and subsequent quantification of MeDIP-seq data. As the central part of the workflow we have developed a Bayesian statistical model that transforms the enrichment read counts to absolute levels of methylation and, thus, enhances interpretability and facilitates comparison with other methylation assays. We suggest several calibration strategies for the critical parameters of the model, either using additional data or fairly general assumptions. By comparing the results with bisulfite sequencing (BS) validation data, we show the improvement of QSEA over existing methods. Additionally, we generated a clinically relevant benchmark data set consisting of methylation enrichment experiments (MeDIP-seq), BS-based validation experiments (Methyl-seq) as well as gene expression experiments (RNA-seq) derived from non-small cell lung cancer patients, and show that the workflow retrieves well-known lung tumour methylation markers that are causative for gene expression changes, demonstrating the applicability of QSEA for clinical studies. QSEA is implemented in R and available from the Bioconductor repository 3.4 (www.bioconductor.org/packages/qsea).

A field guide for cancer diagnostics using cell-free DNA: From principles to practice and clinical applications.

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

Hepatitis D virus replication is sensed by MDA5 and induces IFN-ß/λ responses in hepatocytes.

BACKGROUND & AIMS: Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication. METHODS: HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs. RESULTS: Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2NTCP cells. HDV strongly activated IFN-ß and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I (DDX58) or TLR3 knock-down, but were almost completely abolished upon MDA5 (IFIH1) depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in HuH7.5NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2NTCP and HepaRGNTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication. CONCLUSION: Active replication of HDV induces an IFN-ß/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN-activated state. LAY SUMMARY: In contrast to hepatitis B virus, infection with hepatitis D virus induces a strong IFN-ß/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of hepatitis D virus replicative intermediates. An IFN-activated state did not prevent hepatitis D virus replication in vitro, indicating that hepatitis D virus is resistant to self-induced innate immune responses and therapeutic IFN treatment.

Spatial distribution of EGFR and KRAS mutation frequencies correlates with histological growth patterns of lung adenocarcinomas.

Multiregional analysis provided first indications for morphological and molecular heterogeneity in lung adenocarcinomas (ADCs), but comprehensive morpho-molecular comparisons are still lacking. The purpose of our study was to investigate the spatial distribution of EGFR and KRAS alterations systematically throughout whole tumor cross-sections in correlation with the tumor cell content and the histopathological patterns. Central sections of 19 ADCs were subdivided into 467 segments of 5 mm × 5 mm. We determined the predominant histological growth pattern and the allele frequencies of driver gene mutations by digital PCR in every segment. We further quantified the absolute cell counts and proportions of tumor and non-neoplastic cells in all segments to normalize the mutant allele frequencies. Driver gene mutations could be detected in >99% of the tumor containing segments, with high levels of inter- and intratumor heterogeneity regarding the mutant allele frequency (range: 0.04-19.36). Different patterns for the distribution of the variant allele frequency within a tumor were recognizable. While some cases showed ubiquitously low or high levels, others revealed regions with focally elevated frequencies. Differences between KRAS and EGFR alterations were not significant. The great majority of the analyzed tumor sections (16/19) exhibited two or more morphological growth patterns. Mutant allele frequencies were significantly higher in segments with a predominant solid pattern compared to all other histologies (p < 0.01). Our data indicate that driver gene mutations are present with high levels of inter- and intratumor heterogeneity throughout the whole tumor, with a correlation between the allele frequencies and histological growth patterns.

Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.

Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS: Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS: To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS: Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS: Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

microRNA biomarkers in body fluids of prostate cancer patients.

The abundance of miRNAs - small non-coding RNAs involved in posttranscriptional regulation of gene expression - in tissues and body fluids of cancer patients hold great promise to identify specific biomarkers, which may be useful for early diagnosis as well as to predict the clinical outcome and treatment response. For the extraction and quantification of miRNAs from cells and tissues, present technologies for transcriptome analyses like microarrays, quantitative real-time PCR or next generation sequencing can be applied. However, the analyses of miRNAs in body fluids like serum or urine is still a challenge with respect to the nucleic acid recovery from very limited sources of biomaterial, normalization strategies and validation using independent technologies. The presence of specific miRNA patterns in body fluids like serum of cancer patients suggests a promising role of these molecules as surrogate markers. However, the majority of miRNA studies were addressed in relatively small patient cohorts limiting the validity and the clinical application of potential miRNA biomarkers or signatures. We reflect the critical steps to translate miRNA biomarker into clinical routine diagnostics and present future aspects for the fast, robust and standardized quantification of miRNAs in body fluids.

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs.

RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs generated from SARS-CoV-1 and SARS-CoV-2 genomic RNAs remained largely unexplored, possibly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA sequencing datasets, we observed that human Calu-3 cells infected by SARS-CoV-1 or SARS-CoV-2 accumulate multiple previously unreported short viral RNAs. In addition, we verified the presence of the five most abundant SARS-CoV-2 short viral RNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by quantitative PCR. Interestingly, the copy number of the observed SARS-CoV-2 short viral RNAs dramatically exceeded the expression of previously reported viral microRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 short viral RNAs could serve as biomarkers for early infection stages due to their high abundance. Furthermore, unlike SARS-CoV-1, the SARS-CoV-2 infection induced significant (Benjamini-Hochberg-corrected p-value <0.05) deregulation of Y-RNA, transfer RNA, vault RNA, as well as more than 300 endogenous short RNAs that aligned predominantly to human protein-coding and long noncoding RNA transcripts. In particular, more than 20-fold upregulation of reads derived from Y-RNA (and several transfer RNAs) have been documented in RNA-seq datasets from SARS-CoV-2 infected cells. Finally, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various human genome (hg38) sequences, suggesting opportunities to investigate regulatory roles of short viral RNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.

miR-147b mediated suppression of DUSP8 promotes lung cancer progression.

Dual-specificity phosphatase 8 (DUSP8) plays an important role as a selective c-Jun N-terminal kinase (JNK) phosphatase in mitogen-activated protein kinase (MAPK) signaling. In this study, we found that DUSP8 is silenced by miR-147b in patients with lung adenocarcinoma (LUAD), which correlates with poor overall survival. Overexpression of DUSP8 resulted in a tumor-suppressive phenotype in vitro and in vivo experimental models, whereas silencing DUSP8 with a siRNA approach abrogated the tumor-suppressive properties. We found that miR-147b is a posttranscriptional regulator of DUSP8 that is highly expressed in patients with LUAD and is associated with lower survival. NanoString analysis revealed that the MAPK signaling pathway is mainly affected by overexpression of miR-147b, leading to increased proliferation and migration and decreased apoptosis in vitro. Moreover, induction of miR-147b promotes tumor progression in vitro and in vivo experimental models. Knockdown of miR-147b restored DUSP8, decreased tumor progression in vitro, and increased apoptosis via JNK phosphorylation. These results suggest that miR-147b plays a key role in regulating MAPK signaling in LUAD. The link between DUSP8 and miR-147b may provide novel approaches for the treatment of lung cancer.

Genome-wide DNA methylation-analysis of blastic plasmacytoid dendritic cell neoplasm identifies distinct molecular features.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) constitutes a rare and aggressive malignancy originating from plasmacytoid dendritic cells (pDCs) with a primarily cutaneous tropism followed by dissemination to the bone marrow and other organs. We conducted a genome-wide analysis of the tumor methylome in an extended cohort of 45 BPDCN patients supplemented by WES and RNA-seq as well as ATAC-seq on selected cases. We determined the BPDCN DNA methylation profile and observed a dramatic loss of DNA methylation during malignant transformation from early and mature DCs towards BPDCN. DNA methylation profiles further differentiate between BPDCN, AML, CMML, and T-ALL exhibiting the most striking global demethylation, mitotic stress, and merely localized DNA hypermethylation in BPDCN resulting in pronounced inactivation of tumor suppressor genes by comparison. DNA methylation-based analysis of the tumor microenvironment by MethylCIBERSORT yielded two, prognostically relevant clusters (IC1 and IC2) with specific cellular composition and mutational spectra. Further, the transcriptional subgroups of BPDCN (C1 and C2) differ by DNA methylation signatures in interleukin/inflammatory signaling genes but also by higher transcription factor activity of JAK-STAT and NFkB signaling in C2 in contrast to an EZH2 dependence in C1-BPDCN. Our integrative characterization of BPDCN offers novel molecular insights and potential diagnostic applications.

A Cancer-Indicative microRNA Pattern in Normal Prostate Tissue.

We analyzed the levels of selected micro-RNAs in normal prostate tissue to assess their potential to indicate tumor foci elsewhere in the prostate. Histologically normal prostate tissue samples from 31 prostate cancer patients and two cancer negative control groups with either unsuspicious or elevated prostate specific antigen (PSA) levels (14 and 17 individuals, respectively) were analyzed. Based on the expression analysis of 157 microRNAs in a pool of prostate tissue samples and information from data bases/literature, we selected eight microRNAs for quantification by real-time polymerase chain reactions (RT-PCRs). Selected miRNAs were analyzed in histologically tumor-free biopsy samples from patients and healthy controls. We identified seven microRNAs (miR-124a, miR-146a & b, miR-185, miR-16 and let-7a & b), which displayed significant differential expression in normal prostate tissue from men with prostate cancer compared to both cancer negative control groups. Four microRNAs (miR-185, miR-16 and let-7a and let-7b) remained to significantly discriminate normal tissues from prostate cancer patients from those of the cancer negative control group with elevated PSA levels. The transcript levels of these microRNAs were highly indicative for the presence of cancer in the prostates, independently of the PSA level. Our results suggest a microRNA-pattern in histologically normal prostate tissue, indicating prostate cancer elsewhere in the organ.