Recent strategies for electrochemical sensing detection of miRNAs in lung cancer.

MicroRNAs (miRNAs) associated with lung cancer are diversifying. MiR-21, Let-7, and miR-141 are common diagnostic targets. Some new lung cancer miRNAs, such as miR-25, miR-145, and miR-126, have received increasing attention. Although various techniques are available for the analysis of lung cancer miRNAs, electrochemistry has been recognized for its high sensitivity, low cost, and rapid response. However, how to realize the signal amplification is one of the most important contents in the design of electrochemical biosensors. Herein, we mainly introduce the amplification strategy based on enzyme-free amplification and signal conversion, including non-linear HCR, catalytic hairpin assembly (CHA), electrochemiluminescence (ECL), and Faraday cage. Furthermore, new progress has emerged in the fields of nanomaterials, low oxidation potential, and simultaneous detection of multiple targets. Finally, we summarize some new challenges that electrochemical techniques may encounter in the future, such as improving single-base discrimination ability, shortening electrochemical detection time, and providing real body fluid samples assay.

Deep whole-genome ctDNA chronology of treatment-resistant prostate cancer.

Circulating tumour DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring1. However, owing to past emphasis on targeted and low-resolution profiling approaches, our understanding of the distinct populations that comprise bulk ctDNA is incomplete2-12. Here we perform deep whole-genome sequencing of serial plasma and synchronous metastases in patients with aggressive prostate cancer. We comprehensively assess all classes of genomic alterations and show that ctDNA contains multiple dominant populations, the evolutionary histories of which frequently indicate whole-genome doubling and shifts in mutational processes. Although tissue and ctDNA showed concordant clonally expanded cancer driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after clinical progression on potent inhibitors of the androgen receptor (AR) pathway, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally, we leverage nucleosome footprints in ctDNA to infer mRNA expression in synchronously biopsied metastases, including treatment-induced changes in AR transcription factor signalling activity. Our results provide insights into cancer biology and show that liquid biopsy can be used as a tool for comprehensive multi-omic discovery.

Systematic comparative analysis of strand-specific RNA-seq library preparation methods for low input samples.

Despite the recent precipitous decline in the cost of genome sequencing, library preparation for RNA-seq is still laborious and expensive for applications such as high throughput screening. Limited availability of RNA generated by some experimental workflows poses an additional challenge and increases the cost of RNA library preparation. In a search for low cost, automation-compatible RNA library preparation kits that maintain strand specificity and are amenable to low input RNA quantities, we systematically tested two recent commercial technologies-Swift RNA and Swift Rapid RNA, presently offered by Integrated DNA Technologies (IDT) -alongside the Illumina TruSeq stranded mRNA, the de facto standard workflow for bulk transcriptomics. We used the Universal Human Reference RNA (UHRR) (composed of equal quantities of total RNA from 10 human cancer cell lines) to benchmark gene expression in these kits, at input quantities ranging between 10 to 500 ng. We found normalized read counts between all treatment groups to be in high agreement. Compared to the Illumina TruSeq stranded mRNA kit, both Swift RNA library kits offer shorter workflow times enabled by their patented Adaptase technology. We also found the Swift RNA kit to produce the fewest number of differentially expressed genes and pathways directly attributable to input mRNA amount.

Epitachophoresis is a novel versatile total nucleic acid extraction method.

Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

Comparative study of bioinformatic tools for the identification of chimeric RNAs from RNA Sequencing.

Chimeric RNAs are gaining more and more attention as they have broad implications in both cancer and normal physiology. To date, over 40 chimeric RNA prediction methods have been developed to facilitate their identification from RNA sequencing data. However, a limited number of studies have been conducted to compare the performance of these tools; additionally, previous studies have become outdated as more software tools have been developed within the last three years. In this study, we benchmarked 16 chimeric RNA prediction software, including seven top performers in previous benchmarking studies, and nine that were recently developed. We used two simulated and two real RNA-Seq datasets, compared the 16 tools for their sensitivity, positive prediction value (PPV), F-measure, and also documented the computational requirements (time and memory). We noticed that none of the tools are inclusive, and their performance varies depending on the dataset and objects. To increase the detection of true positive events, we also evaluated the pair-wise combination of these methods to suggest the best combination for sensitivity and F-measure. In addition, we compared the performance of the tools for the identification of three classes (read-through, inter-chromosomal and intra-others) of chimeric RNAs. Finally, we performed TOPSIS analyses and ranked the weighted performance of the 16 tools.

MACE-Seq-based coding RNA and TrueQuant-based small RNA profile in breast cancer: tumor-suppressive miRNA-1275 identified as a novel marker.

INTRODUCTION: Disruption of cellular processes in the breast by abnormally expressed miRNA is characterized to develop cancer. We aimed to identify the differential expression of small RNAs (sRNAs) and mRNAs in formalin-fixed paraffin-embedded (FFPE) tissue of the breast cancer (BC) and normal adjacent tissue (NAT). Another aim is to determine the differential expression of miR-1275 as a novel biomarker for BC and also identify its target genes. METHODS: TrueQuant method for analysis of sRNA expression and MACE-sequencing method for analysis of gene expression were used analyzing. The RT-qPCR technique was used to confirm miR-1275 down expression. Target genes of miR-1275 were computationally identified using target prediction sites and also the expression level of them was experimentally determined among the expressed genes. RESULTS: TrueQuant findings showed that 1400 sRNAs were differentially expressed in the FFPE tissue of two Kurdish cases with BC, as compared to NAT. Among the sRNAs, 29 small RNAs were shown to be significantly downregulated in BC cells. The RT-qPCR results confirmed that miR-1275 was significantly down-expressed in 20 Kurdish cases with BC compared to NAT. However, Overall survival (OS) analysis revealed that the correlation between the expression level of miR-1275 and clinical significance was highly corrected in cases with BC (OS rate: P = 0.0401). The MACE-seq results revealed that 26,843 genes were differentially expressed in the BC tissue compared to NAT, but 7041 genes were displayed in a scatter plot. Furthermore, putative target genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were computationally identified as direct targets of miR-1275 in several target predicted sites. The MACE-seq results revealed that the expression level of these targets was increased in BC tissue compared to NAT. The level of these targets was negatively associated with miR-1275 expression. Finally, the role of down-regulated miR-1275 on its targets in biological mechanisms of BC cells was identified; including cell growth, proliferation, movement, invasion, metastasis, and apoptosis. CONCLUSION: Down-expressed miR-1275, a tumor suppressor, is a novel biomarker for early detection of BC. DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA are newly identified to be targeted by miR-1275.

A genomic-clinicopathologic Nomogram for the preoperative prediction of lymph node metastasis in gastric cancer.

BACKGROUND: Preoperative evaluation of lymph node (LN) state is of pivotal significance for informing therapeutic decisions in gastric cancer (GC) patients. However, there are no non-invasive methods that can be used to preoperatively identify such status. We aimed at developing a genomic biosignature based model to predict the possibility of LN metastasis in GC patients. METHODS: We used the RNA profile retrieving strategy and performed RNA expression profiling in a large GC cohort (GSE62254, n = 300) from Gene Expression Ominus (GEO). In the exploratory stage, 300 GC patients from GSE62254 were involved and the differentially expressed RNAs (DERs) for LN-status were determined using the R software. GC samples in GSE62254 were randomly allocated into a learning set (n = 210) and a verification set (n = 90). By using the Least absolute shrinkage and selection operator (LASSO) regression approach, a set of 23-RNA signatures were established and the signature based nomogram was subsequently built for distinguishing LN condition. The diagnostic efficiency, as well as the clinical performance of this model were assessed using the decision curve analysis (DCA). Metascape was used for bioinformatic analysis of the DERs. RESULTS: Based on the genomic signature, we established a nomogram that robustly distinguished LN status in the learning (AUC = 0.916, 95% CI 0.833-0.999) and verification sets (AUC = 0.775, 95% CI 0.647-0.903). DCA demonstrated the clinical value of this nomogram. Functional enrichment analysis of the DERs was performed using bioinformatics methods which revealed that these DERs were involved in several lymphangiogenesis-correlated cascades. CONCLUSIONS: In this study, we present a genomic signature based nomogram that integrates the 23-RNA biosignature based scores and Lauren classification. This model can be utilized to estimate the probability of LN metastasis with good performance in GC. The functional analysis of the DERs reveals the prospective biogenesis of LN metastasis in GC.

Liquid Biopsies in the Clinical Management of Germ Cell Tumor Patients: State-of-the-Art and Future Directions.

Liquid biopsies constitute a minimally invasive means of managing cancer patients, entailing early diagnosis, follow-up and prediction of response to therapy. Their use in the germ cell tumor field is invaluable since diagnostic tissue biopsies (which are invasive) are often not performed, and therefore only a presumptive diagnosis can be made, confirmed upon examination of the surgical specimen. Herein, we provide an overall review of the current liquid biopsy-based biomarkers of this disease, including the classical, routinely used serum tumor markers-the promising microRNAs rapidly approaching the introduction into clinical practice-but also cell-free DNA markers (including DNA methylation) and circulating tumor cells. Finally, and importantly, we also explore novel strategies and challenges for liquid biopsy markers and methodologies, providing a critical view of the future directions for liquid biopsy tests in this field, highlighting gaps and unanswered questions.

Interleukin-33 modulates immune responses in cutaneous melanoma in a context-specific way.

Controversial roles of interleukin-33 (IL-33) have been reported in melanoma from animal studies. We aimed to investigate the role of IL-33 in human cutaneous melanoma. RNA-seq data of 471 cases of cutaneous melanoma were retrieved from The Cancer Genome Atlas. The tumor microenvironment (TME) was deconstructed by the xCell algorithm using RNA-seq data. We evaluated the prognostic value of IL-33 and the relationship between IL-33 and immune components in TME. We also inferred the potential cellular sources of IL-33. All the analyses were conducted separately in three sub-cohorts, which are based on the biopsy sites of samples: primary melanoma; lymph node (LN) metastases; other metastases, including metastases to skin/soft tissue, or visceral sites. In the two metastasis sub-cohorts, IL-33 is associated with better prognosis and more active immune responses in the tumor. However, IL-33 is not a prognostic factor in the primary melanoma sub-cohort. Furthermore, we found that IL-33 is mainly derived from stromal cells in the metastasis sub-cohorts, and from epithelial cells/keratinocytes in the primary melanoma sub-cohort. These findings provide evidence for the context-specific anti-tumor effects of IL-33 in melanoma. And the distinct effects of IL-33 may be determined by the cellular sources of IL-33.

Specific Detection of Prostate Cancer Cells in Urine by RNA In Situ Hybridization.

PURPOSE: Noninvasive tests that can accurately detect prostate cancer are urgently needed for prostate cancer diagnosis, surveillance and prognosis. Exfoliated prostate cells captured in urine represent a promising resource for noninvasive detection of prostate cancer. We investigated performance of a novel cell-based urine test for detection of clinically significant prostate cancer. MATERIALS AND METHODS: We previously developed a multiplex RNA in situ hybridization assay targeting NKX3-1, PRAC1 and PCA3 that enables identification and quantification of malignant and benign prostate cells released into urine. We investigated application of the assay for prostate cancer detection in a cohort of 98 patients suspected of harboring prostate cancer. Urine was collected following digital rectal examination, and the sediment was isolated and evaluated by RNA in situ hybridization. Samples were scored based on cellular expression of RNA in situ hybridization targets. Cells of prostate origin were defined by positivity for NKX3-1 and/or PRAC1, and prostate cancer cells by positivity for PCA3. RESULTS: Prostate cells (NKX3-1/PRAC1+ cells) were detected in 69 samples, among which 20 were positive for PCA3 (ie positive for prostate cancer cells). Comparison of RNA in situ hybridization results with biopsy outcome and clinical variables revealed that positivity for cancer by RNA in situ hybridization significantly correlated with intermediate/high risk cancer (p=0.003), PSA density (p=0.022), significant disease (p <0.0001) and Gleason score (p=0.003). The test was 95% specific and 51% sensitive for detection of clinically significant prostate cancer. CONCLUSIONS: Identification of exfoliated prostate cancer cells in urine by RNA in situ hybridization provides a novel tool for highly specific and noninvasive detection of prostate cancer.

A novel method for detection of exfoliated prostate cancer cells in urine by RNA in situ hybridization.

BACKGROUND: In the current study, we explore the feasibility of detecting exfoliated prostate cancer cells in urine using an RNA in situ hybridization (RISH) assay. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved in post-digital rectal examination (DRE) urine samples using RISH. METHODS: We focused on method development, optimization, and analytical evaluation of RISH-based detection of prostate cancer in urine. We optimized a sample collection, processing, and target detection workflow for urine cytology specimens in conjunction with RNA target detection by RISH. We screened a panel of 11 prostate-specific RNA targets, and selected NKX3-1 and PRAC1 as markers for cells of prostate origin and PCA3 as a marker of prostate malignancy. Following analytical validation of a multiplexed NKX3-1/PRAC1/PCA3 assay, we evaluated whether prostate cancer cells can be detected in a pilot cohort of 19 post-DRE specimens obtained from men diagnosed with prostate cancer. RESULTS: Using cytology specimens prepared from spiked urine samples, we established the analytical validity of the RISH assay for detection and visualization of prostate cells in urine. Cells of prostate origin could be readily and specifically identified and separated into benign and malignant cell populations based on the multiplex test that consisted of markers specific for prostate cells (NKX3-1, PRAC1) and prostate cancer cells (PCA3). Upon evaluation of post-DRE urine from a pilot cohort of prostate cancer patients, we identified 11 samples in which prostate cells were present, 6 of which were also positive for prostate cancer cells. CONCLUSIONS: Multiplex RISH enables the direct visualization and molecular characterization of individual exfoliated prostate cells in urine. This proof-of-principle study provides evidence supporting the application of RISH as a potential noninvasive tool for prostate cancer detection.

Cross-Site Concordance Evaluation of Tumor DNA and RNA Sequencing Platforms for the CIMAC-CIDC Network.

PURPOSE: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study. EXPERIMENTAL DESIGN: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms. RESULTS: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed. CONCLUSIONS: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network.

Neoantigen prediction in human breast cancer using RNA sequencing data.

Neoantigens have attracted attention as biomarkers or therapeutic targets. However, accurate prediction of neoantigens is still challenging, especially in terms of its accuracy and cost. Variant detection using RNA sequencing (RNA-seq) data has been reported to be a low-accuracy but cost-effective tool, but the feasibility of RNA-seq data for neoantigen prediction has not been fully examined. In the present study, we used whole-exome sequencing (WES) and RNA-seq data of tumor and matched normal samples from six breast cancer patients to evaluate the utility of RNA-seq data instead of WES data in variant calling to detect neoantigen candidates. Somatic variants were called in three protocols using: (i) tumor and normal WES data (DNA method, Dm); (ii) tumor and normal RNA-seq data (RNA method, Rm); and (iii) combination of tumor RNA-seq and normal WES data (Combination method, Cm). We found that the Rm had both high false-positive and high false-negative rates because this method depended greatly on the expression status of normal transcripts. When we compared the results of Dm with those of Cm, only 14% of the neoantigen candidates detected in Dm were identified in Cm, but the majority of the missed candidates lacked coverage or variant allele reads in the tumor RNA. In contrast, about 70% of the neoepitope candidates with higher expression and rich mutant transcripts could be detected in Cm. Our results showed that Cm could be an efficient and a cost-effective approach to predict highly expressed neoantigens in tumor samples.

Differential Expression of miR-21, miR-23a, and miR-27a, and Their Diagnostic Significance in Egyptian Colorectal Cancer Patients.

Background: Colorectal cancer (CRC) rates are affected by genetics, ethnicity, and environmental factors; it is considered one of the most aggressive human malignancies with high mortality and morbidity rates worldwide due, in part, to its asymptomatic nature during the early stages of disease. Objective: Owing to the impact of microRNA (miRNA) dysregulation on CRC development and progression, this study was conducted to explore the expression levels of mir-21, -23a, and -27a in the sera and tissues of Egyptian CRC patients and to evaluate their diagnostic efficacy based on circulating levels. Methods: In the test phase, the relative expression levels of the studied miRNAs were evaluated in the sera of 70 participants (35 CRC patients and 35 healthy controls) using quantitative real-time-polymerase chain reaction and to verify their diagnostic value. The exploratory phase was designed to validate the tumor-derived trait by comparing the miRNA levels in the cancerous and adjacent noncancerous tissues. Results: The relative expression levels of the studied miRNAs were significantly upregulated in both serum and tumor tissues of the patients compared to their corresponding controls. In addition, significant positive correlations were found between the relative expression levels of the studied miRNAs in serum samples and their levels in the matched CRC tissues. The serum expression levels of mir-21 and -23a were more predictive of CRC than mir-27a. Conclusion: Circulating mir-21, -23a, and -27a expression levels appear to be valuable diagnostic biomarkers for CRC, especially when combined.

Tyrosine kinase inhibitor discontinuation in the management of chronic myeloid leukemia: a critical review of the current practice.

Introduction: Tyrosine kinase inhibitors (TKIs), which target BCR-ABL1 kinase activity, have significantly prolonged the overall survival of patients affected by chronic myeloid leukemia (CML) and changed drastically the outcome. Evidences from several studies suggest that in patients who have achieved a sustained, stable and deep molecular response, TKI treatment can be safely discontinued with a close subsequent monitoring. Thus, a stable deep molecular response (DMR) has become a feasible treatment goal in CML. Areas covered: In this review, the main findings extrapolated from sponsored and real-life evidences regarding TKI discontinuation were discussed, through a broad research on Medline, Embase and archives from EHA and ASH congresses (including words such as discontinuation, treatment-free remission, TFR, etc). Moreover, suggestions emerged from international guidelines about treatment-free remission (TFR) are presented. Expert opinion: With the growing availability of clinical trials and real-life data on TFR, in recent years the possibility of offering to CML patients a safe, informed and shorter path to TFR, through the achievement of a stable deep molecular response (DMR), has become an increasing option. However, many controversial aspects remain regarding treatment choices and timings, predictive factors, patient communication and optimal strategies aimed at achieving a successful TFR.

A single-cell survey of cellular hierarchy in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate integrative analysis on the hierarchy of different AML subtypes. METHODS: Using Microwell-seq, a high-throughput single-cell mRNA sequencing platform, we analyzed the cellular hierarchy of bone marrow samples from 40 patients and 3 healthy donors. We also used single-cell single-molecule real-time (SMRT) sequencing to investigate the clonal heterogeneity of AML cells. RESULTS: From the integrative analysis of 191727 AML cells, we established a single-cell AML landscape and identified an AML progenitor cell cluster with novel AML markers. Patients with ribosomal protein high progenitor cells had a low remission rate. We deduced two types of AML with diverse clinical outcomes. We traced mitochondrial mutations in the AML landscape by combining Microwell-seq with SMRT sequencing. We propose the existence of a phenotypic "cancer attractor" that might help to define a common phenotype for AML progenitor cells. Finally, we explored the potential drug targets by making comparisons between the AML landscape and the Human Cell Landscape. CONCLUSIONS: We identified a key AML progenitor cell cluster. A high ribosomal protein gene level indicates the poor prognosis. We deduced two types of AML and explored the potential drug targets. Our results suggest the existence of a cancer attractor.

Deep Sequencing of Small RNAs from Neurosurgical Extracellular Vesicles Substantiates miR-486-3p as a Circulating Biomarker that Distinguishes Glioblastoma from Lower-Grade Astrocytoma Patients.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM; astrocytoma grade IV) biology and are novel sources of biomarkers. EVs released from GBM tumors can cross the blood-brain-barrier into the periphery carrying GBM molecules, including small non-coding RNA (sncRNA). Biomarkers cargoed in circulating EVs have shown great promise for assessing the molecular state of brain tumors in situ. Neurosurgical aspirate fluids captured during tumor resections are a rich source of GBM-EVs isolated directly from tumor microenvironments. Using density gradient ultracentrifugation, EVs were purified from cavitron ultrasonic surgical aspirate (CUSA) washings from GBM (n = 12) and astrocytoma II-III (GII-III, n = 5) surgeries. The sncRNA contents of surgically captured EVs were profiled using the Illumina® NextSeqTM 500 NGS System. Differential expression analysis identified 27 miRNA and 10 piRNA species in GBM relative to GII-III CUSA-EVs. Resolved CUSA-EV sncRNAs could discriminate serum-EV sncRNA profiles from GBM and GII-III patients and healthy controls and 14 miRNAs (including miR-486-3p and miR-106b-3p) and cancer-associated piRNAs (piR_016658, _016659, _020829 and _204090) were also significantly expressed in serum-EVs. Circulating EV markers that correlate with histological, neuroradiographic and clinical parameters will provide objective measures of tumor activity and improve the accuracy of GBM tumor surveillance.