BackgroundGene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material.MethodsWe describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples.ResultsReproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies.ConclusionThis methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4736-4) contains supplementary material, which is available to authorized users.
Currently the standard practice in tumor biomarker research still relies on invasive tumor biopsy from local sites following by molecular testing or NGS assay. However, this only provides a very limited characterization of tumor composition. Recent studies in non-invasive biomarker research have demonstrated the potential advantages of using cell-free nucleic acids isolated from blood plasma to study genetic heterogeneity of tumor population and dissect the complex cancer clonal architecture. However, it has been challenging to apply to practical research due to low sample yield and sensitivity of detection approaches. Moreover, presence of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each plasma sample and characterize the comprehensive spectrum of mutations including single nucleotide variants, gene amplifications, and structural variants. Recently developed Oncomine™ Pan-Cancer Cell-free Assay employs an amplification-based approach from Ion Torrent NGS technology and achieves exceptional sensitivity and specificity with input amount as little as 20 ng. It includes the most comprehensive genetic content to simultaneously interrogate both cfDNA and cfRNA. Multiple libraries were pooled together for templating on Ion Chef™ and sequenced on Ion GeneStudio S5 systems. In this study, a panel of cancer cell lines harboring multiple variant types were selected and cultured for extended time after apoptosis. We were able to extract cell-free nucleic acids that were released into the cell media and mimic the circulating DNA profile of liquid biopsy samples using in vitro model. Using Oncomine™ Pan-Cancer Cell-free Assay, we successfully detected all the expected variants in these cancer cell lines including gene amplification (MET, ERBB2, CDK4) and fusion variants (ALK fusion and MET exon skipping). Subsequently, we applied this assay to a set of longitudinal liquid biopsy samples collected from a human subject with NSCLC during the course of 15 months. The results showed that a well-known TP53 mutation R248Q was consistently detected in the longitudinal samples with varying allelic frequencies. Interestingly, additional gene amplifications including MET, CDK4 and FGFR3 were identified at late time points. Furthermore, these observations were confirmed by digital PCR and concordant with FISH analyses in solid tumor. Overall, this study demonstrates that Pan-Cancer assay provides a unique and complete NGS solution for comprehensive genetic mutation assessment using in vitro and in vivo liquid biopsy models. Citation Format: Ru Cao, Kris Lea, Madhu Jasti, Jeff Schageman, Khalid Hanif, Yanchun Li, Jian Gu, Varun Bagai, Priyanka Kshatriya, Harriet Wikman, Sonja Loges, Kelli Bramlett. Characterization of genetic mutation spectra and identification of gene amplification and fusion variants in cell-free nucleic acid from cultured cancer cell media and liquid biopsy specimens using Oncomine™ Pan-Cancer Cell-Free Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5123.
e23065 Background: Advances in non-invasive tumor biomarker research have shown that tumor cells release fragments of DNA called circulating tumor DNA (ctDNA) into peripheral blood. Somatic mutations representing the tumors could be successfully detected from isolated ctDNA, providing new potential for tumor sample assessment in addition to traditional tissue biopsy methods. However, the low amount of ctDNA in the blood, which can be less than 1% allelic frequency, presents significant challenges for reliable variant detection with NGS assays. Improvement of sequencing accuracy at low allelic frequency is a critical factor in the implementation of NGS in ctDNA liquid biopsy research. Methods: We demonstrate the technical feasibility for a sample-to-variant NGS workflow that utilizes a broad multi-gene panel to survey a comprehensive list of variants relevant to multiple tumor types for liquid biopsy research. The method includes novel library preparation and analysis reporting for Ion Torrent™ sequencing platforms. 20ng of input cell-free DNA was subjected to the library generation protocol. Prepared libraries were templated on Ion Chef™ and sequenced on Ion S5™. Results: We successfully optimized an NGS workflow that enables the simultaneous examination of more than 360 driver and resistance hotspot mutations in a single-pool assay panel, achieving high sensitivity and specificity with limit of detection at 0.1% allelic frequency. The targeted regions span genes and variants relevant to multiple tumor types for comprehensive variant detection across high-value content reviewed by industry experts and researchers. Sequencing on the Ion S5™ delivered > 95% on-target reads and uniform amplification across targeted regions with deep sequencing depth ( > 40,000x). The workflow is compatible with single or multiple pooled samples on Ion Torrent™ sequencing chips. Conclusions: We demonstrate the ability to accurately detect high-value variants implicated in multiple tumors at 0.1% allelic frequency on Ion Torrent™ NGS. (For Research Use Only. Not for use in diagnostic procedures.)
Introduction Osteosarcoma (OS) is the most common primary bone sarcoma that mainly occurs in children and adolescents. The existence of drug resistant cancer stem cells (CSCs) with progenitor properties is responsible for OS relapse and metastasis. Thus, development of specific therapies targeting OS-CSCs is necessary to increase the long-term survival rate. Although ascorbic acid (AA) has controversial history as anticancer agent, recently it has been re-evaluated revealing more cytotoxic effect to cancer than normal cells. The aim of the study was to analyse AA as potential therapeutic for selective targeting of OS-CSCs. Material and methods To establish primary tumour cultures, tumour samples were mechanically dissected and enzymatically digested. Sarcosphere assay was used to isolate OS-CSCs. The cytotoxic effect of AA was determined by MTT assay as well as relationship between cell concentration and AA. OS-CSCs were treated with different concentrations of AA (2.5-55 mg/ ml) during 72 hour. Concentrations of AA used for further experiments were 30 mg/ml and 40 mg/ml, respectively. Effect of AA on sarcosphere-forming ability was measured under low-attachment condition during 28 days. Cell death type was determined by Annexin V/PI staining using flow cytometry. Levels of GAPDH were determined by western blot while ROS were measured by DCFH-DA assay. Seahorse XF analyzer was used to measure glycolysis and oxidative phosphorylation. Results and discussions While AA did not have any effect on hMSCs, U2OS and Hek 293, respectively, AA efficiently induced dose-dependent viability reduction of OS-CSCs. Further, it can be concluded that IC 50 values of AA depend on the number of seeded OS-CSCs. AA successfully reduced sarcosphere formation on 6th day. High cytotoxicity of AA was further confirmed by Annexin V/PI staining. Prevalent death mode induced by AA was apoptotic since more than 70% of Annexin V-positive cells were detected. In addition, AA inhibited the activity of the key glycolytic enzyme GAPDH and induced ROS levels. Following the treatment with AA, extracellular acidification rate as a measure of glycolysis, was reduced significantly. Moreover, AA increased metabolic potential of OS-CSCs implying cells' ability to meet an energy demand via respiration and glycolysis. Conclusion Based on the obtained results, it can be concluded that AA selectively targets OS-CSCs. The death mechanism is based on the blockage of glycolytic cycle and increased intracellular levels of ROS. Introduction The female mammary gland is a very dynamic organ that undergoes continuous tissue remodelling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy increase the risk of breast cancer, the reasons are unclear. Clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy and that miR-424 (322)/503...
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