Session 5.3 Levy

Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogene...

The enzyme Telomerase maintains telomeres at the ends of chromosomes. The Telomerase Reverse Transcriptase (TERT) gene codes for the enzyme’s catalytic domain and is not expressed in normal somatic cells. As a consequence, normal cells acquire senescence by shortening of their telomeres during cell division and eventually undergo apoptosis. In contrast to normal somatic cells, expression of TERT is reinstated in cancer cells causing escape from senescence and apoptosis by maintaining the telomeres. It has recently been shown that mutations in the TERT promoter region play a key role in regulating and reinstating TERT expression. Up to 90% of cancers carry a mutation in the TERT promoter region. Mutations like C228T and C250T create new binding sites for the E26 transformation-specific (ETS) transcription factor that regulates TERT expression (1,2). Experimental evidence showed that the ETS factor GA-binding protein, alpha subunit (GABPA) binds to the de novo ETS motif and activates TERT transcription in cancer cells.

TaqMan dPCR Liquid Biopsy Assays targeting the TERT promoter region

This year's 3rd Annual TCGC: The Clinical Genome Conference, held June 10-12, 2014 in San Francisco, is a three-day event that weaves together the science of sequencing and the business of implementing genomics in the clinic. It uniquely illustrates the mutual influence of those areas and the need to therefore consider the needs, challenges and opportunities of both - from next-generation sequencing and variant interpretation to insurance reimbursement and electronic health records - throughout the entire research process.Learn more at http://www.clinicalgenomeconference.com

CRISPR /Cas9

RatherIrfan

Clinical molecular diagnostics for drug guidance

Nikesh Shah

Production and purification of Viral vectors for gene and cell therapy appli...

Dr. Priyabrata Pattnaik

The Clinical Genome Conference 2014

Nicole Proulx

The tumor microenvironment (TME) is the intersection between tumor cells and surrounding non-transformed cells. It contains immune cells, signaling molecules, stromal and extracellular matrix. Research has shown the TME is often associated with tumor growth. However, the function and regulatory mechanism of each constituent is still poorly understood. The presence of PD-L1 is a promising marker to predict positive response for T cell checkpoint therapy. Current IHC methods to measure PD-L1 are subjective and highly variable. A higher-throughput and standardized method that can systematically measure gene expression of cells present in the TME has emerged to be a more desirable solution. We applied the OncomineTM Immune Response Research Assay to measure the expression of 395 genes in non-small cell lung cancer (NSCLC) research samples from 40 matched FFPE and fresh frozen sample types. This assay covers genes involved in checkpoint pathway, T cell regulation, cytokine and interferon signaling pathways, and markers of different tumor infiltrating lymphocyte (TIL) subsets, as well as tumor markers. With an input requirement of 10 ng of total RNA, libraries were generated, templated on the Ion ChefTM and sequenced on the Ion S5TM System. Sequencing data was analyzed and mapped with Torrent Suite Software and differential expression analysis was conducted with AffymetrixTM Transcriptome Analysis Console.

Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...

Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and Characterization

John Blue

mRNA-based Therapeutics - Creative Biolabs

Creative-Biolabs

Viral Based Gene Delivery System for Car-t Cell Engineering

Creative-Biolabs

Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...

Advanced prostate cancer is highly heterogeneous but this inter-patient heterogeneity has until recently not been understood. We have through an international research effort dissected the molecular landscape of advanced castration resistant prostate, elucidating key molecular targets in this group of diseases. We have also shown that PARP inhibitors have antitumor activity against a significant proportion of these cancers, mainly in men whose cancers harbor DNA repair defects.

Víctor M. González - Introducción a la tecnología de aptámeros y posibles apl...

ponenciasexpoquim11

Lepow Day Poster 2

David Dornblaser

Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2

Noninvasive detection of rare mutations in blood could allow tumor monitoring for research purposes. Research studies have suggested that cfDNA contains DNA from tumor cells with somatic mutations that could inform on tumor progression and therapeutic resistance. Here, we demonstrate a complete workflow from a single tube of blood through data analysis for research samples down to a 0.1% allelic frequency. The low abundance tumor mutations found in cfDNA requires sensitive and accurate mutation detection. We have developed two panels that utilize an amplificationbased assay that generates tagged DNA copies, which allows detection of low abundance tumor mutations found in cfDNA. The two panels allow multiplex interrogation of primary driver and resistance mutations specific to ctDNA from breast and colon cancer. The Oncomine™ Colon cfDNA panel targets 236 hotspots within 14 genes while the Oncomine Breast cfDNA panel covers 157 hotspot mutations in 10 genes. This workflow was validated from matched single blood tubes, Streck and K2EDTA. Additionally, the utility for cancer research was demonstrated with concordance studies using matched FFPE and plasma from oncology samples.

Genome Editing CRISPR-Cas9

Ek Han Tan

An NGS workflow to detect down to 0.1% allelic frequency in cfDNA

Single-cell microRNA expression profiling is a challenging workflow. From cell lysis, reverse transcription, preamplificatin to real-time PCR, every step involves technical pitfalls. Therefore it is critical to have a robust system that facilitates universal cDNA synthesis and universal amplification of all miRNAs in one workflow without introducing bias. Here we present a new poster – introducing a robust real-time PCR workflow and protocol for profiling miRNA expression from a single cell and how we analyze the single cells by using the free data analysis software.

miScript Single Cell Poster

Catherine Kibirige, Clinical Research Scientist at Imperial College London, discusses: • The development of PCR assays that detect down to 3 copies of HIV-1 DNA/RNA with >90% reliability. • A pilot study using archival cells (PBMCs) from HIV- infected men with <20 copies HIV-1 RNA/ml plasma. HIV- 1 RNA was detected significantly more frequently in cells from a high inflammation group (21/27; 78%) compared to a low inflammation group (7/27; 26%; p=.0003). • Using the assays to characterize the viral-inhibition assay (VIA) used in IAVI vaccine clinical trials to assess the impact of anti-HIV CD8 responses in the context of CD4 T-cell HIV transcription. Assessing HIV levels in archival samples to establish and correlate the HIV levels measured by these assays to previous assessments, thus enhancing the vaccine design efforts.

Lentiviral mediated CRISPR/Cas9

XinLu52

Developing Ultra-Sensitive PCR Protocols for HIV Vaccine Research

Kate Barlow

What's hot (20)

Applications of Single Cell Analysis

Targeted Single Cell Sequencing for Accurate Mutation Detection in Heterogene...

TaqMan dPCR Liquid Biopsy Assays targeting the TERT promoter region

CRISPR /Cas9

Clinical molecular diagnostics for drug guidance

Production and purification of Viral vectors for gene and cell therapy appli...

The Clinical Genome Conference 2014

Gene expression profile of the tumor microenvironment from 40 NSCLC FFPE and ...

Dr. Ben Hause - Metagenomic Sequencing for Virus Discovery and Characterization

mRNA-based Therapeutics - Creative Biolabs

Viral Based Gene Delivery System for Car-t Cell Engineering

Detection and Surveillance of Antibiotic Resistance Genes From Food and Ferti...

Víctor M. González - Introducción a la tecnología de aptámeros y posibles apl...

Lepow Day Poster 2

Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2

Genome Editing CRISPR-Cas9

An NGS workflow to detect down to 0.1% allelic frequency in cfDNA

miScript Single Cell Poster

Lentiviral mediated CRISPR/Cas9

Developing Ultra-Sensitive PCR Protocols for HIV Vaccine Research

Similar to Session 5.3 Levy

Recent studies in non-invasive biomarker research have demonstrated the potential of using cell-free nucleic acids isolated from blood plasma to serve as surrogates for solid tumors. Somatic mutations representing the tumors could be successfully detected from cell-free DNA (cfDNA) and cell-free RNA (cfRNA), providing new tumor assessment methods in addition to tissue biopsy. However, the low amount of circulating tumor fragments in the blood presents significant challenges for accurate variant detection with NGS assays. Moreover, utilization of both cfDNA and cfRNA requires methods capable of interrogating both types of analytes to maximize the utility of each blood sample.

An Efficient NGS Workflow for Liquid Biopsy Research Using a Comprehensive As...

With recent advances in next-generation sequencing (NGS) technologies, it is now possible to detect somatic mutations with allele frequencies in blood samples as low as 0.1% from circulating tumor DNA. A natural extension to this achievement is adding the ability to simultaneously detect copy number variants and gene fusions. A panel such as this addresses a full repertoire of variant classes found to be linked with certain tumors and would enable researchers additional to profile cancer samples more dynamically thus enriching current diagnostic tool sets. Here, we present progress on such an approach and apply current NGS technology to achieve our goals.

PCR, RT-PCR, FISH

tcha163

Avs molecular diagnostic techniques for detection of plant pathogens

AMOL SHITOLE

Development of a Breast and Lung Cancer Research Panel To Target Therapeutica...

Circulating cell free DNA is a potential tumor marker in a non-invasive blood test for the treatment and evaluation of cancer and recurrence monitoring. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing on the Ion Torrent™ platform is an optimal tool or mutation detection with very little sample input required. Here, we demonstrate a complete workflow from isolation through molecular characterization of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We have completed molecular characterization of circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples. Our results verify the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.

targeting

Fisiopatologia Bicocca

Chimeric Antigen Receptor (car) Technique-Creative Biolabs

Creative-Biolabs

Epi tect methylation qpcr arrays 2013

Elsa von Licy

IJAA

Dr. Amar Anandrao Telke

Discover Therapeutic Aptamers For Vegf165 And Egfr

Jessica Myers

NEW AGE ADC IN LUNG CANCER 2022.pptx

madurai

Circulating Cell Free DNA Targeted Sequencing Workflow | ESHG 2015 Poster PM1...

In recent years, advances in next-generation sequencing (NGS) technologies have enabled faster and cheaper methods for uncovering the genetic basis of disease. For cancer, NGS based screening for known tumour subtypes can inform diagnosis and allow the clinician to tailor a specific therapy based on testing outcome. Here we present the validation of one such NGS based kit approved for CE-IVD* use to screen for specific chromosomal translocations in non-small cell lung cancer (NSCLC) samples by targeting specific breakpoints in known fusion transcripts. The kit tested (Oncomine™ Solid Tumour Fusion Transcript Kit) included a single primer poolcontaining amplicon designs to simultaneously screen for over 75 specific rearrangements involving the receptor tyrosine kinase (RTK) genes ALK, RET and ROS1 as well as NTRK1. The panel was compatible with formalin-fixed paraffin-embedded (FFPE) lung tumour samples and achieved high sensitivity down to 10 ng of RNA input. In addition, amplicon assays designed at the 5’ and 3’ ends the RTK genes provide non-specific evidence that a translocation exists in a sample by comparing expression imbalance between the two ends. Validation testing was carried out at three external clinical laboratories (CLIA, CAP, INAB). In addition to positive and negative control samples, each site contributed FFPE lung tumour samples for which ALK fusion status was known prior to NGS library preparation carried out using the Ion AmpliSeq workflow. For site-specific samples (n=144, 16 samples per sequencing run), high concordance, sensitivity and specificity were measured at 97.2%, 90.5% and 98.4%, respectively.

High Sensitivity Detection of Tumor Gene Mutations-v3

Michael Powell

3targeting

Fisiopatologia Bicocca

Western Blotting Of Camkii Œí And T 287

Beth Salazar

ICAAC 2013: Resumen

PROANTIBIOTICOS

6. Molek tech.pptx [Repaired].pptx

ArdiansyahPrayitno2

Aptamers

Rakhi Gangil

EACR-P0ster-17

mastaneh zohri

Clinical Validation of an NGS-based (CE-IVD) Kit for Targeted Detection of Ge...

Blood screening to track tumor recurrence and resistance may improve treatment selection and monitoring. Virtually all tumors carry somatic DNA mutations, serving as biomarker in blood. Circulating cell-free DNA (cfDNA) is one source of tumor DNA in blood. Tumor DNA comes from different tumor clones, and its abundance in plasma can be very low at critical stages such as early recurrence or development of resistance. This enables interest in detecting mutation biomarkers at very low frequency from cfDNA. We present a research use only analysis workflow for detection of low frequency DNA variants. Our variant calling method enables sensitive and specific detection of somatic mutations to 0.1% frequency.

Computational Methods for detection of somatic mutations at 0.1% frequency fr...

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An Efficient NGS Workflow for Liquid Biopsy Research Using a Comprehensive As...

PCR, RT-PCR, FISH

Avs molecular diagnostic techniques for detection of plant pathogens

Development of a Breast and Lung Cancer Research Panel To Target Therapeutica...

targeting

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Epi tect methylation qpcr arrays 2013

IJAA

Discover Therapeutic Aptamers For Vegf165 And Egfr

NEW AGE ADC IN LUNG CANCER 2022.pptx

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High Sensitivity Detection of Tumor Gene Mutations-v3

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