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Join Pillar Biosciences at
The Association for Molecular Pathology's Second Global Congress on Molecular Pathology

Learn about the latest advancements in custom NGS assay development and precision medicine for oncology and unmet diagnostic needs.

Poster P087 | Evaluation of a Targeted NGS Panel Using Single-Vial Amplification of Candidate Genes in Solid Tumors

Meet author Helen Fernandes on Friday, May 17, 10:00-10:45am

Devon H, Endre H, Freeman C, Hsiao S, Mansukhani M and Fernandes H
Laboratory of Personalized Genomic Medicine, Department of Pathology and Cell Biology, Columbia University Medical Center, New York.

Introduction Targeted Next Generation Sequencing (NGS) assays are largely used for tumor profiling and identification of biomarkers with clinical relevance. The use of such assays for detection of variants with therapeutic, diagnostic and prognostic potential is well established. Recent investigations have documented the association of variants in genes leading to DNA damage repair deficiency with response to immunotherapy. We customized a 52 gene targeted using single tube Stem-Loop Inhibition-Mediated Amplification (SLIMamp) technology (Pillar Biosciences) for detection of therapeutically informative variants.

Methods Thirty previously tested FFPE and cytology samples harboring 28 different clinically relevant variants present in 12 genes were included in the evaluation. Variants present were previously detected using either, TruSeq Amplicon Cancer Panel (Illumina) (N=18) or a 467 targeted Comprehensive Cancer Panel (CCP) (N=12). NGS libraries, using the customized multi-cancer panel were prepared with DNA input ranging from 10 – 80 ng. A biosynthetic multiplexed control containing multiple variants including SNVs and indels (Seracare) was used to assess the reproducibility of the assay. For each run, up to 16 samples were normalized, pooled and run on the MiSeq (Illumina). Data analysis including sequence alignment, variant calling and annotation was performed using FASTQ files, with the Pillar Variant Analysis Toolkit (PiVAT). FASTQ files were also analyzed on NextGENE for comparison.

Results All 30 samples were successfully sequenced. Overall, there was 100% concordance between the 47 variants identified using the SLIMamp technology and previously validated technologies. The alterations included missense variants (N=39), indels (N=6), two splice variants. The mutant allele fraction (MAF) percentage in the samples, ranged from 3% to 80%. Correlation of MAF obtained by the two different methodologies was excellent (R2=0.925). The on target percentage was >99% and average coverage obtained across the samples was greater than 2000X. The uniformity of coverage across all target regions was excellent.

Conclusions Interrogation of variants in solid tumors with a customized multi-gene panel, using SLIMamp technology can identify actionable alterations, including missense variants and indels in tumors with low input DNA. The panel can reliably identify tumor biomarkers that potentially respond to targeted therapy and immunotherapy. Targeted panels using SLIMamp technology have extended applications for cancer therapy.

Pillar booth 33

Stop by to meet with Pillar Biosciences CEO Gang Song along with the rest of the Pillar team to find out more about custom NGS assays and SLIMamp® enrichment chemistry.

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