Abstract 679: Orthogonal confirmation using the Pacbio®two step universal adapter amplicon protocol to validate a comprehensive genomic profiling assay for hematologic malignancies
Authors: Grant Hogg, Kimberly A. Holden, John Howitt, Adib Shafi, Amanda Willliamson, Tong Liu, Helen Cao, Xiaojun Guan, Michael Mooney, Qian Zeng, Stan Letovsky, Li Cai, Eric A. Severson, Shakti Ramkissoon, Anjen Chenn, Marcia Eisenberg, Eyad Almasri, Taylor J. Jensen, Jonathan Williams
Journal: Cancer Research (2025)

Abstract Next generation sequencing (NGS) using targeted panels is standard of care to aid diagnosis, prognosis and treatment selection for hematologic diseases such as acute myeloid leukemia. We have validated the Labcorp Pan-Heme NGS (LPH) panel that targets 141 clinically relevant genes to detect single nucleotide variants (SNVs), insertions/deletions (indels), FLT3 internal tandem duplicates (FLT3-ITDs) and copy number aberrations (CNAs) in myeloid and lymphoid malignancies. In total, 677 samples were assessed including 315 clinical samples. Analytical sensitivity and specificity experiments estimated that the limit of detection (LoD) for the LPH panel was 3-5% variant allele frequency (VAF). Determining accuracy of SNV/indels in clinical samples requires an independent confirmation. Sanger is the gold standard but has a sensitivity of ∼15% VAF. We developed and utilized a custom PacBio multiplexed two-step amplicon sequencing protocol using Sanger sequencing products as template. We determined it to be a sensitive, cost-effective and easily customizable approach to detect variants with VAF &amp;lt;15%. The LPH panel interrogates all coding exons of the 141 genes using a custom hybrid capture-based assay of genomic libraries from 250 ng blood or bone marrow genomic DNA followed by sequencing on Illumina® sequencers. For PacBio confirmation, M13 tagged amplicons with length ranging from 214-678bp were generated using 10-50 ng input DNA, amplified with asymmetric barcodes, and processed for PacBio HiFi sequencing on a Sequel II. PacBio variants were called using Mutect2 (GATK) and/or visual inspection of .bam files by Integrated Genome Viewer (IGV). A subset of variants was called using the PacBio Amplicon Analysis tool (pbAA). 217 amplicons from 126 unique clinical samples were assessed by PacBio with a VAF ranging from 3-100% and comprising 140 SNVs, 63 indels &amp;lt;25bp and 14 indels ≥ 25bp. Concordance between PacBio and the LPH panel was 84.8% (184/217) and 69.4% (66/95) for the subset of variants with VAF between 3-5%. 9 of 184 required pbAA analysis to make a final consensus call. 63 variants were assessed with PacBio by processing the same M13 amplicons previously taken through Sanger analysis. This "double-dip" approach showed an 96.7% agreement (59/61) between PacBio and Sanger. The 2 variants not in agreement had VAF &amp;lt;15%.The PacBio variant calls in this study were incorporated into the results of the LPH panel analytical validation as orthogonal information, yielding an overall PPA for SNVs/indels of 99.1% (1250/1272) and FDR of 0.8% (10/1260) using VAF thresholds of 3% for SNVs and 5% for indels. In conclusion, the PacBio amplicon protocol can utilize amplicons generated for Sanger sequencing with minimal additional wet lab processing offering a flexible approach to confirmation for variants below the threshold of Sanger sensitivity. Citation Format: Grant Hogg, Kimberly A. Holden, John Howitt, Adib Shafi, Amanda Willliamson, Tong Liu, Helen Cao, Xiaojun Guan, Michael Mooney, Qian Zeng, Stan Letovsky, Li Cai, Eric A. Severson, Shakti Ramkissoon, Anjen Chenn, Marcia Eisenberg, Eyad Almasri, Taylor J. Jensen, Jonathan Williams. Orthogonal confirmation using the Pacbio®two step universal adapter amplicon protocol to validate a comprehensive genomic profiling assay for hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 679.