Analytical validation and implementation of a pan cancer next-generation sequencing panel, CANSeqTMKids for molecular profiling of childhood malignancies

Kala F Schilter; Brandon A Smith; Qian Nie; Kathryn Stoll; Juan C Felix; Jason A Jarzembowski; Honey V Reddi

doi:10.3389/fgene.2023.1067457

Analytical validation and implementation of a pan cancer next-generation sequencing panel, CANSeq^TMKids for molecular profiling of childhood malignancies

Front Genet. 2023 Feb 9:14:1067457. doi: 10.3389/fgene.2023.1067457. eCollection 2023.

Authors

Kala F Schilter¹, Brandon A Smith¹, Qian Nie¹, Kathryn Stoll¹, Juan C Felix^{1

2}, Jason A Jarzembowski², Honey V Reddi¹

Affiliations

¹ Precision Medicine Laboratory, Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, United States.
² Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI, United States.

Abstract

Next-Generation Sequencing (NGS) allows rapid analysis of multiple genes for the detection of clinically actionable variants. This study reports the analytical validation of a targeted pan cancer NGS panel CANSeq^TMKids for molecular profiling of childhood malignancies. Analytical validation included DNA and RNA extracted from de-identified clinical specimens including formalin fixed paraffin embedded (FFPE) tissue, bone marrow and whole blood as well as commercially available reference materials. The DNA component of the panel evaluates 130 genes for the detection of single nucleotide variants (SNVs), Insertion and Deletions (INDELs), and 91 genes for fusion variants associated with childhood malignancies. Conditions were optimized to use as low as 20% neoplastic content with 5 ng of nucleic acid input. Evaluation of the data determined greater than 99% accuracy, sensitivity, repeatability, and reproducibility. The limit of detection was established to be 5% allele fraction for SNVs and INDELs, 5 copies for gene amplifications and 1,100 reads for gene fusions. Assay efficiency was improved by automation of library preparation. In conclusion, the CANSeq^TMKids allows for the comprehensive molecular profiling of childhood malignancies from different specimen sources with high quality and fast turnaround time.

Keywords: assay validation; childhood cancer; clinical implementation; molecular profiling; next generation sequencing panel; pan cancer assay.