CANSeq^TMKids is a comprehensive molecular profiling assay that evaluates relevant DNA mutations (SNVs, indels and CNVs) across 130 key genes and RNA fusions across 91 fusion transcript driver genes associated with pediatric cancer, in a single NGS assay (Table 1).

A total of 65 samples including FFPE tissue (n = 32), cell blocks (n = 2), whole blood (n = 8), bone marrow (n = 4), cell lines (n = 7) and commercial controls (n = 12) were used in the validation (Table 2). The size of the sample cohort was established based on recommended guidelines (Jennings et al., 2017). This study was performed using retrospective specimens with known molecular profiling results, known diagnoses and represented different tumor types (Table 3). Specimens were de-identified per IRB guidelines prior to inclusion in the study. Due to the diverse nature of childhood cancers, the CANSeq^TMKids panel has been designed to evaluate both solid tumors and hematological tissues. An analytical validation plan outlining sample cohort, validation strategy and processes involved, was reviewed and approved prior to study start. This study was approved by the Medical College of Wisconsin Institutional Review Board.

DNA and RNA from all specimens was extracted per established protocols. FFPE specimens were macro dissection-enriched prior to extraction. DNA quantification and quality was evaluated using the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA) and considered acceptable if the resultant A260/A280 absorbance ratio was between 1.8 and 2.1. RNA quantification was evaluated using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA) and was considered acceptable if sufficient quantity of RNA to ensure a 10 ng input was obtained for downstream processing.

Libraries were prepared by both manual and automated Ion Chef process. For the DNA portion of library preparation, the manual library preparation requires 8 µL with a concentration of 2.5 ng/μL whereas the automated library preparation requires 15 µL with a concentration of 0.7 ng/μL. The RNA requirements are slightly less with 5 µL with a concentration of 2 ng/μL for manual prep and 10 µL with a concentration of 1 ng/μL for the automated process. The manual process followed the Oncomine™ Childhood Cancer Research Assay (OCCRA) (Thermo Scientific, Waltham, MA) and the Ion AmpliSeq™ Library Preparation user guide. The Automated library preparation used the Oncomine™ Childhood Cancer Research Assay, Chef-Ready kit on the Ion Chef (Thermo Fisher Scientific). Libraries were barcoded with IonCode™ Barcode Adapters 1–384 Kit and normalized to 100 pmol/L by the Equalizer kit (Thermo Scientific, Waltham, MA). DNA and RNA libraries were then combined and diluted at an 80:20 DNA:RNA ratio at ∼50p.m. and templated overnight on the Ion 540 chip using Ion 540™ Kit—Chef (Thermo Scientific, Waltham, MA).

Sequencing was performed using 540 chips on the Ion GeneStudio™ S5 Prime Sequencer (Thermo Fisher Scientific, Waltham, MA). Raw reads from sequencing were processed and aligned to the reference genome hg19 on Ion Torrent Suite Software versions 5.12 and 5.14 (Thermo Fisher Scientific, Waltham, MA) and the run metrics of the Ion Torrent Suite used to determine quality control of sequencing runs. The minimum cutoff of ISP (Ion Sphere™ Particle) loading was 80% and the maximum of polyclonal ISPs was 50%, with threshold for total reads at 60M. The minimum percent usable reads were set to be 30%, and the minimum raw accuracy was 99%.

Variant calling and fusion detection was performed on Ion Reporter™ versions 5.14 and 5.16 server system by the OCCRA - w2.5 - IR workflow. The quality control and variant calling analysis were performed on the Ion Reporter™ (IR) software package. Tertiary analysis and report generation was established using the GO Pathology Workbench (GenomOncology, Cleveland, OH).

Analytical validation studies were carried out per guidelines from the Association for Molecular Pathology (AMP) and College of American Pathologists for the validation of Next-Generation Sequencing–Based Oncology Panels (Jennings et al., 2017). Details of the validation addressing STARD guidelines is presented in Supplementary Table S1.

The run metrics of the Ion Torrent Suite were used to determine quality control of sequencing runs which included base score, average sequencing depth, fusion panel control reads, minimum sequencing depth for variant calls, uniformity of coverage (ISP Loading), and strand bias of SNV and INDEL (Table 4; Figure 1). The thresholds of DNA mapped reads were 3M with mean depth ≥800x. The minimum mean read length was 75bp with uniformity ≥80% and mean raw accuracy ≥99%. The minimum RNA mapped reads was 20,000 with mean read length of 60bp.

Analytical accuracy was established using the reference Coriell cell line NA12878 with a mean raw accuracy of 99.8% (Table 5). The Seraseq Tri-Level mix control targets variants at different allelic frequencies (4%, 7% and 10%) establishing the limit of detection to be ≥5% allele frequency (AF) for SNVs and INDELs since variants in the 3%–5% allele frequency range are detectable but display variable reproducibility. The minimum AF for small deletions (6–15 nt) was 3.4% and for small insertions (3-4 nt) was 3.8%. and SNVs were detected at 3.5% AF (Table 6). CNVs were detected at about 4.86–6.64 copies, depending on the cancer type (Table 7). All 14 fusions of the Seraseq Fusion v3 Mix control covered by the OCCRA, were detected at 43 reads (Table 8), establishing the cutoff to be 45 for clinical implementation. Automation of library prep resulted in the fusion detection cut-off being increased to 1,100 fusion spanning reads reducing the sensitivity for fusion detection, no impact was observed on the detection of DNA variants. SNVs, INDELs and fusions were able to be detected with 1 ng DNA and RNA input respectively. Gene amplifications were only detected with 5 ng of DNA (Table 9). Results from the AOHC established a PPA of 97% and a PPV of 100% (Table 10), with the combined PPA and PPV of all variants type at 97.2% and >99% with a 95% CI of 93.3%–99%, respectively (Table 11).

A total of 3,640 negative variants were identified in both NA12878 and NA19240 samples with 772 INDELs and 2,869 SNVs/MNVs. Total 1820 negative variants were identified in NA18507 sample with 386 INDELs and 1,434 SNVs/MNVs. There were no positive variants detected across all hotspots, giving a specificity of ≥99% for all HapMap DNA samples (Table 12). Two normal colon and lung RNA samples were used to establish specificity of fusion detection of the assay. There was one false positive non-targeted fusion FHIT-TIRAP. F8T4 detected at 2,827 reads in one of the normal colon RNA replicates, resulting in the specificity greater than 99% in fusion detection (Table 13).

A total of 39 true positive variants of SNV/MNV, INDEL, CNV and fusions were detected across the samples and all replicates, resulting in an overall combined variant repeatability of >99% (95% CI of 91.0%–100%) (Table 14). A total of 73 true positive variants of SNV/MNV, INDEL, CNV and fusions were detected in the combined samples and all replicates resulting in an overall combined variant reproducibility of >99% (Table 15).