Abstract
To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform whole-genome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Our analysis pipeline achieves high accuracy by cross-validating variants between sequencing types, thereby removing the need for confirmatory testing, and facilitates comprehensive reporting in a clinically-relevant timeframe. Three-platform sequencing has a positive predictive value of 97–99, 99, and 91% for somatic SNVs, indels and structural variations, respectively, based on independent experimental verification of 15,225 variants. We report 240 pathogenic variants across all cases, including 84 of 86 known from previous diagnostic testing (98% sensitivity). Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity. These results emphasize the critical need for incorporating WGS in pediatric oncology testing.
Highlights
To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform wholegenome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory
We show that three-platform sequencing achieves both high sensitivity of 98% of known pathogenic variants and a high positive predictive value (PPV) of 97–99, 99, and 91% for somatic single nucleotide variations (SNVs), indels and structural variations, respectively
We demonstrated experimentally that threeplatform sequencing had near perfect PPV and sensitivity, producing a high yield of clinically relevant P/LP variants
Summary
To evaluate the potential of an integrated clinical test to detect diverse classes of somatic and germline mutations relevant to pediatric oncology, we performed three-platform wholegenome (WGS), whole exome (WES) and transcriptome (RNA-Seq) sequencing of tumors and normal tissue from 78 pediatric cancer patients in a CLIA-certified, CAP-accredited laboratory. Combined WES and RNA-Seq, the current standard for precision oncology, achieved only 78% sensitivity These results emphasize the critical need for incorporating WGS in pediatric oncology testing. Traditional workflows to capture these diverse genetic abnormalities in the clinic are complex and multimodal, often including a combination of karyotyping, fluorescent in situ hybridization, copy-number microarray, quantitative RT-PCR, and Sanger sequencing Such workflows lack the scalability and flexibility necessary to incorporate recently discovered lesions and are of limited use for studying cases with complex or nonstandard findings[2,3,4]. Clinical NGS tests using targeted gene panels or whole exome sequencing (WES) have been used to identify pathogenic sequence mutations including single nucleotide variations (SNVs) and small insertion-deletions (indels) in both adult[6,7,8] and pediatric cancer studies[8,9,10]. Clinical adoption of WGS has been limited to pilot studies involving very small numbers of cases (mostly < 10)[12,13,14,15,16,17] and the median turnaround time for generating the final clinical report may exceed 100 days due to mandatory secondary validation of variants by a CLIA-certified lab[12,18]
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