Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

Selection, optimization and validation of ten chronic disease polygenic risk scores for clinical implementation in diverse US populations.

Polygenic risk scores (PRSs) have improved in predictive performance, but several challenges remain to be addressed before PRSs can be implemented in the clinic, including reduced predictive performance of PRSs in diverse populations, and the interpretation and communication of genetic results to both providers and patients. To address these challenges, the National Human Genome Research Institute-funded Electronic Medical Records and Genomics (eMERGE) Network has developed a framework and pipeline for return of a PRS-based genome-informed risk assessment to 25,000 diverse adults and children as part of a clinical study. From an initial list of 23 conditions, ten were selected for implementation based on PRS performance, medical actionability and potential clinical utility, including cardiometabolic diseases and cancer. Standardized metrics were considered in the selection process, with additional consideration given to strength of evidence in African and Hispanic populations. We then developed a pipeline for clinical PRS implementation (score transfer to a clinical laboratory, validation and verification of score performance), and used genetic ancestry to calibrate PRS mean and variance, utilizing genetically diverse data from 13,475 participants of the All of Us Research Program cohort to train and test model parameters. Finally, we created a framework for regulatory compliance and developed a PRS clinical report for return to providers and for inclusion in an additional genome-informed risk assessment. The initial experience from eMERGE can inform the approach needed to implement PRS-based testing in diverse clinical settings.