Table 2.

Key open questions for clinical NGS testing in oncology

Application	Examples	NGS method	Clinical research questions
Oncogenic driver identification	BRAF V600E, KIT, EGFR, ERBB2, FGFR3, PIK3CA, AKT1, TSC1, ROS1 mutations, ERBB2 amplifications, ALK translocations	WES, or targeted sequencing of tumor DNA or ctDNA	Is it useful to sequence large panels of genes vs actionable drivers?
Characterization of resistance	EGFR T790M, ESR1 mutations	WES, or targeted sequencing of tumor DNA or ctDNA	Is it useful to detect genetic mechanisms of resistance earlier, using ctDNA?
Identification of patients sensitive to immunotherapy	Mutational burden; neoantigens; Gene expression profiles	WES, or targeted (large panel) sequencing of tumor DNA; RNA‐Seq of tumor	Can identification of mutational process and clonality improve prediction based on mutational burden? Can personalized cancer vaccines boost responses to immune checkpoint inhibitors? Can gene expression signatures identify immune‐responsive tumors?
Germline mutations	BRCA1, BRCA2, PTEN, TSC1, CDH1	WGS or WES of normal DNA	Should the number of patients screened for germline mutations be expanded? Is it useful to interrogate a broad panel of genes ?
Quantification of intratumor heterogeneity	none	Whole exome sequencing of multiple tumor sites and metastatic lesions, or plasma	Is ITH associated with resistance to therapy and worse outcome? Is it targetable?
Quantification of pathway activation	ER, AR, mTOR, and CDK4 signatures	RNA‐Seq of tumors	Can gene expression signatures identify responsive patients for targeted therapies?
Cancer screening	none	Ultradeep sequencing of plasma	Can ultradeep sequencing on ctDNA identify some cancers early? Is it useful?
Regulatory approval	none	Any	How can clinical validity of NGS tests be established for previously approved biomarkers? What level of evidence is required for biomarkers that are rare in an indication? How will approval be maintained as the field advances?