Detection of Circulating Tumor Cells of Nonepithelial Origin: A Bench to Bedside Approach

Abstract

Purpose/Objective(s): No validated biomarkers exist for minimal residual disease (MRD) detection in non-small cell lung cancer (NSCLC). Tumor DNA is continually shed into the circulation, where it can be accessed for analysis by next-generation sequencing (NGS). We aimed to design a NGS-based method for quantifying circulating tumor-derived DNA in NSCLC and other cancers. Materials/Methods: We developed CAPP-Seq (CAncer Personalized Profiling by deep Sequencing), a novel method for noninvasive and sensitive measurement of cancer burden from cfDNA. Our strategy leverages hybrid affinity capture to enrich recurrently mutated genomic regions from tumor genomic DNA and plasma cfDNA. We used mixtures of NSCLC cell line DNA in cfDNA to benchmark CAPP-Seq. We then analyzed a set of NSCLC tumor/normal genomic DNA pairs. Finally, we studied serial plasma samples collected from patients before and after treatment with radiation therapy, chemotherapy, and/or surgery. Results: We show that CAPP-Seq detects multiple types of cancer-specific somatic mutations within NSCLC DNA, including single nucleotide variants, insertions/deletions, and rearrangements. Benchmarking analyses demonstrate that CAPP-Seq accurately enumerates somatic mutations within cfDNA at a fractional abundance of 0.025%. Applying CAPP-Seq to tumor/normal genomic DNA pairs from 11 NSCLC patients uncovered a median of 3 somatic mutations per patient (range, 1-16). Fifteen plasma samples from 7 patients were profiled by CAPP-Seq. The abundance of patient-specific cancer markers within plasma cfDNA was readily measured by CAPP-Seq in both earlyand late-stage NSCLC. Importantly, circulating tumor DNA responded appropriately to anti-cancer therapies, becoming undetectable in 2 patients (after surgery and chemoradiation therapy, respectively) and declining but remaining detectable in 5 patients (after chemotherapy in 4 patients and surgery in 1 patient). CAPP-Seq measurements correlated with tumor volume (R Z 0.97) and allowed for the detection of resistance mutations in patients being treated with targeted therapies. Conclusions: In this study, we described CAPP-Seq, a novel NGS-based approach to MRD testing in cancer patients. CAPP-Seq could have clinical utility for monitoring disease burden and response to therapy, early detection of relapse, distinguishing radiation pneumonitis from relapse, and risk stratification in early-stage NSCLC following surgery or stereotactic body radiation therapy. Ultimately, we envision CAPP-Seq becoming a routine clinical assay for measuring tumor burden in diverse cancers from plasma and other body fluids. Author Disclosure: S.V. Bratman: None. A.M. Newman: None. J. To: None. J.F. Wynne: None. J.W. Neal: None. H. Wakelee: None. J. Shrager: None. B.W. Loo: None. A.A. Alizadeh: None. M. Diehn: None.