Background: The acquisition of suitable tumor tissue is a challenge for a significant fraction of late-stage NSCLC patients who require EGFR testing to inform choice of therapy. An alternative for these patients could be the assessment of EGFR mutations in circulating tumor DNA (ctDNA). In this study, we examined the detection of EGFR T790M mutation in ctDNA from urine, assessed urine sample requirements, and compared the results with contemporaneously matched tumor tissue and plasma in TIGER-X (NCT01526928), a Phase 1/2 clinical study of rociletinib in previously treated mutant EGFR patients with advanced NSCLC. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR.

Methods: 63 Stage IIIB/IV NSCLC patients enrolled in either Phase 1 or 2 components of TIGER-X and representing all therapeutic dose groups consented to optional urine collection. Maximum sample volumes were 100 mL for urine and 2 mL for plasma. To maximize assay sensitivity in urine, samples containing the recommended sample volume of ≥90 mL (≥ 90% of maximum in this study) were evaluated; all samples received were processed to assess this recommendation. Urinary and plasma ctDNA were tested for mutations by the same EGFR assays using a sensitive and quantitative short footprint assay method that employs a mutation enrichment step followed by next generation sequencing.

Results: Urine volumes ranged from 8-100 mL with a median DNA yield of 313 ng (N = 63). The median DNA yield was 299 ng for urine specimens with volume <90 mL (N = 44) and 392 ng for specimens with the previously recommended volume of ≥90 mL (N = 19). 45/63 patients (71%) were positive for T790M by urine in comparison to 47/63 (75%) by central lab testing of tissue. Using tissue as the reference, the positive percent agreement (PPA) between urine and tumor T790M test results was 93% (13/14) using urine samples with volumes ≥90 mL and 73% (34/47) when the urine volume was <90 mL. The PPA between plasma and tissue T790M test results was 80% (42/51). Overall, there were 13 cases that were tumor T790M+/urine T790M-, and 7 cases that were urine T790M+/tumor T790M-. Six urine T790M+/tumor T790M- cases were also T790M+ in plasma. Two additional urine T790M+ cases could not be analyzed by the tumor test due to insufficient material. Serially matched urine and plasma assessments were performed on a subset of patients (N = 15). In most cases, the urine and plasma serial profiles mirrored one another. A rapid drop in urine and plasma mutant EGFR levels was seen in almost all patients who experienced clinical benefit from rociletinib.

Conclusions: The analysis of ctDNA from urine identified a similar proportion of T790M+ patients as tissue-based testing with highest PPA amongst patients with urine volumes ≥90 mL. Discordant samples between urine and tissue that were not identified by the tumor test may be explained by tumor heterogeneity and/or inadequate biopsy. EGFR mutation detection from urine increases with urine volume and DNA yields and should be considered as a viable approach, particularly when tumor tissue is not available. Lastly, monitoring urine ctDNA T790M mutations longitudinally with baseline and post-therapy sampling could be clinically useful to determine benefit from therapy.

Citation Format: Shirish Gadgeel, Chris Karlovich, Vlada Melnikova, Lecia V. Sequist, D. Ross Camidge, Heather Wakelee, Maurice Perol, Geoffrey R. Oxnard, Karena Kosco, Cecile Rose T. Vibat, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Mark G. Erlander, Karen Reckamp. Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients treated with rociletinib (CO-1686). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A31.