Abstract B4: Use of urinary circulating tumor DNA (ctDNA) KRAS for monitoring treatment response in patients with metastatic colorectal cancer (mCRC)
Authors: Barzi Afsaneh , Melnikova Vlada , Vibat Cecile Rose T. , Hancock Saege , Ning Yan , Agafitei Dana , Erlander Mark G. , Lenz Heinz-Josef 
Published: May 2016

Background: Radiographic imaging in patients with mCRC is the current standard of care for monitoring responses. At least 40% of patients with mCRC have tumor associated KRAS mutations, and these may be detected in tumor DNA in plasma and urine. The aim of this study was to correlate the dynamics of KRAS mutational load in urinary and plasma ctDNA with clinical responses in patients with mCRC.

Methods: In a blinded biomarker study of 30 metastatic patients CRC receiving chemotherapy or surgical treatment for resectable liver metastases, interim analysis was conducted in 13 patients (7 harboring KRAS G12/13 mutation in the tumor tissue, 6 wild-type for KRAS by tissue). For KRAS-positive patients, urine and plasma specimens were obtained pre-treatment, 2 weeks after initiation of chemotherapy, and subsequently at monthly intervals. Urine ctDNA was extracted using a method that preferentially isolates short fragmented ctDNA. Quantitative analysis of KRAS G12/13 mutation load in both urine and plasma was performed with the same validated KRAS G12/13 assay, which utilizes a wild-type blocker PCR enrichment followed by a next generation sequencing-based detection (MiSeq) with standardized reporting of mutant copies per 10^5 genome equivalents.

Results: In an interim analysis, patients enrolled had pre-treatment urine and plasma specimens. In addition, a subset of patients (6) had longitudinal collections for up to 17 months. Eleven of 13 patients had liver-dominant metastatic disease. All KRAS tissue positive patients were receiving FOLFOX; all KRAS tissue-negative patients were receiving FOLFIRI plus cetuximab. In all KRAS G12/13 tissue positive patients with liver metastatic disease, a urinary ctDNA KRAS mutation concordant with tissue was identified; in 2 patients with non-liver metastatic disease, KRAS mutation was discordant or unidentifiable suggesting different biology in these patients. Plasma ctDNA KRAS mutation concordant with tissue was identified in 6 of 7 patient. Overall, the dynamics of ctDNA KRAS mutation burden in urine showed stronger concordance with clinical course and lack of temporal fluctuations as compared to the dynamics of ctDNA KRAS in plasma. A significant decrease in urinary ctDNA KRAS signal was observed as early as 2 weeks on chemotherapy, and this molecular response correlated with subsequently documented radiographic response. In 1 patient with documented progressive disease, an increase in urinary ctDNA KRAS G12/13 signal was observed 2 months prior to radiographic progression. Finally, 5 of 6 patients who tested negative for KRAS G12/13 mutations by tumor biopsy also tested negative in urine and plasma; 1 additional tissue KRAS wild-type patient tested positive for KRAS G12D mutation by urine and plasma ctDNA.

Conclusions: In a proof of concept study, urine ctDNA was shown to carry a concordant KRAS mutation with tissue of mCRC patients. ctDNA analysis identified additional KRAS G12/13 mutation positive patient in whom KRAS mutations were undetected by the tumor test suggesting tumor heterogeneity and/or inadequate biopsy. The dynamics of ctDNA KRAS mutational load in urine correlated with radiographic responses, especially in liver-dominant metastatic patients, suggesting that urinary ctDNA may be a valuable method for monitoring treatment responses in patients with mCRC.

Citation Format: Afsaneh Barzi, Vlada Melnikova, Cecile Rose T. Vibat, Saege Hancock, Yan Ning, Dana Agafitei, Mark G. Erlander, Heinz-Josef Lenz. Use of urinary circulating tumor DNA (ctDNA) KRAS for monitoring treatment response in patients with metastatic colorectal cancer (mCRC). [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 B4.