Background: Cell-free (cf) DNA and circulating tumor cells (CTC) in the blood of cancer patients offer an easily obtainable, low-risk, inexpensive, and repeatedly available source of biologic material for mutation analysis and monitoring of molecular changes throughout cancer therapy.
Methods: DNAs from plasma and CTC from patients with advanced cancers who progressed on systemic therapy were tested for BRAF V600 and KRAS G12/G13 mutations using the ICE-COLD-PCR platform. ICE COLD-PCR, "Improved and Complete Enrichment COamplification at Lower Denaturation” selectively amplifies mutant DNA by exploiting differences in denaturation temperatures between mutant DNA duplexes and normal “wild-type” DNA duplexes. KRAS Exon 2 and BRAF Exon 15 ICE COLD-PCR was performed on plasma samples, and from matched CTCs collected using ScreenCell® MB kits (ScreenCell, Sarcelles, France). Amplicons were analyzed by Sanger sequencing methods and results were compared to the mutation status of the archival primary or metastatic tumor tissue as determined in a CLIA-certified lab.
Results: Blood samples from 59 patients with advanced cancers (colorectal cancer, n=32; melanoma, n=12; non-small cell lung cancer, n=7; other cancers, n=8), were obtained before treatment and, if possible, sequentially during therapy and tested for BRAF (30), KRAS (29) or BRAF and KRAS (1) mutations. BRAF mutations were detected in 97% (30/31) of archival tumor samples compared to 65% (20/31) of cfDNA samples (agreement 68%) and to 3% (1/31) of CTC samples (agreement 6%). KRAS mutations were detected in 90% (26/29) of archival tumor samples compared to 86% (25/29) of cfDNA samples (agreement 83%) and to 10% (3/29) of CTC samples (agreement 21%); however, CTCs had different KRAS mutation subtypes than those in tumor tissue. Of interest, in 3 patients with serial blood collection for cfDNA BRAF mutations, 2 (melanoma, Erdheim-Chester disease) had BRAF V600E cfDNA mutations at baseline, which disappeared during the response to subsequent therapy. Another patient (melanoma) did not have a BRAF V600E cfDNA mutation immediately after being taken off BRAF inhibitor therapy due to intolerance, but a BRAF V600E cfDNA mutation emerged when the patient was treated with non-BRAF targeting therapy. In 2 patients with serial blood collections for cfDNA KRAS mutations, 2 (colorectal cancer) did not have KRAS cfDNA mutations at baseline, but KRAS cfDNA mutations (G13D, G12D, respectively) emerged following disease progression and subsequent therapy. Conclusions: Detection of BRAF and KRAS mutations in cfDNA can provide a fast and noninvasive alternative to mutation testing in tumor tissue. Although these mutations can be also detected in CTC, the level of concordance with tumor tissue results is low.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C203.
Citation Format: Filip Janku, Ben Legendre, Katherine Richardson, Gerald S. Falchook, Aung Naing, Veronica R. Holley, David S. Hong, Ralph G. Zinner, Siqing Fu, Apostolia M. Tsimberidou, Vivek Subbiah, Daniel D. Karp, Sarina A. Piha-Paul, Jennifer J. Wheler, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Funda Meric-Bernstam, Amy Kruempel, Jaclyn Pope, Courtney Cubrich, Grant Wu, Yanggu Shi, Marcia Lewis, Razelle Kurzrock. BRAF and KRAS mutation testing in cell-free DNA and circulating tumor cells from blood of patients with metastatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C203.