Abstract 5688: Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients
Authors: Ibañez Mónica Garzón , casas Clara Mayo de las , Ariza Nùria Jordana , Bel Ariadna Balada , Garcia Beatriz , Villatoro Sergio , Bertrán-Alamillo Jordi , Martinez-Bueno Alejandro , Ramirez Santiago Viteri , Rosado Ana Pérez , Espinosa Daniela Morales , Catalán Maria José , Karachaliou Niki , Molina-Vila Miguel Ángel , Rosell Rafael 
Published: July 2017

Abstract
Background: Mutations in the KRAS proto-oncogene, GTPase (KRAS) are driver alterations in several tumors, such as non-small-cell lung (NSCLC) and colorectal cancer (CRC). However, their role as a prognostic marker in liquid biopsies is unclear. We studied the status of KRAS mutations in the peripheral blood of advanced NSCLC and colorectal cancer patients visited in our hospital and evaluated its potential value as a monitoring marker in the clinical practice.
Methods: We developed a sensitive TaqMan assay, in the presence of a PNA clamp, for the determination of KRAS mutations (codons 12, 13 and 61) in circulating-free DNA (cfDNA). The assay detected 2.5 pg mutated DNA/µL and a ratio of KRAS mutated versus wild type allele of 1:20000 and was validated in 80 cancer patients previously genotyped in tumor tissue showing a clinical sensitivity of 72.5% and specificity of 100%. cfDNA was isolated from serum and plasma specimens, using an automatic extractor and mutational analyses were performed in quadruplicates samples. Serum and plasma samples were drawn at diagnosis, during follow-up and progression.
Results: 239 NSCLC and 11 colorectal cancer patients were screened for KRAS mutations in cfDNA at presentation in the period of 2013 to 2016. In addition, 160 cfDNA samples collected during disease monitoring of 61 patients were analyzed. Paired biopsies were available from 110/250 p at diagnosis (60 positive for KRAS in FFPE and 50 WT). The KRAS mutation was detected in the cfDNA of 76.6% (46/60) p positive in tumor tissue vs. only in 1/50 WT patients where a G12C was detected in cfDNA in a consistent manner, probably associated with a heterogeneity of the tumor. In patients with no biopsy available, we detected KRAS mutations in cfDNA in 14.3% (20/140) cases at diagnosis. The most prevalent mutations observed in cfDNA were G12C (44.7 %), G12V (22.4%) and G12D (10.4%) for NSCLC patients and G13D (5.9%) for colorectal patients. The Q61H mutation was detected in the cfDNA of one NSCLC and one CRC p. Of the 67 patient’s positive in cfDNA at presentation, KRAS mutations were detected in serum and plasma in 38 patients, only in plasma in 24 and only in serum in 5. Regarding the 61 patients with serial blood samples, we observed a correlation between the KRAS mutation load in cfDNA and the course of the disease, with a decrease in patients responding to chemotherapy and an increase during disease progression.
Conclusions: Our results demonstrate the feasibility of KRAS mutation analysis in the cfDNA of advanced NSCLC and colorectal patients. Analysis of KRAS mutations in the blood of mutated patients can have a value in patients with no biopsy available and to monitor the course of the disease.
Citation Format: Mónica Garzón Ibañez, Clara Mayo de las casas, Nùria Jordana Ariza, Ariadna Balada Bel, Beatriz Garcia, Sergio Villatoro, Jordi Bertrán-Alamillo, Alejandro Martinez-Bueno, Santiago Viteri Ramirez, Ana Pérez Rosado, Daniela Morales Espinosa, Maria José Catalán, Niki Karachaliou, Miguel Ángel Molina-Vila, Rafael Rosell. Routine testing for KRAS mutations in cfDNA from blood of advanced cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5688. doi:10.1158/1538-7445.AM2017-5688