Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non‐small‐cell lung cancer and melanoma patients. Cell‐free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA‐Q‐PCR or next‐generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty‐three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine–kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies.

Collection of tumor samples is not always feasible in non-small cell lung cancer (NSCLC) patients, and circulating free DNA (cfDNA) extracted from blood represents a viable alternative. Different sensitive platforms have been developed for genetic cfDNA testing, some of which are already in clinical use. However, several difficulties remain, particularly the lack of standardization of these methodologies. Areas covered: Here, the authors present a review of the literature to update the applicability of cfDNA for diagnosis and monitoring of NSCLC patients. Expert commentary: Detection of somatic alterations in cfDNA is already in use in clinical practice and provides valuable information for patient management. Monitoring baseline alterations and emergence of resistance mutations is one of the most important clinical applications and can be used to non-invasively track disease evolution. Today, different technologies are available for cfDNA analysis, including whole-genome or exome sequencing and targeted methods that focus on a selection of genes of interest in a specific disease. In the case of Next Generation Sequencing (NGS) approaches, in depth coverage of candidate mutation loci can be achieved by selecting a limited number of targeted genes.

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Background: With the advent of precision medicine, screening for clinically relevant mutations and gene fusions is mandatory in many tumor types. However, in a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis. In addition, repeated tissue biopsies for monitoring the course of the disease and the emergence of mechanisms of resistance to targeted therapies are not feasible. Liquid biopsies constitute the only alternative available in these cases. The nCounter technology has been adapted to detect mutations and gene fusions in FFPE biopsies from cancer patients with a minimum requirement of tumor material and sample handling, a short turnaround time and a straightforward data analysis. However, nCounter has not been tested in liquid biopsy samples. Methods: For mutation analysis, the SNV Solid Tumor Panel was used, which allows for detection of 97 driver mutations in 24 genes. For fusions, a customized panel for ALK, ROS1, RET, and NTRK1 fusion transcripts was used with a 14-cycles preamp step. First, proof-of-concept experiments were run by spiking plasma samples with a mixture of genomic DNAs or RNAs from positive cell lines. Next, 65 circulating-free DNA (cfDNA) samples from advanced cancer patients, previously genotyped by other techniques, were analyzed using the SNV panel. Of those, 60 had been purified from plasma, 4 from ascites and 1 from the pleural effusion. Nineteen were positive for EGFR mutations, 20 for KRAS, 13 for BRAF, 5 for PIK3CA, 2 for NRAS, and 6 were pan-negative. Finally, 8 circulating cell-free RNA samples isolated from plasma were tested with the nCounter Low RNA Input Kit and the lung fusion panel. Of those, 6 corresponded to lung cancer patients harboring ALK or ROS1 rearrangements in tumor tissue, but previous RT-PCR only detected fusions transcripts in 2. Results: Spiking experiments revealed that the nCounter SNV Panel was able to detect mutations at allelic fractions as low as 0.2% for most of the drivers. When testing liquid biopsies, 63/65 cfDNA samples from cancer patients were evaluable, despite having DNA concentrations lower than 1 ng/µL. The SNV Panel successfully detected EGFR, KRAS, BRAF, PIK3CA and NRAS mutations with a concordance rate of 97.5% with previous genotyping by NGS, Therascreen® or Taqman® with PNA, corresponding to a Cohen’s kappa of 0,913. In the case of the lung fusion panel, ALK, ROS1 and RET fusion transcripts were detected in all spiked plasma cfRNA. Two samples from lung cancer patients with positive RT-PCR results were also detected by the nCounter low-input lung fusion panel. Research is ongoing to further improve the performance of the nCounter low-input fusion panel in liquid biopsy samples. Conclusions: Our results demonstrate the feasibility of mutation analysis in the cfDNA of advanced cancer patients using nCounter. The nCounter technology also shows promise for the detection of gene fusions in cfRNA Citation Format: Ana A. Giménez-Capitán, Chung-Ying Huang, Jill Bracht, Rich Boykin, Clara Mayo-de-las-Casas, Joseph M. Beechem, Cristina Teixidó, Ariadna Balada-Bel, Beatriz Garcia-, Sergio Villatoro, Monica Garzón, Nuria Jordana-Ariza, Cristina Aguado, Santiago Viteri, Juan José García, Rafael Rosell, Jay Gerlach, Noemi Reguart, Miguel Angel Molina-Vila. nCounter for detection of clinically relevant alterations in liquid biopsies of solid tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1384.

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