Abstract 3072: Prospective study of genetic mutations in matched tumor and plasma specimens in advanced cancer patients referred to phase I trials
Authors: Perkins Geraldine , Dukes Juliet , Pope Lorna , Clark Jeremy , Yap Timothy Anthony , Riisnaes Ruth , Cassidy Amy , Denholm Katie Ann , Kaye Stan , Bono Johann Sebastian De 
Published: June 2012

Abstract
Background: Circulating free DNA (cfDNA) represents a minimally-invasive resource for detecting mutations in advanced cancer patients. The primary objective of this study was to determine if cfDNA is representative of tumor tissue for multiplex mutation detection utilizing Sequenom MassARRAY.
Methods: Metastatic colorectal, breast, melanoma and ovary cancer patients referred to the Drug Development Unit at the Royal Marsden Hospital between 9/09-8/10 gave their consent to provide DNA from matched archival formalin fixed paraffin embedded (FFPE) tumor and plasma. Samples mutation genotyping using the Sequenom MassARRAY technology and the OncoCartaTM Panel allowed the parallel analysis of 238 simple and complex mutations across 19 selected key oncogenes. Dilutions (10ng/µl to 0.01ng/µl) of HCT116 DNA containing KRAS G13D mutation were used to determine assay sensitivity and specificity.
Results: Serial dilution spiking experiments revealed that the KRAS G13D mutation was reproducibly detectable to 40ng/ml of HCT116 DNA; 74 patient samples were then analyzed. The median quantity of cfDNA was 305ng/ml (range 10-32000). Of the 25 colorectal patients, the median quantity of cfDNA was 361ng/ml (range 189-4603); KRAS, BRAF and PIK3CA mutations were detected in 8 (31%), 3 (12%) and 3 (12%) tumor specimens respectively; concordant data for matched plasma tumor DNA was found in 88% of patients for KRAS, 100% for BRAF mutations and 33% for PIK3CA (1 patient had a PIK3CA mutation in primary and plasma; one patient had a mutation detected only in the primary; one patient had no mutation in the primary or plasma, but a mutation detected in a metastatic liver deposit). The recently reported oncogenic AKT1 E17K mutation was detected in 1 colorectal patient in tissue and plasma. Of the 19 breast cancer patients, the median cfDNA concentration was 354ng/ml (range 10-32000); PIK3CA H1047R mutation was detected in 4 (21%) FFPE tumor specimens; concordance between matched FFPE and cfDNA was 75% (3 out of 4 patients). The AKT1 E17K mutation was detected in 1 patient in both tissue and plasma. In 15 melanoma patients the median cfDNA concentration was 141ng/ml (range 46-1002); BRAF V600E, NRAS codon 61 and MET R970C mutations were detected in 4 (27%), 3 (20%) and 1 (7%) FFPE tumor specimens respectively. Concordance between matched FFPE and cfDNA was 50% for BRAF, 75% for NRAS and 100% for MET mutations. Another MET mutation, T992I, was found in plasma but not in tissue for one patient. Of the 15 ovary patients, the median cfDNA concentration was 261ng/ml (range 79-957). Two KRAS mutations and 1 PIK3CA H1047R mutation were detected in FFPE, but no mutation was found in any plasma sample.
Conclusions: A high concordance in detected mutations was observed between FFPE tumor and matched cfDNA. cfDNA is representative of tumor DNA and may be used for the prospective selection of cancer patients for targeted therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2011-3072