Abstract 488: Detection of somatic mutations in plasma allows for non-invasive real-time therapy response monitoring of lung cancer patients
Authors: Costa Jose L. , Fernandes Gabriela , Reis Joana , Silva Miguel , Hespanhol Venceslau , Machado José C. 
Published: July 2016

Abstract
Tumor-specific (somatic) mutations in plasma can serve as biomarkers for tumor detection, monitor tumor response to specific therapies, detect residual disease after surgery, and long-term follow-up. The intrinsic low abundance of circulating cell-free tumor DNA (cfDNA) makes the detection and quantification of such mutations in plasma a challenging task. This study aimed to establish a comprehensive strategy to be used in the detection of clinically relevant somatic mutations in plasma of lung cancer patients both at diagnosis and during follow-up of disease.
Plasma samples from healthy controls were used to isolate cfDNA and to optimize the experimental approach. A custom multiplex PCR panel was designed to target 420 unique genetic variants. To cover a broad range (0.0 -100%) of variant allele fractions (VAF), as expected for somatic mutations in the plasma of cancer patients, the cfDNA samples from the controls were mixed at five different ratios and sequenced using five different levels of coverage. In order to evaluate our strategy, plasma samples obtained at different stages of disease were collected from a group of 100 lung cancer patients and used to isolate cfDNA. The mean treatment follow up time was 6 months with a minimum of 1 and a maximum of 23 months. Different enrichment strategies where tested. All amplified products were used to prepare libraries and sequenced using the Ion PGM system. The QuantStudio 3D Digital PCR System was used to confirm selected results.
Our controlled data set enabled us to define VAF, allele depth and allele quality score cutoffs for a reliable detection of low VAF in cfDNA. For variants present at VAF ≥5%, a sensitivity of 100% was achieved at ≥500x coverage. At ≥1000x coverage, more than 94% of the variants present at VAF &lt;5% were successfully detected with an average sequencing quality score &gt;Q30. In the plasma isolated from lung cancer patients, tumor derived genetic mutations could be identified in as little as 10ng of cfDNA. Mutations identified in cfDNA mirrored the mutations identified in tumor biopsies. Mutations with VAF as low as 0.1% could be detected in cfDNA and confirmed using Digital PCR. The changes in tumor-specific mutations in cfDNA was then correlated with the clinical follow up of the patients. Response to therapy, including resistance, could be observed by tracking specific mutations in cfDNA and the detection of tumor relapse could be anticipated up to two months using cfDNA when compared to standard methodologies.
In conclusion, the comprehensiveness and speed of the NGS methodology, combined with the high sensitivity of detection, delivered an experimental protocol for the detection of somatic mutations in cfDNA for non-invasive real time therapy response monitoring of cancer patients. This strategy can be implemented in any molecular pathology laboratory with clear benefits for the management of cancer patients.
Citation Format: Jose L. Costa, Gabriela Fernandes, Joana Reis, Miguel Silva, Venceslau Hespanhol, José C. Machado. Detection of somatic mutations in plasma allows for non-invasive real-time therapy response monitoring of lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 488.