PURPOSE:Concurrent chemoradiation therapy (CRT) is the principal treatment modality for locally advanced lung cancer. Cell death due to CRT leads to the release of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) into the bloodstream, but the kinetics and characteristics of this process are poorly understood. We hypothesized that there could be clinically meaningful changes in cfDNA and ctDNA during a course of CRT for lung cancer. METHODS AND MATERIALS:Multiple samples of plasma were obtained from 24 patients treated with CRT for locally advanced lung cancer to a mean dose of 66 Gy (range, 58-74 Gy) at the following intervals: before CRT, at weeks 2 and 5 during CRT, and 6 weeks after treatment. cfDNA was quantified, and a novel next generation sequencing (NGS) technique using enhanced tagged/targeted-amplicon sequencing was performed to analyze ctDNA. RESULTS:Patients for whom specific mutations in ctDNA were undetectable at the baseline time point had improved survival, and potentially etiologic driver mutations could be tracked throughout the course of CRT via NGS in multiple patients. We quantified the levels of cfDNA from patients before CRT, at week 2, week 5, and at 6 weeks after treatment. No differences were observed at weeks 2 and 5 of therapy, but we noted a significant increase in cfDNA in the posttreatment follow-up samples compared with samples collected before CRT (P = .05). CONCLUSIONS:Dynamic changes in both cfDNA and ctDNA were observed throughout the course of CRT in patients with locally advanced lung cancer. Specific mutations with therapeutic implications can be identified and tracked using NGS methodologies. Further work is required to characterize the changes in cfDNA and ctDNA over time in patients treated with CRT and to assess the predictive and prognostic potential of this powerful technology.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

Introduction Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. Materials and methods We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. Results The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. Conclusions These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.

Abstract Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA. The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots. The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965). This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients. Citation Format: Samuel Woodhouse, Vincent Plagnol, Karen Howarth, Stefanie Lensing, Matt Smith, Michael Epstein, Mikidache Madi, Sarah Smalley, Catherine Leroy, Jonathan Hinton, Frank de Kievit, Esther Musgrave-Brown, Colin Herd, Katherine Baker-Neblett, Will Brennan, Peter Dimitrov, Nathan Campbell, Nitzan Rosenfeld, James Clark, Davina Gale, Jamie Platt, John Calaway, Greg Jones, Tim Forshew. Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 939.

Molecular profiling of tumors using circulating tumor DNA (ctDNA) in the blood of cancer patients, as a liquid biopsy, is rapidly becoming established as a useful source of information to aid clinical decision-making when a solid tumor biopsy is not available, or is limited in amount or quality. DNA alterations are often found in a small fraction of the total cell free DNA in plasma, and their detection requires specially designed assays that are sensitive and reproducible. Individual hotspot mutations can be assayed using technologies such as droplet/digital PCR, but multiplexing such assays is limited by the small amount of clinical material. This can be addressed by assays based on next generation sequencing (NGS), to create sensitive panels for ctDNA analysis. For clinical application, it is essential that such NGS assays be standardized and reproducible, both intra-and inter-laboratory. Standardization for tissue-based NGS assays has only recently been implemented, after much discussion. We describe a strategy for validation and standardization of a high sensitivity NGS-based ctDNA assay between two laboratories, based in the US and UK. The enhanced TAm-Seq® assay (eTAm-Seq™) uses efficient library preparations and bespoke algorithms to identify cancer mutations within a panel of 34 genes, covering cancer hotspots as well as entire coding regions of selected genes. To ensure this complex process is standardised and controlled, a high level ISO and CLIA quality management system is implemented. To perform analytical validation of this assay, we used reference standards and plasma controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We compared performance of the assay between two laboratories, finding a high rate of concordance and reproducibility. Using DNA quantities typical of those found in up to 4ml of plasma from cancer patients, our assay provides high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Standard dilution curves of well-characterized reference samples show that the accuracy of the eTAm-Seq® assay is predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. Additional data with associated clinical data will be presented at the meeting. Our data demonstrates eTAm-SeqTM assy's robustness and performance in two labs, supporting its use as a basis for clinical applications globally, allowing a high degree of standardization and comparability for molecular profiling of tumors using liquid biopsy.