Abstract 3097: Analytical development of the RaDaRTM assay, a highly sensitive and specific assay for the monitoring of minimal residual disease
Authors: Marsico Giovanni , Sharma Garima , Perry Malcolm , Hackinger Sophie , Forshew Tim , Howarth Karen , Platt Jamie , Rosenfeld Nitzan , Osborne Robert 
Published: September 2020

Abstract
Background
5%-30% of patients with primary non-metastatic cancer eventually relapse and die of metastatic disease, even though no macroscopic disease remains after initial curative-intent treatment. Adjuvant therapy is often administered to target minimal residual disease (MRD) that may be present but does not change outcome for most patients. Detecting MRD by liquid biopsy analysis after initial treatment and in advance of overt relapse could help identify patients who may benefit from adjuvant therapy or enrolment in clinical trials.
Methods
We developed InVision®MRD, a highly sensitive and specific method for detection of trace levels of tumor DNA in plasma cell-free DNA. Tumor-specific variants are identified using whole exome sequencing of tumor tissue samples, and a bespoke process designs primer panels targeting mutated loci. A multiplex high-fidelity PCR using InVision® technology captures and amplifies up to 48 patient–specific tumor variants in cell-free DNA, followed by high depth next generation sequencing. Proprietary algorithms compare patient-specific sequencing data to controls,and integrate information across variants and replicates to detect trace levels of tumor-derived signal. The method is currently available for research use only (RUO).
Results
We evaluated the performance of the InVision®MRD assay using samples from cancer patients and cell-line dilutions. We diluted three different tumor cell-lines into their matched normal counterparts. Primer panels were designed against 48 variants, specific to the tumor-cell line. Dilutions were amplified by multiplex PCR and sequenced on the Illumina NovaSeq to depth &gt;100,000 reads per locus. Using 48 variants, the InVision®MRD assay had a mean sensitivity of 100% at 20 parts per million (20 ppm, equivalent to 0.002%) and sensitivity of 70% at 10 ppm (0.001%) with an overall specificity of 100%. To evaluate the effect of the number of variants on sensitivity and specificity we performed bootstrapping experiments selecting subsets of variants. When using only 16 variants, the I InVision®MRD assay had a mean sensitivity of 95% at 40 ppm (0.004%), 64% at 20 ppm (0.002%) and 22% at 10 ppm (0.001%) with specificity 99.99%. We tested the InVision®MRD assay on samples from cancer patients and detected tumor DNA in patient plasma and diluted samples with estimated tumor fractions at and below 20 ppm (0.002%).
Conclusions
The InVision®MRD assay provides a highly sensitive and specific method to detect MRD in plasma samples of cancer patients. Our results demonstrate excellent sensitivity with analysis of 16 variants which is further enhanced when 48 variants are analyzed. The high sensitivity of our approach is key to providing early information to guide treatment decisions after initial treatment by surgery or chemo/radiotherapy. The approach is generalisable to multiple tumor types and specimen types.
Citation Format: Giovanni Marsico, Garima Sharma, Malcolm Perry, Sophie Hackinger, Tim Forshew, Karen Howarth, Jamie Platt, Nitzan Rosenfeld, Robert Osborne. Analytical development of the RaDaRTM assay, a highly sensitive and specific assay for the monitoring of minimal residual disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3097.