Abstract 4542: Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay

Abstract

Introduction: Liquid biopsy-based detection and monitoring of tumor-specific somatic mutations in cell-free DNA has great potential to improve patient care by detecting cancer early, assisting adjuvant therapy decision-making, monitoring residual disease, determining treatment effects, assessing relapse and the consequent need for follow-up intervention. We have developed a novel highly sensitive and specific approach for the detection and quantification of circulating tumor DNA (ctDNA) by tracking personalized cancer signatures in plasma. We recently described our ability to detect ctDNA presence longitudinally in patients with non-small cell lung cancer (NSCLC) and have validated this personalized approach for detection of tumor DNA in plasma, and made it available for research use only (RUO). Summary of Experimental Procedures: This study describes results of the Signatera RUO assay's analytical validation test titrations of cancer cell lines and their matched normal counterparts. Multiplex-PCR (mPCR) assay pools specific to the tumor mutational signatures were derived from whole-exome data analysis of the tumor cell line and its corresponding matched normal cell line. Tumor DNA spike-in mixtures were amplified using the mPCR assays and then barcoded and sequenced at ultra-high depth on the Illumina HiSeq instrument (average > 100,000 reads per target). This study also demonstrates the ability to detect ctDNA using Signatera RUO assay's personalized somatic mutations in patients with epithelial and non-epithelial ovarian cancer. Results/Data Summary: The Signatera RUO analytical validation results demonstrate a variant-level sensitivity of ~60% at spiked-in tumor DNA concentrations of 0.03% along with an assay specificity of 99.9% in wild-type DNA. By targeting 16 somatic mutations present in the tumor, an estimated LOD of 0.01% tumor DNA in a patient's plasma is achieved. The Signatera RUO assay also demonstrates the ability to detect ctDNA in plasma down to less than 0.1% VAF at baseline (prior to treatment) in epithelial and non-epithelial ovarian cancer. Conclusion: The Signatera RUO assay provides a unique method to noninvasively detect ctDNA by assaying for personalized cancer signatures in plasma by ultra-high-depth sequencing of custom-made multiplex PCR assays, with high sensitivity and high specificity. Our study demonstrates the potential of this technology to improve the current standard of care by enabling early recurrence detection and the monitoring of treatment effectiveness across several tumor types. Citation Format: Himanshu Sethi, Raheleh Salari, Samantha Navarro, Prashanthi Natarajan, Ramya Srinivasan, Scott Dashner, Tony Tin, Mustafa Balcioglu, Ryan Swenerton, Bernhard Zimmermann. Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay [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 4542.

Prognostic Gene Expression Profiling in Melanoma: Necessary Steps to Incorporate into Clinical Practice

“…Thus, in contrast to other GEP tests, Signatera directly detects presence of metastatic disease (rather than risk of metastasis) and can be repeated over time to assess CM recurrence or response to therapy. Signatera has demonstrated the ability to detect molecular residual disease up to 2 years earlier than radiographic imaging in breast, bladder, colorectal and non-small-cell lung cancers with a 98% positive predictive value for relapse [17][18][19][20][21]. In a Phase II trial (INSPIRE) conducted in 70 patients with advanced cancers (including head and neck, triple-negative breast, ovarian cancers and CM), Signatera was used to assess ctDNA at baseline and after the third cycle of treatment with pembrolizumab.…”

Use of Circulating Tumour DNA to Assess Minimal Residual Disease in Gastrointestinal Cancers

“…For example, the Signatera ctDNA test, which is commercialized and has received US Food and Drug Administration (FDA) Breakthrough Device Designation, requires WES of a patient's primary tumour and the buffy coat to identify 16 clonal tumour-specific somatic mutations that are not driver mutations under selection pressure and that are not present in the germline. 50,51 Sixteen PCR primers are designed for the identified single-nucleotide variants, and a patient's peripheral blood is drawn at pre-specified times and then tested against the multiplex PCR with ultradeep sequencing. If at least two mutations are detected, this results in a positive read.…”

Detecting liquid remnants of solid tumors treated with curative intent: Circulating tumor DNA as a biomarker of minimal residual disease (Review)

“…Signatera™, which has received three US Food and Drug Administration (FDA) breakthrough device designations for MRD testing (one in May, 2019 and two in March, 2021), utilizes a patient's top 16 somatic variants from the primary tumor detected by WES to personalize a 16-plex multiplex PCR-based NGS approach for the detection of MRD in plasma. Each target can achieve an average depth of 100,000× with ultra-deep sequencing, and this approach has exhibited a sensitivity >98% at 0.01-0.02% ctDNA concentrations ( 60 ). The total TAT of the Signatera™ assay is between 4–5 weeks, including ~2 weeks for tumor sequencing results, 1 week for designing the patient-specific PCR primers and assay, and 1–2 weeks for post-treatment blood sample testing.…”