Personalized Cancer Monitoring Assay for the Detection of ctDNA in Patients with Solid Tumors

Abstract

Background: Highly sensitive molecular assays have been developed to detect plasma-based circulating tumor DNA (ctDNA), and emerging evidence suggests their clinical utility for monitoring minimal residual disease and recurrent disease, providing prognostic information, and monitoring therapy responses in patients with solid tumors. The Invitae Personalized Cancer Monitoring^™ assay uses a patient-specific, tumor-informed variant signature identified through whole exome sequencing to detect ctDNA in peripheral blood of patients with solid tumors.

Methods: The assay's tumor whole exome sequencing and ctDNA detection components were analytically validated using 250 unique human specimens and nine commercial reference samples that generated 1349 whole exome sequencing and cell-free DNA (cfDNA)-derived libraries. A comparison of tumor and germline whole exome sequencing was used to identify patient-specific tumor variant signatures and generate patient-specific panels, followed by targeted next-generation sequencing of plasma-derived cfDNA using the patient-specific panels with anchored multiplex polymerase chain reaction chemistry leveraging unique molecular identifiers.

Results: Whole exome sequencing resulted in overall sensitivity of 99.8% and specificity of > 99.9%. Patient-specific panels were successfully designed for all 63 samples (100%) with ≥ 20% tumor content and 24 (80%) of 30 samples with ≥ 10% tumor content. Limit of blank studies using 30 histologically normal, formalin-fixed paraffin-embedded specimens resulted in 100% expected panel design failure. The ctDNA detection component demonstrated specificity of > 99.9% and sensitivity of 96.3% for a combination of 10 ng of cfDNA input, 0.008% allele frequency, 50 variants on the patient-specific panels, and a baseline threshold. Limit of detection ranged from 0.008% allele frequency when utilizing 60 ng of cfDNA input with 18-50 variants in the patient-specific panels (> 99.9% sensitivity) with a baseline threshold, to 0.05% allele frequency when using 10 ng of cfDNA input with an 18-variant panel with a monitoring threshold (> 99.9% sensitivity).

Conclusions: The Invitae Personalized Cancer Monitoring assay, featuring a flexible patient-specific panel design with 18-50 variants, demonstrated high sensitivity and specificity for detecting ctDNA at variant allele frequencies as low as 0.008%. This assay may support patient prognostic stratification, provide real-time data on therapy responses, and enable early detection of residual/recurrent disease.

Fig. 1

Workflow for the Invitae Personalized Cancer Monitoring assay. This tumor-informed assay utilizes whole exome sequencing of matched tumor and germline specimens to identify tumor-specific variants. A proprietary algorithm selects tumor-specific variants to design a patient-specific panel. The designed panel is then used to generate next-generation sequencing libraries, featuring anchored multiplex polymerase chain reaction chemistry, and unique molecular identifiers, from cell-free DNA (cfDNA) extracted from the patient’s plasma specimen. From the sequencing data, stringent data quality-control measures and a proprietary circulating tumor DNA (ctDNA)-calling algorithm are used to assess the ctDNA status in the patient’s specimen. FFPE formalin-fixed paraffin-embedded

Fig. 2

Effect of tumor content on variants suitable for patient-specific panel (PSP) design. Samples diluted to mimic low tumor cellular content that range from 5 to 35% were sequenced with whole exome sequencing at DNA input levels of (A) 50 ng, (B) 100 ng, and (C) 200 ng. The sequencing output was analyzed by the proprietary panel design algorithm to determine the expected range of variants suitable for the PSP design at the different tumor contents and DNA input levels. Results showed that the number of variants suitable for the PSP design was correlated with tumor content but not with the DNA input level

Fig. 3

Precision and reproducibility of variants in whole exome sequencing. Reproducibility and precision were evaluated using three reference samples: Seraseq Tumor Mutation DNA Mix AF10, Seraseq Myeloid Mutation DNA mix, and NA12878. To evaluate reproducibility, next-generation sequencing libraries of these samples were prepared in triplicate by two operators in three batches. Overall precision and reproducibility concordance by overall variant types and four different variant types (single nucleotide variation [SNV], deletions, insertions, and duplications) are shown in the left panels, breakdowns to variant allele frequency (AF) to 5–10% is shown in the middle panels and 10–15% is shown in the right panels

Fig. 4

Assay sensitivity with different panel sizes. Patient-specific panels with 16–50 variants were assessed along with cell-free DNA input amounts (10–60 ng) and analytical thresholds (baseline and monitoring). A. The y-axis shows sensitivity (%) and the x-axis shows variant allele frequency (AF) [%]. Larger panel size, higher input amount, and baseline threshold correlated with detection at lower AFs. B. Heat plot of sensitivity data points