Onsite serial monitoring of high grade serous ovarian cancer (HGSOC) mutations in circulating tumor DNA (ctDNA) via droplet digital PCR.

Abstract

5603 Background: Comprehensive Genomic Profiling (CGP) of tumor and blood samples is increasingly employed for ovarian cancer diagnosis. We used diagnostic CGP sequencing results (523 genes) to identify mutations in HGSOC patients for surveillance of tumor treatment response by an onsite ddPCR assay (Bio-Rad, Hercules, CA). We compared ctDNA mutations to imaging (computed tomography & MRI) and CA125 values across a cohort of HGSOC patients undergoing surgery followed by platinum-based therapy with diverse outcomes. Methods: CGP mutations from matched index tumor, plasma and buffy coat cell samples (TSO-500, Illumina) were used to identify tumor- and patient-specific, coding mutations for interrogation of serial blood samples. Concordance was high for tumor and blood (MED = 97.1%, Q1: 93.3%, Q3: 98.8%) and 15 patients with inclusive longitudinal samples were selected for retrospective analysis of ctDNA samples versus CA-125, imaging and outcomes. Mutation variant allele frequency (VAF) suitable for primer construction ranged in variant allele frequency (VAF) from 0.5 to 24.4% (MED = 2.5%, Q1: 1.2; Q3: 3.8%) in cfDNA. A lower limit of detection (LLOD) of 1 to 2 copies/nanogram cell free DNA (cfDNA) was established using synthetic gene Block targets and FFPE tumor dilutions. Results: There was a significant correlation (r 2 = 0.85, p<0.0001) of initial target cfDNA VAF and numbers of mutation copies detected per nanogram cfDNA. There was no significant correlation of mutation copies with index or serial CA125 values (r 2 =0.11, p=0.38) over the wide range of CA125 values across the patient cohort. However, all patients displayed parallel dynamic changes in multiplex ctDNA mutations and CA125 values with variations in disease status and outcome over 1-3 years. Platinum-sensitive patients with a sustained response to therapy (n=3) based on imaging and CA 125 values displayed rapid elimination of tumor mutations via ddPCR prior to stabilization of “normal” CA125 values. Absence of mutations was further validated by interrogating 5X concentrated patient cfDNA aliqouts. Three patients (platinum-resistant) that initially responded to treatment by imaging and declining CA 125 values never achieved ctDNA mutation levels below the LLOD. These patients subsequently displayed tumor escalation with correlative increases in ctDNA mutation levels and CA125 values. Conclusions: Patient mutation monitoring via ddPCR of blood samples collected and processed onsite, provided rapid, iterative and quantitative results consistent with radiographic imaging and CA 125 assays. In addition, durable remission cases were characterized by rapid loss of mutations detected by ctDNA ddPCR assays prior to imaging and diminution of CA125 values. Low levels of ctDNA mutations above LLOD persisted in responders who otherwise achieved normal CA125 values but later developed disease progression.