Cell-free DNA analysis as a molecular tool to monitor response to immune checkpoint inhibition in endometrial cancer.
Authors: Manning-Geist Beryl , Patel Juber Ahamad A , Marra Antonio , Da Cruz Paula Arnaud , Hanlon Etta J , Abu-Rustum Nadeem , Shah Ronak H. , Berger Michael F. , Hensley Martee Leigh , Zamarin Dmitriy , Weigelt Britta , Friedman Claire Frances 
Published: June 2022

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Background: Immune checkpoint inhibitors (ICI) targeting PD-1 have meaningful activity in microsatellite instability high (MSI-H), hypermutated or mismatch repair deficient (dMMR) advanced endometrial cancer (EC). We investigated if high-depth circulating cell free (cf)DNA sequencing can be used to assess MSI status and monitor ICI response in EC patients enrolled in a phase II trial (NCT03241745). Methods: Patients with recurrent/persistent MSI-H/dMMR/hypermutated EC with measurable disease and ≥1 prior lines of cytotoxic therapy were treated from 06/2018-01/2022 with nivolumab until progression of disease (PD) or unacceptable toxicity. Radiologic tumor response was assessed every 12 wks by RECIST 1.1 criteria.Pre-treatment ECs and matched normal blood-derived DNA were subjected to whole-exome sequencing (WES); cfDNA from plasma at baseline, 2 wks and every 6 wks thereafter was subjected to high-depth MSK-ACCESS sequencing (129 genes). MSI in WES and cfDNA was calculated by MSIsensor and ADMIE, respectively. Results: Ten patients with ≥10 ng cfDNA at baseline and 2 weeks after nivolumab initiation were included. Most (80%) had grade 3 endometrioid EC; 70% had MLH1 hypermethylated EC. Two patients had partial response (PR), 3 had stable disease (SD) and 5 had PD on nivolumab. A high-depth sequencing assay captured somatic mutations in cfDNA at baseline in all patients (median, 29.5; range, 2-75). Median circulating tumor (ct)DNA fraction in cfDNA was 13.1% at baseline (range, 0.0-86.0%). In 8 cases, ctDNA fraction was sufficient to perform MSI assessment. Liquid biopsy MSI status matched tumor MSI status assessed by WES: in 7 patients with MSI-H disease by tumor WES, cfDNA MSI status was also MSI-H ( > 0; range, 0.21-5.46); in 1 patient with MSI-low but hypermutated tumor, cfDNA MSI was 0.0. In all cases, changes in ctDNA fractions reflected ICI response. Median ctDNA fraction percent change from week 0 to week 8 was -51.6% (IQR, -6.2, -85.1) in patients with PR and SD vs +17.7% (IQR, -6.6, +356.6) in patients with PD (p = 0.08). In patients with PR, ctDNA fraction decreased at week 2 and stayed low at week 8 and weeks 32-55 of nivolumab in concordance with ongoing response. In 3 patients with SD, ctDNA fractions decreased by week 2. In 1 patient with SD at week 8 and PD at week 22, ctDNA fraction increased from week 8 to week 20. In 5 patients with PD, 4 had increased ctDNA fraction by week 8. The last patient had a stable ctDNA fraction but increasing allele frequency of 2 truncating B2M alterations, possibly associated with ICI resistance. Conclusions: In patients with advanced hypermutated ECs, cfDNA sequencing can be used to accurately detect MSI status. Early changes in ctDNA fraction may be associated with durable response to ICI or may anticipate radiological progression. Future studies may use ctDNA to assess mechanisms of ICI resistance and offer opportunities for adaptive therapy intervention. Clinical trial information: NCT03241745.