Circulating tumor DNA analysis as a real-time method for monitoring tumor burden in melanoma patients undergoing treatment with immune checkpoint blockade

Abstract

Background: Assessment of therapeutic activity of drugs blocking immune checkpoints such as CTLA-4 and PD-1/PD-L1 can be challenging, as tumors may seem to enlarge or appear anew before regressing, due to intratumoral inflammation. We assessed whether circulating tumor DNA (ctDNA) levels could serve as an early indicator of true changes in tumor burden in patients undergoing treatment with these agents.

Findings: Tumors from 12 patients with metastatic melanoma undergoing treatment with checkpoint blocking drugs were analyzed for the presence of hotspot somatic mutations in BRAF, cKIT, NRAS, and TERT. Plasma was collected serially from each patient and levels of ctDNA were compared with radiologic and clinical outcomes. In 5 of 10 patients studied, mutations were detected in BRAF(1), NRAS(2), TERT(1) and ALK(1). Analysis of plasma from 4 of 5 patients identified mutations identical to those found in tumor specimens. Plasma ctDNA levels ranged from undetectable (<0.01%) to 5.5% of total circulating cell-free DNA. In 3 patients, increasing ctDNA levels correlated with progressive disease assessed by radiography. In one patient, ctDNA levels increased after undergoing a needle biopsy of a tumor deposit. In another patient, ctDNA levels increased initially as lymphadenopathy progressed by examination, but then became undetectable 3 weeks prior to clinical improvement.

Conclusions: Levels of ctDNA correlated with clinical and radiologic outcomes, and, in one case, preceded eventual tumor regression. Further prospective analysis is required to assess the utility of ctDNA as an early biomarker of clinical outcomes in patients receiving immune checkpoint blocking drugs.

Keywords: Anti-PD-1; Biomarker; Checkpoint blockade; Circulating tumor DNA; Immunotherapy; Ipilimumab.

Figure 1

Correlation of ctDNA measurements with clinical course. A) Increasing levels of ctDNA (NRAS A182G) correlate with progressive disease assessed by radiography in patient #08, a 52-year-old man with metastatic melanoma who received BMS-936559 (anti-PD-L1). B) Levels of ctDNA (BRAF V600E) in patient #03, a 69-year-old woman with metastatic melanoma who received BMS-936559, increased substantially after a needle biopsy of a lower extremity soft tissue metastasis on treatment day 155 (red arrow). SLD, sum of longest tumor diameters.

Figure 2

Clinical course and ctDNA measurements for patient #01, a 68-year-old woman with biopsy-proven unresectable melanoma of the left neck and left supraclavicular regions. She received ipilimumab as first-line therapy, to which she had an “immune-related” response. A) Treatment timeline. Ipilimumab (anti-CTLA-4, 3 mg/kg) was administered intravenously every 3 weeks for 3 doses. ctDNA levels (TERTmut) increased initially as metastatic lymph nodes enlarged on physical examination (week 3), but ctDNA became undetectable at week 6 even though metastatic lymph nodes were still palpable. Significant disease regression was noted clinically 3 weeks later and complete disease resolution was demonstrated on CT and FDG-PET scans performed 4 months after treatment initiation. B) CT and FDG-PET images demonstrating the resolution of palpable cervical lymphadenopathy after administration of ipilimumab.