Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression

Abstract

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

Keywords: BRAF; Biomarker; Circulating tumor DNA (ctDNA); Lactate dehydrogenase (LDH); Melanoma; NRAS.

Figure 1

Overview of patient enrollment, sample collection and analysis. Of 43 eligible patients, 31 had tumors with BRAF V600E, V600K, NRAS Q61R, L, or K mutations. See text for tumor genotyping details.

Figure 2

Comparison of ctDNA and LDH sensitivity to detect measurable disease pre‐treatment. Panel A ctDNA and LDH results are categorized by patient pre‐treatment RECIST score at the time of sample collection. All samples and radiographic scans were obtained within 45 days prior to treatment initiation. The percentage of elevated results for each marker, stratified by RECIST score, is shown along with the average marker level for each RECIST category. Panel B displays a bar graph of the overall sensitivity to detect measurable disease by biomarker, based on the data from panel A.

Figure 3

Paired comparison of ctDNA and LDH sensitivity to detect measurable disease at progression. Panel A shows a 2 × 2 table of matched pairs. LDH and ctDNA levels were obtained within 15 days of a disease progression event. If more than one LDH value was available in this time window, the LDH values were averaged. Panel B provides more detail of the results used for panel A. Specifically, ctDNA and LDH results are categorized by patient progression event, including the RECIST score at the time of sample collection, or the nature of a non‐RECIST progression event (e.g. new brain metastases). The percentage of elevated results for each marker, stratified by RECIST score or non‐RECIST progression event, is shown along with the average marker level for each category.

Figure 4

Serial monitoring of ctDNA in melanoma patients undergoing systemic treatment. The graphs display the RECIST scores, LDH levels, ctDNA copies/ml, and clinical course for 2 patients. To enable the plotting of all three data sets on the same graph the left y‐axis plots both RECIST scores (in total cm) and LDH levels (in IU/L/100). The right y‐axis plots ctDNA (copies/ml). Shaded areas represent approximate time period during which the patient underwent the specified treatment. Patient A clinical event markers denote the following: 1. Metastatic brain progression (day 73); 2. Deceased (day 146). Patient B clinical event markers denote the following: 1. New brain metastasis (day 34); 2. Increasing brain metastasis (day 231); 3. Two new brain metastases (day 323); 4. Hospice (day 367).