Prospective blinded study of BRAFV600E mutation detection in cell-free DNA of patients with systemic histiocytic disorders

Abstract

Patients with Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) have a high frequency of BRAF(V600E) mutations and respond to RAF inhibitors. However, detection of mutations in tissue biopsies is particularly challenging in histiocytoses due to low tumor content and stromal contamination. We applied a droplet-digital PCR assay for quantitative detection of the BRAF(V600E) mutation in plasma and urine cell-free (cf) DNA and performed a prospective, blinded study in 30 patients with ECD/LCH. There was 100% concordance between tissue and urinary cfDNA genotype in treatment-naïve samples. cfDNA analysis facilitated identification of previously undescribed KRAS(G12S)-mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAF(V600E) mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a noninvasive biomarker to monitor response to therapy in LCH and ECD.

Significance: Patients with BRAF(V600E)-mutant histiocytic disorders have remarkable responses to RAF inhibition, but mutation detection in tissue in these disorders is challenging. Here, we identify that analysis of plasma and urinary cfDNA provides a reliable method to detect the BRAF(V600E) mutation and monitor response to therapy in these disorders.

Figure 1. BRAFV600E mutant allele burden in cell-free DNA (cfDNA) of urine and plasma based on BRAFV600E tissue genotype result

(A) Pie chart representation of BRAFV600E mutational genotype as determined by initial tissue biopsy (left) or urinary cfDNA analysis (right). Results were recorded as BRAFV600E mutant (yellow), BRAFV600E wildtype (white), or result indeterminate (grey). (B) Ratio of BRAFV600E:BRAF wildtype in urinary cfDNA of patients based on BRAF mutational status as determined from tissue biopsy (BRAFV600E mutant, BRAF wildtype, or BRAF indeterminate). Lines and error bars for BRAFV600E mutant and BRAF wildtype patients represents mean ± standard error of the mean. (C) Ratio of BRAFV600E:BRAF wildtype in plasma cfDNA of patients based on BRAF mutational status as determined from tissue biopsy. Each point represents a single test result from the initial assessment of BRAFV600E:BRAF wildtype allelic ratio in cfDNA. Dotted points represent samples collected during therapy. The red dashed line indicates the cutpoint defining a positive versus negative cfDNA result.

Figure 2. Effect of therapy on BRAFV600E mutant allele burden in cell-free DNA (cfDNA) of systemic histiocytosis patients

(A) Comparison of BRAFV600E allele burden in treatment naïve urine samples compared with urinary samples acquired anytime during therapy. (B) Effect of RAF inhibitors on cfDNA BRAFV600E mutant allele burden in 7 consecutive patients monitored weekly during treatment with RAF inhibitors. The initial sample in each patient is prior to initiation of therapy. The red dashed line indicates the cutpoint defining the presence or absence of the BRAFV600E mutation.

Figure 3. Dynamic monitoring of serial urinary cell-free DNA (cfDNA) BRAFV600E mutant allele burden in systemic histiocytosis patients

(A) Gadolinium-enhanced T1 MRI images of ECD involvement of brain (green arrows), and ¹⁸F-FDG-PET images of disease in the right atrium (asterisk) and testes (asterisk), pre-dabrafenib and after 2 months of dabrafenib. (B) Urinary BRAFV600E cfDNA results throughout this same patient’s therapy. (C) Urinary BRAFV600E cfDNA results of an ECD patient treated with anakinra followed by a period of treatment cessation and then initiation of vemurafenib. (D) Maximal intensity projection (MIP) images of ¹⁸F-FDG-PET scan images (top) demonstrating tibial infiltration by ECD pre-vemurafenib (left scan), following 10 weeks of vemurafenib (middle scan), and then 16 weeks after vemurafenib discontinuation (right scan) in an ECD patient with accompanying urinary cfDNA results for each time point (below).