BRAF-V600E Mutation on Circulating Cell-Free DNA Is a Promising Biomarker of High-Risk Adult Langerhans Cell Histiocytosis
Authors: Kobayashi Masayuki , Ohno Nobuhiro , Fukuyama Tomofusa , Kawamata Toyotaka , Uchimaru Kaoru , Tojo Arinobu 
Published: October 2019

Abstract
Background: Langerhans cell histiocytosis (LCH) is a rare disorder characterized by clonal proliferation of Langerhans cells and significant infiltration of immune cells. Although the precise pathophysiology is to be elucidated, oncogenic BRAF-V600E mutation could be detected in LCH lesions from the majority of patients (Badalian-Very, et al. Blood. 2010). Furthermore, Berres ML et al. found that patients with active, high-risk LCH carried BRAF-V600E in circulating CD11c+/CD14+ cell fractions. In patients with various kinds of cancers, circulating cell-free DNA (cfDNA) in peripheral blood contains cancer-derived genomic DNA and has been applied to non-invasive diagnostic procedure, so called liquid biopsy. In the very recent report, BRAF-V600E was successfully detected on cfDNA from patients with colorectal cancer in 100% sensitivity and specificity (Thierry AR, et al. Nature Med. 2014). Then, in this study, we evaluated BRAF mutation on cfDNA as a potential biomarker of LCH using allele-specific quantitative polymerase chain reaction (ASQ-PCR).
Methods: We cloned normal and mutant BRAF alleles that include exon 15 and neighboring sequences into pCR2.1 to make the standard curve. cfDNA was prepared from plasma of adult LCH patients and was subjected to genotyping BRAF alleles by ASQ-PCR, which was specifically designed for detection of BRAF-V600E with a 3'-phosphate-modified oligonucleotide blocker according to Thierry AR, et al. Mutant BRAF load was estimated from the standard curve in each assay and was expressed as the percentage of mutant alleles to total number of alleles.
Results and Discussion: Plasma cfDNA was prepared from 8 adult patients with LCH listed in Table.1 as well as normal subjects including cancer-free patients. The mean quantity of recovered cfDNA in LCH vs normal was 316.5pg/ml (median, 290.4) vs 92.0pg/ml (median, 91.8). Three high-risk patients with active multiple lesions were positive for BRAF-V600E. In these patients, the mean ratio of mutant BRAF alleles to total was 3.25 % (median, 2.59 %). Next, we compared the sensitivity of ASQ-PCR of BRAF-V600E between cfDNA and cellular DNA in the same blood sample. Naturally, too much more DNA was recovered from mononuclear cells than plasma in the same blood volume, whereas the ratio of mutant to total alleles was more than 10-fold higher in cfDNA, suggesting that LCH-derived genomes are significantly enriched in cfDNA compared with cellular DNA, and that cfDNA is more adequate for liquid biopsy in LCH with BRAF-V600E. Then, in a BRAF-V600E-positive patient, we followed the mutant BRAF load during the course of initial chemotherapy. The ratio of mutant to total alleles was estimated as 1.00% prior to chemotherapy and not detectable after one course of chemotherapy consisting of vinblastine, prednisolone, methotrexate and 6-mercaptopurine. The validity of this ASQ-PCR data was confirmed by a series of routine imaging analysis performed at the same time. Taken together, ASQ-PCR of BRAF-V600E on cfDNA may contribute to planning of risk-based treatment as well as monitoring of treatment efficacy in LCH, especially in an active, high-risk group. A number of BRAF-targeted inhibitors have been approved or under clinical trial for various cancers with BRAF mutant, and one of those, vemurafenib is also active against LCH with BRAF-V600E (Haroche J, et al. Blood. 2013). Hereafter, the utility of BRAF-V600E on cfDNA should be validated in a large cohort of LCH patients.
Conclusion: In spite of a very small cohort, we demonstrated the feasibility of BRAF-V600E on cfDNA as a biomarker of active, high-risk LCH.
Disclosures: This work was supported by grants from "Japan LCH Study Group" (JLSG).
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Disclosures
Kobayashi: Japan LCH Study Group (JLSG): Research Funding. Fukuyama:Pfizer Japan Inc.: Employment. Tojo:Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Celgene: Consultancy; Novartis Pharmaceuticals Japan.: Research Funding, Speakers Bureau.