Abstract B79: Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease
Authors: Simko Stephen J. , Berres Marie-Luise , Lim Karen Phaik-Har , Peters Tricia , Price Jeremy , Lupo Philip J. , Hicks M. John , Shih Albert , Heym Kenneth , McClain Kenneth L. , Merad Miriam , Sarabia Stephen , Lopez-Terrada Dolores , Allen Carl E. 
Published: May 2015

Abstract
Purpose: Langerhans Cell Histiocytosis (LCH) is a clonal disorder characterized by inflammatory lesions with characteristic CD207+ dendritic cells (DCs). LCH has variable clinical presentations ranging from single lesions to potentially fatal multi-system “risk organ” disease. The etiology of LCH remains elusive, with debate of LCH as an inflammatory versus malignant disorder unresolved. The first recurrent somatic genetic mutation in LCH, BRAF-V600E, was recently reported in 57% of LCH lesions (Badalian-Very et al., 2010). Here we investigate the clinical significance of BRAF-V600E as a potential biomarker of risk organ or refractory disease.
Methods: Formalin-fixed, paraffin embedded (FFPE) tissue, peripheral blood, and sorted peripheral monocyte/dendritic cell populations were genotyped for BRAF-V600E mutations with allele-specific, real-time PCR assays. The presence of BRAF-V600E mutations was correlated with clinical variables and analyzed with standard statistical methods. A subsequent validation set of 8 patient peripheral blood samples was identified for quantitative analysis of levels of BRAF-V600E positive cells with the BRAF Rotor-Gene Q (RGQ) PCR assay (Qiagen, Valencia, CA), and concordance with results from Qiagen qBiomarker qPCR assay was determined. Quantitation was performed using a delta Ct method of the BRAF-V600E assay, and results were reported as percentage of mutant cells in a background of wild-type cells using standard curves.
Results: Lesions from 100 patients with LCH were genotyped, and 64% carried the V600E mutation, which localized to the infiltrating CD207+ DCs. In 16 patients with more than one lesion, BRAF status remained fixed, suggesting somatic mutation of BRAF is an early event. BRAF-V600E did not define specific clinical risk groups or impact overall survival, but it was associated with approximately two-fold higher risk of relapse (p=0.04). Furthermore, the cellular compartment carrying the mutation correlated with disease severity: the ability to detect BRAF-V600E in circulating mononuclear cells defined risk organ LCH with 100% sensitivity/87% specificity. The ability to detect BRAF-V600E in circulating blood cells in patients with risk organ LCH defined clinically detectable disease with 97% sensitivity/100% specificity. For development of a clinically reproducible minimal residual disease assay that would be CLIA-compliant and commercially available, a separate validation sample set was identified. With a limit of detection of 0.02% mutant cells in a background of wild-type cells, the RGQ assay correctly detected BRAF-V600E mutations in all 8 validation specimens and in known BRAF-V600E positive cell lines and did not detect mutations in 10 additional BRAF-V600E mutation negative clinical specimens (analytical specificity = 100%). The RGQ quantitative results correlated with the qBiomarker assay results (R2=0.924) with comparable analytical sensitivity.
Conclusions: The molecular foothold of BRAF at the base of LCH pathogenesis will allow therapeutic strategies to move beyond empiric observation to risk-stratified and targeted approaches. Furthermore, effectiveness of therapy may be tested by following BRAF-V600E in peripheral blood cells as a marker of residual disease. Development of validated assays to test for BRAF-V600E in peripheral blood will assist in assigning risk status and assessing therapeutic response.
Citation Format: Stephen J. Simko, Marie-Luise Berres, Karen Phaik-Har Lim, Tricia Peters, Jeremy Price, Philip J. Lupo, M. John Hicks, Albert Shih, Kenneth Heym, Kenneth L. McClain, Miriam Merad, Stephen Sarabia, Dolores Lopez-Terrada, Carl E. Allen. Detectable BRAF-V600E mutation in circulating peripheral blood of patients with Langerhans cell histiocytosis correlates with risk organ involvement and residual disease. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B79.