BRAF p.V600E mutations are detected in greater than 50% of pediatric Langerhans cell histiocytosis (LCH) lesions. However, the use of mutation-specific BRAF V600E immunohistochemistry (IHC) as a surrogate for molecular testing in pediatric LCH is unknown. We tested the mutation-specific BRAF V600E monoclonal antibody (clone VE1) in formalin-fixed, paraffin-embedded LCH samples from 26 pediatric patients (14 males and 12 females, ages 7 mo-17 y) using allele-specific real-time polymerase chain reaction (PCR) with a limit of detection of 0.5% as the comparative gold standard. BRAF VE1 staining was scored for both intensity (0-3+) and percentage of immunoreactive tumor cells (0%-100%). BRAF VE1 immunoreactivity was determined using both lenient (≥1+, ≥1%) and stringent (≥2+, ≥10%) scoring criteria. Using lenient-scoring criteria, we found that the sensitivity and specificity of IHC compared with allele-specific real-time PCR were 100.0% and 18.2%, respectively. The poor specificity of lenient IHC analysis was attributable to weak, 1+ staining in both BRAF-mutated and wild-type LCH. Using stringent-scoring criteria, we found that specificity improved to 100.0% at the expense of sensitivity that decreased to 80.0%. Stringent scoring generated 3 false-negative results, but in all cases, neoplastic tissue comprised less than 5% of the stained section and/or the specimen was decalcified. In conclusion, highly sensitive molecular assays remain the gold standard for BRAF mutation analysis in LCH paraffin-embedded lesions. To avoid false-positive results, unequivocal VE1 staining of 2+ intensity in greater than or equal to 10% neoplastic histiocytes is required. However, negative VE1 results require additional studies to exclude false-negatives, and stringent-scoring criteria may not be optimal for scant or decalcified specimens.

Background Langerhans Cell Histiocytosis (LCH) is a clonal lymphoproliferative 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 “High Risk” 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). In this study, we investigate the clinical significance of BRAF-V600E and identify cells carrying the mutation to determine the origins of LCH. Methods Lesions, peripheral blood, peripheral monocyte/dendritic cell populations, and hematopoietic stem cells were genotyped for the BRAF-V600E mutation with real-time PCR. The presence of the BRAF-V600E mutation was correlated with clinical variables and analyzed with standard statistical methods. Colony-forming unit assays were used to test the hematopoietic potential of CD34+ cells purified from bone marrow aspirate. Results Lesions from 100 patients with LCH were genotyped, and 64% percent 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 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 High-Risk LCH with 100% sensitivity/87%specificity. The ability to detect BRAF-V600E in circulating blood cells in patients with High-Risk LCH defined clinically detectable disease with 97% sensitivity/100% specificity. Analysis of sorted populations localized the BRAF-V600E to CD11c+ and CD14+ fractions in peripheral blood, and to CD34+ cells in bone marrow. Potential of the CD34+ hematopoietic stem cells with the BRAF-V600E mutation to differentiate into myeloid precursors was verified with in vitrocolony-forming unit assays. 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. We hypothesize that High-Risk LCH arises from somatic mutation of an immature myelomonocytic precursor cell, where Low-Risk disease arises from somatic mutation of tissue-restricted DC precursors. We therefore propose classifying LCH as a bone fide myeloid neoplasia in which BRAF-V600E expression in precursor versus mature dendritic cells defines clinically distinct risk-groups. Disclosures: No relevant conflicts of interest to declare.

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.

Introduction Despite indistinguishable histology and the common feature of Birbeck granules in lesion biopsies, clinical presentation of patients with Langerhans Cell Histiocytosis (LCH) is highly variable, from single lesion cured by curretage, to multi-system disease requiring aggressive chemotherapy or stem cell transplant. Risk stratification for Langerhans Cell Histiocytosis has historically assigned clinical risk groups based on anatomic location and extent of LCH lesions, which is the basis for dose and duration of chemotherpy on recent Histiocyte Society trials. In this study, we test the hypothesis that distinct subgroups of patients with LCH may be identified by relative levels of circulating biomarkers. Methods Pre-therapy plasma was collected on 97 patients with LCH (82 Pediatric: 17 High-Risk, 23 Multisystem/Multifocal “Non-risk”, 42 Single Lesion “Non-risk”; 15 Adult: 5 High-Risk, 5 Multisystem/Multifocal “Non-risk”, 5 Single Lesion “Non-risk”) and 49 control subjects (32 Pediatric, 17 Adult). Quantitative levels of plasma proteins (158 analytes) was determined by multiplex analysis with Millipore MagPix kits and the Luminex plate reader. Data were analyzed with both unsupervised and supervised methodologies. Results Consensus clustering with non-negative matrix factorization (NMF) clusters identified three groups which were analyzed along with clinical categories. Significant clinical variables included age (adult samples clustered in NMF group 1) and LCH risk category (High-Risk LCH samples clustered in NMF group 3). Samples from patients with the BRAF-V600Emutation or relapse within 1 year did not cluster into any NMF group with signifiance. Additionally, supervised analysis identified specific molecules that were significantly differentially expressed between different clinical categories after multiple testing correction (FDR<0.10): Pediatric LCH vs Adult LCH (72 molecules significant, largest differences in MMP-3, MMP-2 and osteopontin); Pediatric Control vs Pediatric LCH (66 molecules significant, largest differences in SDF-1a, IL-20, MIP-1d, FGF-2 and sIL-4R); Pediatric Low-Risk vs Pediatric High-Risk (47 molecules significant, largest differences in sTNF-R11, sTNF-RI, I-309, sIL2Ra and osteopontin). While previous studies have analyzed expression differences of cytokines in LCH lesions and plasma, in this study the most striking differences are between control vs LCH samples are chemokine molecules. The largest differences between Low-Risk and High-Risk LCH patients include inflammatory cytokines and receptors. Conclusions Despite mounting evidence supporting pathogenesis of LCH as a myeloid neoplasia arising from immature dendritic cell precursors, these results are consistent with exuberant chemokine and cytokine expression in patients with active LCH, supporting a potential role for inflammation in pathogenesis. This study demonstrates the feasibility of identifying novel LCH sub-groups according to plasma protein profiles with unsupervised analysis, and significant differences can be detected in protein levels between clinical risk groups. Future studies will validate the clinical utility of plasma biomarkers in diagnosis, risk-stratification and determining response to therapy. Finally, feasibility of collecting plasma compared to viable lesions makes plasma studies ideal for prospective collection and analysis in cooperative group studies. Disclosures: No relevant conflicts of interest to declare.