Prognostic Value of BRAFV600E in Peripheral Blood of Children with Langerhans Cell Histiocytosis: A Multicenter Project of the European Consortium for Histiocytosis

Background: Langerhans cell histiocytosis (LCH) is a rare hematological neoplasia of myeloid origin driven by activating mutations in the mitogen-activated protein kinase pathway, most commonly BRAFV600E. The clinical presentation is heterogeneous ranging from single-system (SS) to multi-system (MS) disease, with or without involvement of risk organs (RO), i.e. liver, spleen, bone marrow. Circulating cell free (ccf-) BRAFV600E has been proposed as a prognostic marker, however molecular-based risk stratification is still an unmet need in patients with LCH. Purpose: To assess the prognostic significance of BRAFV600E in different sources of peripheral blood at baseline (before therapy) and at 6 weeks of treatment according to LCH-IV in children with BRAF-mutated LCH. Methods: Peripheral BRAFV600E was measured in five laboratories of the European Consortium for Histiocytosis (Florence-IT, Newcastle-UK, Paris-FR, Amsterdam-NL, Vienna-AU). DNA from circulating-cell-free (ccf, n=307), peripheral blood mononuclear cells (PBMC, n=186), total leukocytes (n=21) or whole blood (n=36) was screened by digital droplet or allele-specific PCR. BRAFV600E levels at baseline and at week 6, in all sources, were correlated to clinical outcome, defined as event (reactivation/treatment intensification) using Mann-Whitney U test. Survival proportions were calculated using the Kaplan-Meier method and Log-rank (Mantel-Cox) test was used to calculate the significance of the difference between survival curves. Pearson correlation coefficient was used to measure the strength of linear associations between ccf and PBMC BRAFV600E. Results: A total of 259 patients were enrolled. Median age was 29 (IQR=13-79) months; disease extension was represented by 49% SS, 27% MS-RO- and 24% MS-RO+. Data at baseline and week 6 of 259 and 121 patients, respectively, were analyzed. At baseline, BRAFV600E levels were highly correlated between ccf and PBMC-DNA in MS-RO- (r=0.97) and MS-RO+ (r=0.87) but less so in single-system (SS) patients (r=0.53). At baseline, higher BRAFV600E levels were detected in ccf- versus PBMC-DNA (median 0.093% vs 0.006%, p=0.0003). BRAFV600E levels were significantly associated with disease stage, with a median of 0% in SS, 0.11% in MS-RO-, 1.69% in MS RO+, in ccf-DNA (p<0.0001); 0% in SS, 0.01% in MS RO-, 0.5% in MS RO+, in PBMC-DNA (p<0.0001). Higher BRAFV600E levels were observed among patients undergoing events within two years, with a median of 0.07% vs 0% in SS (p=0.004), 0.39% vs 0.014% in MS-RO- (p=0.0007), 2.1% vs 0.36% in MS RO+ (p=0.04), in ccf-DNA and 0.006% vs 0% in SS (p=0.003), 0.07% vs 0.002% in MS RO- (p=0.0002), 0.70% vs 0.05% in MS RO+ (p=0.82), in PBMC-DNA, respectively. In all risk groups, it was possible to define a threshold of BRAFV600E positivity that was associated with shorter 2-year event free survival (EFS). Survival analysis showed significant differences among patients on the basis of the selected cut-offs. At baseline, we identified for ccf-DNA a threshold of 0.1% in SS (p=0.0007) and MS RO- (p=0.0004) patients; 1% in MS RO+ patients (p=0.0182); for PBMC, we established a threshold of 0.01% in SS (p=0.0099) and MS RO- (p<0.0001) patients; 1% in MS RO+ patients (p=0.1414). At week 6, longer 2-year EFS was associated with reduction of >50% of ccf-BRAFV600E (p=0.0002) and clearance of PBMC-BRAFV600E (p=0.059). Conclusion: Measurement of BRAFV600E, in ccf- and PBMC-DNA, both offer potential prognostic value in children with LCH. This multicenter international study provides the basis for molecular-based risk stratification at baseline and at early time points of treatment in different disease groups. Incorporation of this approach in next prospective therapeutical trials may confirm its role in guiding treatment decisions.