Concurrent enhancer of Zeste homolog 2 (EZH2) over-expression with BRAF<sub>V600E</sub> mutation predicts a poor outcome in patients with Langerhans cell histiocytosis (LCH): A pilot study.
Authors: Aditi Saha, Junmin Whiting, Jongphil Kim, Felix Sanjay, Lugen Chen, Dahui Qin, Ling Zhang, Lubomir Sokol
Journal: Journal of Clinical Oncology (2024)

e18536 Background: Prior studies demonstrated upregulated EZH2 protein expression and p-ERK1/2 cascade signaling in histiocytic and dendritic cell neoplasms. BRAF V600E is the most common driver mutation reported in LCH patients (34-64%). In melanoma studies, a signaling pathway with BRAF V600E activating mutation resulted in upregulation of EZH2. The frequency of EZH2 co-expression with BRAF V600E is unknown in LCH. Given the limited durable responses after discontinuation of treatment with BRAF inhibitors in histiocytic disorders, we investigated the prognostic role of EZH2 overexpression and BRAF V600E mutation in LCH. Methods: We analyzed a cohort of 27 consecutive patients (pts) diagnosed with LCH at Moffitt Cancer Center and Tampa General Hospital between 2010-2022. Immunohistochemical (IHC) staining using antibodies against EZH2 (D2C9 clone, Cell Signaling) and BRAF V600E (VE1 clone, Ventana medical systems) were performed. Expression was assessed by an IHC score of stain intensity (0, 1+, 2+ and 3+) multiplied by percent positive cells (H score). H score ≥ 30 was considered as positive. Pyrosequencing was performed to detect BRAF V600E mutations and correlated with VE1 staining results. A positive outcome from either IHC or PCR was deemed indicative of mutation positivity, given the robust presence of mutation representation in melanoma studies through IHC. Results: Entire cohort were grouped for pts who had both EZH2 overexpression and BRAF V600E mutation (EZH2/BRAF V600E ) versus those who did not. Out of 27 pts, 25.9% (n=7) had both EZH2/BRAF V600E . EZH2/BRAF V600E group differed from control group by age ( p=0.046). BRAF V600E mutation was identified in 37.03% (10) pts, and 40.7% (11) showed expression of VE1 by IHC study. Discrepancies between PCR and IHC study were observed in 4 pts and potentially attributable to the restricted availability of tissue samples. 63% and 48.1% of pts were positive for EZH2 and BRAF V600E respectively. 6 pts were observed, 8 underwent surgery with or without radiation, 12 received induction chemotherapy, and only 1 received a BRAF inhibitor. After 18.6 months of median follow up, 92.6% pts were alive. Overall response rate (ORR) to the 1st line therapy was 55.6%. In univariate analysis of OS, H score and BRAF V600E mutation trended towards worse OS but were not statistically significant owing to small sample size. However, BRAF V600E mutation and EZH2 over expression demonstrated a poor overall survival (OS) ( p=0.012). Conclusions: Our study indicates that concurrent presence of BRAF V600E mutation and EZH2 overexpression correlates with poor OS in LCH pts. We hypothesize that in a subset of LCH pts combination therapy targeting both EZH2 and BRAF V600E may improve ORR or overcome resistance to BRAF inhibitor monotherapy. However, the results of our pilot study need to be validated on a larger sample set.