Real-Time Genomic Profiling Identifies Novel Mutations and Improved Therapy for Histiocytoses

BACKGROUND: The histiocytic disorders include several heterogeneous diseases including Langerhans cell histiocytosis (LCH,) juvenile xanthogranuloma, Erdheim-Chester disease (ECD,) and Rosai-Dorfman disease. These conditions have variable clinical courses and can be refractory to contemporary therapy, resulting in end-organ damage or even death. The etiopathogenesis of LCH and the other histiocytoses remained unclear for decades, until the identification of recurring BRAF V600E mutations, and more recently mutations in the downstream gene MAP2K1 (encoding the enzyme MEK) in LCH and ECD. Given the high risk of recurrence and the unpredictable response to therapy in some patients, we sought to characterize the genomic landscape of histiocytic lesions in patients in real-time. Our principal goal was to select alternative treatments for patients with inadequate response to standard therapies. As a secondary goal, we aimed to further characterize the biological effects of MAP2K1 mutations found in LCH and ECD, and determine their susceptibility to targeted therapies.

METHODS AND RESULTS:

We used a hybrid capture-based sequencing platform to molecularly profile eighty-five patient samples from patients with one of the above diagnoses. Fifteen patient samples (18%) harbored the BRAF V600E point mutation, and four LCH patients carried a novel 5 or 6 amino-acid in-frame deletion (indel) in BRAF (N486_P490del or N486_T491>K.) Eleven patient samples (13%) harbored activating mutations in MAP2K1. Additional recurrently altered genes included NRAS, KRAS and CDKN2A/B. Transcriptomic profiling also identified several patients with recurrent ALK gene fusions, previously described in other malignancies and recently also identified in histiocytosis not-otherwise-specified. One patient with multisystem LCH with CNS involvement was found to have a BRAF indel. She declined systemic chemotherapy, but agreed to treatment with a targeted agent. Based on the likelihood of resistance to BRAF V600E-specific inhibitors, we started treatment with the MEK inhibitor Trametinib resulting in resolution of disease-associated lymphadenopathy within days, and improvement of CNS symptoms as well. She remains in remission 4 months after the initiation of treatment. In two children, multi-system refractory LCH progressed to secondary HLH (hemophagocytic lymphohistiocytosis). Both demonstrated the presence of BRAF-V600E and their disease promptly responded to the BRAF inhibitor Dabrafenib.

We then characterized the biological behavior of the MAP2K1 mutations using retroviral transduction in order to stably express these mutations in NIH/3T3 and BaF/3 cells. We demonstrate that these mutations all result in constitutive activation at baseline, as evidenced by increased phosphorylation of the target ERK. These mutant forms of MAP2K1 also express sustained activation of ERK in response to EGF stimulation. Additionally, we tested clinically available MEK inhibitors against mutant forms of MAP2K1, and show that all result in a dose-dependent decrease in phospho-ERK levels in vitro, supporting our hypothesis that MEK inhibition is a valid therapeutic approach in the histiocytic neoplasms.

Finally, we demonstrate with an animal model that MAP2K1 is sufficient to induce disease. Using the cre-lox recombinase system in transgenic mice, we selectively express an activated form of MEK in CD11c-positive cells, which is largely restricted to the dendritic cell/macrophage lineage. These mice developed normally, but by a median of 17 weeks of age, mice became moribund and on necropsy exhibited hepatosplenomegaly with extensive infiltration of spleen, liver and lungs with CD68+ macrophages.

DISCUSSION: Genomic profiling identified mutations in majority of patients, including a novel BRAF indel. We further show that these mutations result in activation of the MAP kinase pathway, and that activated MAP2K1 is capable of transforming hematopoietic cells resulting in a multisystem histiocytic disorder in mice. Finally, we demonstrate that available MEK inhibitors efficiently block disease-associated mutations. We propose that all patients with histiocytic neoplasms undergo comprehensive genomic profiling in order to identify potential causal mutations, and clinical trials for histiocytoses include MEK inhibitors in relapsed/refractory disease or even as upfront therapy.