Non-V600E BRAF Gene Alterations in Hairy Cell Leukemia

Background: Hairy cell leukemia (HCL) is an indolent B-cell malignancy typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1, and tartrate-resistant acid phosphatase (TRAP). Treatment with purine analog monotherapy is highly effective, leading to durable complete remission (CR). In contrast, HCL variant (HCLv), lacking CD25, CD123, annexin A1, and TRAP, achieves poor response to purine analogs, with partial responses in <50%, CRs in <10%, and poorer overall survival from diagnosis. Classic HCL is associated with the BRAF V600E mutation in >90% of cases, serving as both a diagnostic marker and target for class I BRAF inhibitors. In contrast, not only HCLv but also a fraction of immunophenotypically classic HCL cases lack BRAF V600E. Some of these cases exhibited unmutated IGHV4-34 immunoglobulin rearrangement, which is associated with mutations in MAP2K1 (MEK1) downstream of BRAF. Reports of non-V600E BRAF mutations have been rare, including 3 patients with D449E, F468C, or F595L, and 2 patients with both V600E and either S602T or W607L. Methods: Looking for unusual mutations, we performed whole exome sequencing (WES) and/or TrueSight Oncology (TSO500) in 213 HCL/HCLv patients, including 88 with HCLv and 21 with IGHV4-34+ HCL. The HCL/HCLv cells constituted >80% of B-cells and were partially purified using CD19 microbeads followed by positive fraction isolation. BRAF V600E was also tested by droplet digital PCR. Results: We found non-V600E BRAF genetic alterations in 18 patients. Surprisingly, all 18 patients had the classic rather than variant phenotype. Of the 18 patients, 12 had a single non-V600E BRAF mutation, and 6 had V600E along with 1 (n=5) or 2 (n=1) non-V600E BRAF co-mutations. These non-V600E mutations included missense mutations, deletions, and insertions located in exons 8, 12, and 15. None of the 19 non-BRAF mutations observed in the 18 patients had been previously reported in HCL/HCLv, some had been reported in other malignancies, and others were novel alterations found in the protein kinase domain. The most common non-BRAF mutation was p.N486_P490del (n=6), always in the absence of BRAF V600E. Those observed in 2 patients each included p.V487_T491del, p.V504_L505insGKT, p.S607P, and p.D587N. One patient had a V600D mutation, which has been sensitive to BRAF inhibition in melanoma. Initial treatment and relapse data was known for 15 patients. Unless patients received rituximab as part of initial purine analog (n=7, none relapsed), patients after purine analog monotherapy (n=8) had all relapsed (p=0.0002) and had inferior relapse-free survival (RFS, 19.9 months vs not reached, p=0.0004). RFS of these 8 patients after purine analog monotherapy was also inferior compared to a control group of 34 patients who received cladribine monotherapy and began prospective follow-up before any relapses (19.9 vs 271.8 months, p<0.0001). Compared to the 8 patients with non-V600E BRAF mutations treated with purine analog monotherapy, all of whom relapsed at 2.5-73 (median 19.8 months), in the control group 5 of 34 patients (p<0.0001) relapsed at 57-272 (median 98.6) months and 29 remain in remission at 25.0-225.8 (median 47.8) months of follow-up. Five of the 18 patients with non-V600E BRAF mutations had IGHV4-34+ HCL which has been associated with poor prognosis if unmutated. Two of these 5 had 100% and 3 had 97.49%, 97.72% and 98.18% homology to germline. One of these patients who had 100% homology began treatment with cladribine monotherapy and relapsed at 20 months, while the other 4 had initial treatment containing rituximab and have not relapsed. Conclusion: Non-V600E BRAF mutations were observed in patients with classic HCL immunophenotype, some of which were co-mutations in BRAF with V600E. In view of the inferior RFS in these patients when purine analog was not combined with rituximab, these mutations may constitute a higher risk for relapse or chemoresistance. While those with BRAF V600 mutations might be candidates for BRAF inhibition, those with other BRAF mutations represent an opportunity for development of other specific inhibitors, not only for HCL/HCLv, but also for other malignancies. We believe more patients with HCL should be tested for non-V600E BRAF mutations.