MAP2K1 and MAP2K2 genes alterations in hairy cell leukemia and hairy cell leukemia variant.

Abstract

Abstract Background: Hairy cell leukemia (HCL) is an indolent B-cell neoplasm characterized by the expression of CD20, CD22, CD25, CD11c, CD103, CD123, and annexin A1. Treatment with purine analog monotherapy is highly effective, resulting in durable complete remissions (CR). In contrast, the HCL variant (HCLv), which typically lacks CD25, CD123, and annexin A1, responds poorly to purine analogs with poorer overall response rate (ORR) <50% and CR <10%, and median 6-9 year overall survival (OS). Classic HCL is driven by the BRAF V600E mutation or non-V600E BRAF mutations in >95% of cases, constituting both a diagnostic biomarker and a therapeutic target for BRAF inhibitors. Notably, both HCLv and some cases immunophenotypically consistent with classic HCL lack BRAF mutation. BRAF wild-type cases often harbor unmutated IGHV rearrangements with significant representation of IGHV4-34 gene, associated with activating mutations in MAP2K1 encoding MEK1, downstream of BRAF. Since 2014, our report and 5 others described a total of 28 patients with 10 different MAP2K1 mutations. These cases were too few to report clinical implications, and MAP2K2 mutations in HCL/HCLv have not been described. Methods We performed whole exome sequencing (WES) and/or TrueSight Oncology (TSO500) and/or other sequencing including next generation sequencing (NGS) in 225 HCL/HCLv patients, including 91 with HCLv and 24 with unmutated IGHV4-34+ HCL. To be evaluable for WES/NGS, HCL/HCLv cells comprised >80% of B-cells and were partially purified using CD19 microbeads followed by positive fraction isolation. Results We identified 26 different MAP2K1 genetic alterations in 52 BRAF V600E-negative HCL/HCLv patients. 21 patients had HCL and 31 patients HCLv immunophenotype. Unmutated IGHV rearrangements were observed in 19/21 HCL and 15/31 HCLv patients. One HCLv patient harbored two MAP2K1 mutations, and another carried three. MAP2K1 mutations included both missense mutations and in-frame deletions. The most common mutations in HCL/HCLv were K57N and C121S each found in 9 patients, but K57N was observed in 7 HCL and C121S in 7 HCLv patients (p=0.057). Besides K57N and C121S, the most frequent MAP2K1 mutations in HCL were Q56P (n=4) and F53L (n=3); the most frequent in HCLv were I103N and K55E (n=3 each). MAP2K2 mutations were identified in 7 HCLv and 2 HCL patients, most commonly R231C in 4 HCLv patients. The other 3 HCLv patients had R231L, F57C and F57L, while the 2 HCL patients had A100T and P166T. One HCLv patient harbored concurrent mutations in both MAP2K1 and MAP2K2. BRAF V600E was found in none of 21 HCL patients with MAP2K1 mutations but was found in both HCL patients with MAP2K2 mutations. In comparing evaluable patients with C121S (n=9) vs K57N (n=8) MAP2K1 mutations after 1st-line treatment, there was a trend for shorter median relapse free survival for C121S (RFS 4.0 vs 6.3 months, p=0.091) but no difference in OS (p=0.48). Five HCL patients with MAP2K1 mutations did not have high-risk features such as IGHV4-34 usage or an HCLv phenotype. Compared to 39 classic HCL patients with only BRAF V600E mutation after 1st line purine analog treatment, they had significantly shorter RFS (p<0.0001) and OS (p=0.0034). However, 3 of these 5 patients also had unmutated non-IGHV4-34 rearrangements. To investigate the impact of MAP2K1 mutations on leukemic cell behavior and to explore potential therapeutic strategies, we developed an ex vivo model using the Raji cell line. The four most common MAP2K1 mutations observed in HCL and HCLv, C121S, K57N, K57E, and K57T, were introduced into Raji cells using the TALEN gene editing method. Comparative analysis of the resulting stable mutant clones revealed differences in cell proliferation, gene expression profiles, and activation of kinases. These included MEK and ERK phosphorylation, which are directly involved in the MAPK signaling pathway, as well as other kinases such as CREB and AKT, suggesting crosstalk between MAPK and additional signaling pathways. Conclusion We report the largest series to date of MAP2K1 mutations in HCL/HCLv and the first report of MAP2K2 mutations. HCL/HCLv patients with MAP2K1 mutations constitute a high-risk group associated with other known high-risk features including unmutated and/or IGHV4-34+ rearrangements and/or HCLv phenotype. B-cell lines edited to carry MAP2K1 mutations are being studied to test existing and new therapies for these high-risk mutations.