New Generation Sequencing of Targeted Genes in Hairy Cell Leukemia Highlighted Mutations Associated to BRAF^V600E anda New Recurrent Altered Gene, KDM6A, in Hairy Cell Leukemia Variant Patients

Introduction: BRAF^V600E detected in more than 80% of Hairy cell leukemia (HCL) cases, was described as a driver mutation but additional genetic abnormalities seem necessary for the progression of the disease. Within HCL harboring BRAF^WT, the differential diagnosis with the variant form of HCL (HCL-v) is complex.Sequencing of targeted genes by next generation sequencing (NGS) has proved its interest for classification and prognosis of lymphoid neoplasms and opens the lead to new perspectives for personalized medicine.

Methods : We selected a panel of 21 relevant genes (defined as Trichopanel) based on literature review of whole exome sequencing studies. At diagnosis, we analyzed 20 HCL and 4 HCL-v samples and at relapse 2 HCL and 3 HCL-v. HCL diagnosis was based on hairy cell morphology with the co-expression of CD103, CD123, CD25 and CD11c, and HCL-v on hairy cell morphology with a prominent nucleolus and the lack of CD25 and CD123 expression. DNA was extracted from peripheral blood mononuclear cells isolated from blood (17 samples), bone marrow aspirates (10 samples) or biopsies (spleen: 1 and cutaneous nodule: 1 case). NGS was performed on the Ion Torrent PGM^TM device according to the manufacturer' recommendations. The Trichopanel covered 71,020 bases using 712 amplicons. Analyzed genes belong to different gene ontology families: MAPK pathway (BRAF, MAP2K1, DUSP2, MAPK15), epigenetic regulation (ARID1A, ARID1B, EZH2, KDM6A, CREBBP), cell cycle/apoptosis ( TP53, CDKN1B, XPO1), homing (KLF2, CXCR4), NOTCH pathway (NOTH1, NOTCH2), NF-kB pathway (MYD88), inflammation (ANXA1), splicing (U2AF1), differentiation (BCOR) and extracellular transport (ABCA8). Data analysis was performed with the Torrent suite^TM software, then variant analysis was performed using an in-house bioinformatics pipeline detailed as previously described¹. The ratio of variant allele frequency (rVAF) was pondered according to the % of tumor cells estimated by Flow Cytometry.

Results : The Trichopanel was informative for 96% (23/24) of patients and revealed single nucleotide variants (SNVs) in BRAF (18 pts), KLF2 (4 pts), MAP2K1 (3 pts), KDM6A (2 pts), CDKN1B (2 pts), ARID1A (2 pts), CREBBP (2 pts) NOTCH1 (1 pts) and ARID1B (1 pts). BRAF^V600E was found in 90% (18/20) of HCL patients and was not detected in HCL-v patients. In one BRAF^WT HCL patient, we found a mutation on MAP2K1. In the last case, we found no SNV in the genes sequenced by the Trichopanel. In HCL BRAF^V600E patients, other mutations were found in 39% (7/18) of cases: KLF2 (3 pts), CDKN1B (2 pts), NOTCH1 (1 pt), ARID1B (1 pt) and CREBBP (1 pt). All 4 HCL-v patients had SNVs in epigenetic regulation genes e.g.: KDM6A (2 pts), CREBBP (1 pt) or ARID1A (1 pt). KDM6A SNVs were either frameshift deletions or splicing variations with a loss of exon (confirmed by RNA sequencing). We also analyzed at diagnosis and relapse serial samples from 5 patients (2 HCL and 3 HCL-v). In one HCL, patient we observed at diagnosis and at first relapse the same mutational pattern and in the second case, we observed the presence of 2 news sub-clonal mutations (BCOR^E1430X and XPO1^E571K). In the 3 HCL-v patients (2 treated and 1 untreated), no new mutation was observed but variations of the rVAF were identified in 2 cases.

Discussion : In addition to BRAF^V600E mutations, one third of HCL patients had mutations with a rVAF close to BRAF^V600E rVAF. KLF2, which is mutated in 3 patients, is known to play a key role for B-cell homing in lymph nodes and inhibition of NF-kB pathway. KLF2^275N, KLF2^275T, KFL2^T271I variants found in those patients have been previously described in splenic zone marginal lymphoma (SMZL) as inhibitory mutations, possibly suggesting a genetic similarity between both diseases². We also describe new mutations targeting KDM6A, a lysine demethylase protein, in HCL-v disease. Stop-gain KDM6A mutations have been already described in 2 HCL patients with atypical clinical features^3,4. Those mutations and the 2 mutations found in our series leads to the loss of the highly-conserved C-terminal region (included Jumomji and Zinc binding domains) which is essential for its demethylase activity. Loss of KDM6A activity may sensibilise tumor cells to demethylating agents such as 5-azacytidine or EZH2 inhibitors⁵.

Conclusion : the Trichopanel is a useful tool for the diagnosis, relapse and prognosis of HCL and HCL-v.

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