Genomic analysis of hairy cell leukemia identifies novel recurrent genetic alterations

Abstract

Classical hairy cell leukemia (cHCL) is characterized by a near 100% frequency of the BRAFV600E mutation, whereas ∼30% of variant HCLs (vHCLs) have MAP2K1 mutations. However, recurrent genetic alterations cooperating with BRAFV600E or MAP2K1 mutations in HCL, as well as those in MAP2K1 wild-type vHCL, are not well defined. We therefore performed deep targeted mutational and copy number analysis of cHCL (n = 53) and vHCL (n = 8). The most common genetic alteration in cHCL apart from BRAFV600E was heterozygous loss of chromosome 7q, the minimally deleted region of which targeted wild-type BRAF, subdividing cHCL into those hemizygous versus heterozygous for the BRAFV600E mutation. In addition to CDKN1B mutations in cHCL, recurrent inactivating mutations in KMT2C (MLL3) were identified in 15% and 25% of cHCLs and vHCLs, respectively. Moreover, 13% of vHCLs harbored predicted activating mutations in CCND3 A change-of-function mutation in the splicing factor U2AF1 was also present in 13% of vHCLs. Genomic analysis of de novo vemurafenib-resistant cHCL identified a novel gain-of-function mutation in IRS1 and losses of NF1 and NF2, each of which contributed to resistance. These data provide further insight into the genetic bases of cHCL and vHCL and mechanisms of RAF inhibitor resistance encountered clinically.

Figure 1.

Genomic alterations in cHCL and vHCL. (A) Histogram of mutations in cHCL cohort (n = 53 patients) present in ≥2 patients. (B) CN analysis of the cHCL cases. Curated segmentation data for 53 cHCL samples. In the red-blue scale, white corresponds to a normal (diploid) CN log ratio, blue is a deletion, and red is a gain. (C) CN variation plots of peripheral blood MNCs from a single patient with cHCL at initiation, remission, and relapse from BRAF inhibitor treatment illustrating deletion of 7q and 13q [del(7q) and del(13q), respectively] regions at times of treatment initiation and relapse but not in disease remission. Genes mapped in the region of del(7q) with representative fluorescence in situ hybridization (FISH) with 7q deletion (white arrows; probes: red = 7q31; green = centromeric probe chromosome 7 [CEP7]) (D) and del(13q) with representative FISH with 13q14 deletion (white arrows; probes: red = 13q14; green = 13q34) (E). (F) BRAFV600E variant allele frequency (VAF) in patient cases with or without del(7q). (G) CN analysis of 8 cases of vHCL.

Figure 2.

Gain-of-function mutation in IRS1 and NF1/2 loss result in de novo vemurafenib resistance in cHCL. (A) Serial flow cytometric analyses of cHCL cells (CD103⁺/CD25⁺) in the peripheral blood of a patient with cHCL before vemurafenib initiation and during the first month of treatment (top), as well as a different patient before vemurafenib initiation until time of death (bottom). Mutations (B) and CN alterations (C) detected in pretreatment cHCL sample from the vemurafenib-resistant patient. (D) Western blot analysis of human (hIRS1) and mouse IRS1 (mIRS1) complementary DNA constructs in wild-type (WT) or mutant forms on AKT and ERK phosphorylation related to empty vector. (E) Cell growth of IRS1-expressing Ba/F3 cells from (D) after interleukin-3 withdrawal. (F) Quantitative reverse transcription polymerase chain reaction of Nf1 and Nf2 expression after anti-Nf1 or -Nf2 short hairpin (shRNA) knockdown. The numbers below each shRNA indicate the shRNA oligonucleotide sequence (as shown in supplemental Methods). (G) IC50, 50% inhibitory concentration (IC₅₀) of BRAFV600E-expressing Ba/F3 cells to vemurafenib with or without knockdown of mNf1 or mNf2.