Key Points• KMT2C mutations occur in 15% and 25% of patients with cHCL and vHCL, respectively, along with CCND3 and U2AF1 mutations each in 13% of vHCLs.• NF1, NF2, N/KRAS, and IRS1 alterations contribute to clinical resistance to vemurafenib treatment in patients with cHCL.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 5 53) and vHCL (n 5 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. (Blood. 2017;130(14):1644-1648

Minimal residual disease (MRD) in acute myeloid leukemia (AML) is typically measured using multiparameter flow cytometry (MFC). Detection of leukemia mutations using multigene next-generation sequencing (NGS) can potentially be used to measure residual disease. We used a targeted 28-gene NGS panel to detect mutations and different-from-normal 10-color MFC to measure MRD in AML patients before allogeneic hematopoietic stem cell transplantation (HCT). Residual disease was defined when any abnormal blast population was detected using MFC and when any leukemia allele was detected with a variant allele frequency (VAF)  ≥ 5% using NGS. We tracked the clearance of leukemia alleles between AML diagnosis and immediately before HCT and found that mutations in DNMT3A, TET2, and JAK2 were less likely to be cleared than NPM1, IDH 1/2, and FLT3-ITD. Despite varying sensitivities, the concordance rate of residual disease detection before HCT using the 2 assays was 44 of 62 (71%) evaluable cases. Discordance could be explained by residual mutations in DNMT3A and TET2 that were not detected by MFC and presence of residual leukemia mutations with VAF below the established thresholds for mutation calling. Presence of flow MRD and residual mutations immediately before HCT using the 2 assays was associated with relapse risk (MFC: hazard ratio,  4.62; 95% confidence interval [CI], 1.32 to 16.09; P = .016 and NGS: hazard ratio,  4.35; 95% CI, 1.63 to 11.6; P = .003) and survival (MFC: hazard ratio,  2.44; 95% CI, 1 to 5.97; P = .05 and NGS: hazard ratio, 2.1; 95% CI, .97 to 4.55; P = .059) after HCT. Residual disease detected concurrently by MFC and NGS conferred the highest relapse risk compared with patients who were either negative by both assays or had discordant status (overall, P = .008). Although MFC is universally applicable, a multigene NGS approach to measuring residual disease in AML provides additional information on differential clearance of disease alleles and can assess clonal architecture before transplantation.

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The genetic aberrations that drive mixed phenotype acute leukemia (MPAL) remain largely unknown, with the exception of a small subset of MPALs harboring BCR-ABL1 and MLL translocations. We performed clinicopathologic and genetic evaluation of 52 presumptive MPAL cases at Memorial Sloan Kettering Cancer Center. Only 29 out of 52 (56%) cases were confirmed to be bona fide MPAL according to the 2016 World Heath Organization classification. We identified PHF6 and DNMT3A mutations as the most common recurrent mutations in MPAL, each occurring in 6 out of 26 (23%) cases. These mutations are mutually exclusive of each other and BCR-ABL1/MLL translocations. PHF6- and DNMT3A-mutated MPAL showed marked predilection for T-lineage differentiation (5/6 PHF6 mutated, 6/6 DNMT3A mutated). PHF6-mutated MPAL occurred in a younger patient cohort compared with DNMT3A-mutated cases (median age, 27 years vs 61 years, P < .01). All 3 MPAL cases with both T- and B-lineage differentiation harbored PHF6 mutations. MPAL with T-lineage differentiation was associated with nodal or extramedullary involvement (9/15 [60%] vs 0, P = .001) and a higher relapse incidence (78% vs 22%, P = .017) compared with those without T-lineage differentiation. Sequencing studies on flow-cytometry–sorted populations demonstrated that PHF6 mutations are present in all blast compartments regardless of lineage differentiation with high variant allele frequency, implicating PHF6 as an early mutation in MPAL pathogenesis. In conclusion, PHF6 and DNMT3A mutations are the most common somatic alterations identified in MPAL and appear to define 2 distinct subgroups of MPAL with T-lineage differentiation with inferior outcomes.

Summary Repeated therapy of hairy cell leukaemia (HCL) with treatments that have potential long-term toxicities has raised concerns regarding increased risk for younger patients. We compared clinical outcomes and disease complications in 63 patients with HCL aged ≤40 years at diagnosis with 268 patients >40 years treated at Memorial Sloan Kettering Cancer Center. The rate of complete remission following initial therapy was 87% and 83% (P=0.71) and estimated 10-year overall survival was 100% and 82% (p=0.25) in younger and older patients, respectively. Younger patients required therapy earlier and had a significantly shorter time between first and second therapy (median: 63 v 145 months) (P=0.008). Younger patients required significantly more lines of therapy during follow-up. The 10-year cumulative incidence of secondary malignancies in young and old patients was 0.205 and 0.287, respectively (P=0.22). The incidence of secondary cancers in patients aged >40 years at diagnosis increased with the number of treatments for HCL (P=0.018). These results highlight that young patients with HCL have shorter responses to treatment and require more lines of therapy to maintain disease control, while attaining similar long-term survival. This has implications in the design of future clinical trials given our findings that secondary malignancies increase with more chemotherapy exposure.