ALBERT

Description: Background: The pathogenesis of chronic lymphoproliferative disorder of NK-cells (CLPD-NK) is poorly understood. Mutations in the JAK/STAT pathway (especially STAT3) are found in 30% of patients, but the genetic or exogenous drivers responsible for other cases are unknown. Here we comprehensively define the genetic drivers of CLPD-NK by integrated genome and transcriptome sequencing (WGS/WTS) of a large cohort of cases and complementary functional analyses. Methods: We studied 63 CLPD-NK patients (M/F: 42/21, median age: 71 years [35-89 y]) by WGS, WTS and flow cytometry. A validation cohort of 67 patients (M/F: 43/24, median age: 64 years [7-91]) was analyzed by targeted sequencing. To study the role of CCL22 in CLPD-NK pathogenesis, we examined internalization of the CCL22 receptor, CCR4, and cell chemotaxis in response to exogenous wild type (wtCCL22) or mutant CCL22 (mtCCL22: L45R, P79R, IL87_88NF) in CCR4-expressing Ba/F3 cells (Ba/F3-CCR4). To examine potential autocrine/paracrine activity, we exchanged supernatants of Ba/F3-CCR4-wtCCL22 and -mtCCL22 cells and examined CCR4 expression. To examine the in vivo effects of the mutations on proliferation and phenotype, GFP-tagged empty vector, wtCCL22, or mtCCL22-transduced NK-92 cells were engrafted into IL15-transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG) mice. Human NK-92 cells isolated from spleens of moribund mice were analyzed by WTS and immunophenotyping. Results: WGS of 63 CLPD-NK identified STAT3 mutations in 18 (29%) cases, with mutually exclusive CCL22 mutations (mtCCL22) in 14 (22%) patients. WGS of 4500 hematological malignancies showed that mtCCL22 were only found in CLPD-NK. Recurrent co-mutations in both groups were found in ATM (n=3), FAS (n=2) and TET2 (n=5). Of the remaining patients, 23/31 had one or more mutation including epigenetic regulators (n=12), signaling components (n=7) or TP53 (n=4). Our findings of CCL22 mutations were confirmed in an independent validation cohort with STAT3 mutations in 19/67 (28%) and mtCCL22 in 13/67 (19%). CCL22 mutations were clustered at the conserved leucine 45 and proline 79 residues (Fig. 1A). Sequencing of purified CD3+ T and CD56+ NK cells showed that the mtCCL22 were somatic mutations acquired by the CD56+ NK population. mtCCL22 defined a subgroup of CLPD-NK, with high NCAM1 (CD56) positivity by flow cytometry (p