Discovery of STAT5b Mutations and Small Subclones of STAT3 Mutations in Large Granular Lymphocytic (LGL) Leukemia

Large granular lymphocytic (LGL) leukemia is a rare lymphoproliferative disease, characterized by the clonal expansion of cytotoxic CD3+CD8+ T-cells or CD3-CD16/56+ natural killer (NK)-cells. It is often associated with autoimmune phenomena (e.g. cytopenias, rheumatoid arthritis). We recently identified somatic mutations in the STAT3 gene in 40% of monoclonal T-LGL cases (Koskela et al, NEJM, 2012). Here, we report the discovery and functional analysis of novel STAT5b mutations as well as small subclones of STAT3 mutations in other LGL patients, expanding the evidence implicating STAT activation in LGL.

In order to find novel LGL-leukemia associated mutations, exome sequencing was done from untreated STAT3 mutation negative T-LGL leukemia patients using CD8+ LGL-leukemic cells and matched CD4+ control cells. Samples from 158 T-LGL and 40 NK-LGL leukemia patients were further analyzed using both targeted Sanger sequencing and ultra-deep targeted next-gen amplicon sequencing with up to 10,000x coverage (MiSeq, Illumina). Functional analysis of mutated proteins was carried out in Hela cells by Western analysis and luciferase reporter assays.

Exome sequencing revealed a novel somatic missense mutation Y665F in the STAT5b gene in two T-LGL patients diagnosed with WHO2009 criteria with a large CD8+ T-LGL clone (>90%). Only wild-type STAT5b was seen in the matched CD4+ control cells of these patients. Amplicon sequencing of exon 16 of STAT5b (corresponding to the Y665F site) in 158 T-LGL and 40 NK-LGL patients revealed an additional mutation (N642H) in one T-LGL and one NK-LGL patient, resulting in the 2% total frequency (4/198) of STAT5b mutations across all patients. The N642H and Y665F mutations were both located in the Src homology 2 (SH2) domain of STAT5b, which mediates dimerization and activation by trans-phosphotyrosine binding. STAT3 mutations previously reported in T-LGL patients were located in the corresponding domain.

The transcriptional activity of wild-type and mutant STAT5b proteins (N642H and Y665F) was assayed in cells carrying a luciferase reporter with STAT5 binding elements. Luciferase activity of Hela cells transfected with the mutated STAT5b constructs was significantly increased compared to wild-type STAT5b. Furthermore, both the N642H and Y665F variants of STAT5b exhibited higher levels of tyrosine (Y694) phosphorylation than the wild type protein.

The exon 21 in the SH2 domain of the STAT3 gene was also screened by ultra-deep next-gen amplicon sequencing, both from the original T-LGL patient cohort (n=77, patients with monoclonal disease) and 142 additional monoclonal/oligoclonal LGL patients. In the monoclonal cohort, a total of nine new STAT3 mutation positive patients were detected by amplicon sequencing, raising the total number of positive cases to 41 (53%) from the 32 identified by Sanger sequencing. Concomitant to the previously described Y640F, N647I, K658N, D661H, D661V and D661Y STAT3 mutations, several novel mutations in this gene were found: I659L, Q643H, G656C, K658H, K658R and D661I. In the oligoclonal LGL cohort, the mutation frequency was lower (31/142, 22%), suggesting that it may also include patients with reactive polyclonal LGL proliferation.

Our mutational and functional data affirm that STAT family transcription factors play a critical role in the pathogenesis of LGL leukemia. In addition to the previously identified mutations in the STAT3 gene, we found recurrent somatic STAT5b mutations in LGL leukemia. Furthermore, our ultra-deep (10,000x) next-gen sequencing revealed small subclones of STAT3 mutations in patients with oligoclonal LGL. Both STAT3 and STAT5b mutations increased the phosphorylation and transcriptional activity of corresponding proteins. The detection of STAT mutations should be included in the diagnostic assessment of LGL leukemia.

Koskela:Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Kallioniemi:TEKES-FiDiPro: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.