BACKGROUND T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell–receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.)

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs

OBJECTIVETo evaluate the heterogeneity in the clinical expression in a family with glucokinase mature-onset diabetes of the young (GCK-MODY).RESEARCH DESIGN AND METHODSMembers (three generations) of the same family presented either with overt neonatal hyperglycemia, marked postprandial hyperglycemia, or glucosuria. Homeostasis model assessment of insulin resistance (HOMAIR) and insulinogenic and disposition indexes were calculated. Oral glucose tolerance test (OGTT) results in the GCK mutation carriers from this family were compared with those from other subjects with GCK mutations in the same codon (GCK261), with other missense and other types of GCK mutations in different codons from the European MODY Consortium database (GCKm).RESULTSMutation G261R was found in the GCK gene. During the OGTT, glucose (P = 0.02) and insulin (P = 0.009) response at 2 h as well as at the 2-h glucose increment (GCK261 versus other missense GCK mutations, P = 0.003) were significantly higher in GCK261 than in GCKm carriers.CONCLUSIONSDiffering from other GCKm carriers, the glucose and insulin response to oral glucose was significantly higher in GCK261 carriers, indicating clinical heterogeneity in GCK-MODY.

646 Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias, but can also occur in the context of myelodysplastic syndrome (MDS). LGL shares certain pathogenetic similarities with aplastic anemia (AA) and some forms of MDS, in which cytopenias are related to immune suppression of normal hematopoiesis. In these conditions, the inhibition of hematopoietic progenitor and stem cells has been described to be mediated by mostly polyclonal cytotoxic T lymphocytes (CTL). Previously, using molecular analysis of TCR VB repertoire in these diseases we have demonstrated oligoclonal skewing clonal of CTL spectrum that was reminiscent (albeit less pronounced) to that seen in LGL. These observations support the theory that these CTL expansions correspond to a cellular reaction against autologous hematopoietic targets. Detection of STAT3 mutations would substantiate the hypothesis that autoimmune reactions can be due to intrisic genetic lesions in autoimmune cells. The recent discovery of recurrent somatic STAT3 mutations appears to be the key molecular lesion promoting clonal outgrowth of autonomous CTL clones in LGL. This finding raised the hypothesis of whether those mutations could be found in other bone marrow failure (BMF) states and whether they could be diagnostically useful and associated with distinct clinical features. Initially, we have directly sequenced STAT3 exons in 120 T-LGL cases and identified 33 mutations in 32 cases (27%). All mutations were located in the domain of STAT3 (residues 585–688) that shares homology with Src homology 2 (SH2) domains. The STAT3 SH2 domain mediates STAT3 dimerization via binding of phosphotyrosine residue Y705. Two mutations, Y640F and D661Y, accounted for 80% of the somatic variations found, enabling the design of a more sensitive ARMS-PCR method for each of these alterations, suitable for the massive screening we have envisioned for AA and MDS. In BMF, we have first identified 21 MDS patients with known LGL and screened them for the presence of STAT3 mutations: in the CTLs of 6/21 of these patients STAT3 mutations were found and thus less clinically apparent LGL expansion could also be present in more classical MDS. Thus, we extended our screening to CTLs from an additional 368 patients with MDS and no suspected concomitant LGL: we identified 9 additional patients with STAT3 mutated clones. MDS patients carrying STAT3 mutant CTL clones had both advanced and low risk disease (low, n=1; int-1, n=4; int-2, n=8; High, n=2). These patients were characterized by a higher frequency of hypocellular bone marrow (55 vs. 10.5%; p<.001), and neutropenia at diagnosis (p<.04). No significant differences were found in overall survival. By analogy we also searched for STAT3 CTL clones in AA (N=148) and PNH (N=30). In total, we have identified 17 (10%) AA patients with STAT3 mutant CTL clones: all of these patients did not display manifest LGL. Clinically, these patients had a higher proportion of non severe AA (40% vs. 23%) and were more likely to respond to first line immunosuppression (76 vs., 65%) though no statistical significance was reached. In addition, cases with BMF and a subclinical mutant CTL clones were retrospectively tested for the presence of a TCR rearrangement: an oligoclonal (40% of cases) or monoclonal pattern (20% of cases) was seen in most of patients. In sum, the surprising discovery of STAT3 mutated clones in BMF states seems to be predominantly in AA patients and can be also found MDS cases with mainly, hypoplastic features, suggesting that STAT3 mutated self reactive CTL clones may play a role in immune pathogenesis of these conditions. Disclosures: Koskela: Novartis: Honoraria; BMS: Honoraria; Janssen-Cilag: Honoraria. Mufti:Celgene: Consultancy, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.