Novel Somatic Mutations In The Dna-Binding And Coiled-Coil Domain Of The Stat3 Gene In Lgl-Leukemia

Introduction T-cell large granular lymphocyte (T-LGL) leukemia is a rare, clonal disease characterized by the expansion of CD8+ cytotoxic T-cells. We recently discovered that 40% of T-LGL leukemia patients have somatic mutations in the SH2-domain of the STAT3 gene (Koskela et al. NEJM 2012). As aberrant STAT3 activation can be observed in all patients with LGL-leukemia, we now aimed to discover whether patients without mutations in the STAT3 hotspot area harbor mutations in the other parts of the STAT3 gene. Methods Targeted STAT3 sequencing covering all 23 coding exons was done with in-house developed deep amplicon sequencing panel using the Illumina Miseq platform. The data was analyzed with a bioinformatics pipeline, which is based on calling of variants with specific counts/frequencies and filtering out false positives using the estimated error rate and quality data of amplicon reads. All samples with a frequency ratio ≥0.9 were considered to be true mutations after filtering of SNPs and low coverage variants. 111 LGL-leukemia patients with no known STAT3-mutations in the SH2 domain were analyzed. To explore the functional effects of mutations, expression constructs were generated with the identified variants and wild-type STAT3. The variants were expressed in HEK-293 cells carrying a STAT3-responsive SIE-reporter driven luciferase expression sequence to establish increased basal and IL6-stimulated STAT3 activity. Results With targeted amplicon sequencing, 3 patients were discovered to have STAT3 missense mutations in the DNA-binding domain. Two patients presented with the same H410R mutation with a variant allele frequency (VAF) of 49% and 8.8% respectively while another patient had a S381Y mutation (VAF 7%). The mutation H410R occurs in the DNA-binding domain in a highly conserved position, and results in conversion of histidine to arginine, which would predict for a slight increase in hydrophilicity. In addition to STAT3 DNA-binding domain mutations, one T-LGL patient had a novel F174S mutation in the coiled-coil domain of STAT3 (VAF 43%). The coiled-coil domain of STAT3 has previously been shown to be essential in SH2-domain mediated receptor binding and subsequent activation. Luciferase measurements of SIE-reporter HEK-293 cells transfected with constructs expressing either wild-type, variant F174S, H410R or Y640F STAT3 (the most common activating mutation in LGL leukemia) revealed the F174S and H410R variants to be as activating as the Y640F mutation in both unstimulated and IL-6 stimulated conditions. Conclusions T-LGL leukemia patients without STAT3 SH2-domain mutations harbor novel activating mutations in the DNA-binding and coiled-coil domain of STAT3. The frequency of mutations was 3.6% (4 of 111 patients). These findings further highlight the importance of screening the whole STAT3 gene in the diagnostic workup of LGL-leukemia and the central role of STAT3 in the pathogenesis of the disease. Citation Format: Emma I. Andersson, Hanna Rajala, Heikki Kuusanmaki, Arjan van Adrichem, Samuli Eldfors, Sonja Lagstrom, Thomas Olson, Michael Clemente, Pekka Ellonen, Caroline Heckman, Thomas P. Loughran, Jaroslaw P. Maciejewski, Satu Mustjoki. Novel somatic mutations in the DNA-binding and coiled-coil domain of the STAT3 gene in LGL-leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2015-606