Combined SNP Microarray Based Genomic Mapping and Next Generation Transcriptome Sequencing Confirms the Presence of Somatic STAT3 Mutations and Identifies Novel Chromosomal Imbalances and Candidate Genes in T Large Granular Lymphocytic Leukemia (T-LGL)

T-cell large granular lymphocytic leukemia (T-LGL) is a rare lymphoproliferative disease characterized by an expansion of large granular lymphocytes involving blood, bone marrow, spleen and liver. T-LGL cells are mature CD3, CD8 and T-cell receptor (TCR) αβ positive cells exhibiting the immunophenotype of activated cytotoxic T lymphocytes (CTLs). CD4 or TCR γδ positive variants occur rarely. T-LGL affects adults at a median age of 55–60 years and arises commonly in patients with a preexisting autoimmune disorder. Many patients remain asymptomatic for years and do not require treatment. Palliative therapy with immunosuppressant agents such as low dose methotrexate, ciclosporin and fludarabine is used for the correction of severe immune-mediated cytopenias, which often complicates the course of the disease. The molecular pathogenesis of T-LGL remains unclear. No recurrent karyotypic anomalities but several numeric and structural chromosomal alterations have been identified. Recently, activating somatic mutations in the signal transducer and activator of transcription 3 gene (STAT3) have been described by Koskela et al. in approximately 40% of T-LGL patients. STAT3 mutations lead to an increased transcriptional activity and were more prevalent in patients with neutropenia and rheumatoid arthritis than in patients without these conditions. As these findings only explain part of the pathogenesis in the fraction of patients affected by STAT3 mutations, we here aimed to identify novel mutations which may help to better understand the mechanisms of disease development.

We sorted tumor- and non-tumor cells from peripheral blood samples of T-LGL patients by using fluorescence activated cell sorting (FACSDiva®, Becton Dickinson) to perform single nucleotide polymorphism (SNP) chip analysis and next-generation RNA sequencing. SNP chips were analyzed in 10 patients (Affymetrix, Mapping 250K Sty Array®). To identify somatic mutations in patients with T-LGL, we compared CD8/CD57 positive tumor cells with non-tumor cells as germline control. Sample libraries for RNA sequencing of 5 patients were generated with NuGEN Encore®, sequencing was performed on Illumina HiSeq 2000® yielding 100 million 100 basepair single reads, and alignment was realized on TopHat2 against hg19 as reference genome. For quantification and analysis of variants Partek GS 6.6 was used.

High resolution copy number determination employing SNP chips in 10 patients revealed both gains and losses on different chromosomes, among others 1q, 7q, 14q and chromosome X. The affected chromosomal regions included genes with potential relevance to the disease process such as WNT and RASSF gene family members in deleted regions and PIM3 and MAPK family members in gained regions. However, in line with previous reports no recurrent chromosomal aberrations were detected. Preliminary analysis of RNA sequencing data revealed activating STAT3 Y640F mutations in 2 out of 5 patients tested (40%). Interestingly, one of the STAT3 mutated T-LGL clones also exhibited an inactivating mutation of the NFKB inhibitory gene TNFAIP3 (A20), which has been reported to play an important role in the molecular pathogenesis of different B cell lymphomas but has as yet not been described in T-LGL. Detailed analysis of sequencing data is currently ongoing and further results will be presented at the conference. In conclusion, combined RNA sequencing and molecular cytogenetic profiling identified novel specific chromosomal loci and genes that could help to better understand the molecular pathogenesis of T-LGL and develop novel targeted treatment modalities for this disease.

No relevant conflicts of interest to declare.