Somatic STAT3 mutations in Felty syndrome: an implication for a common pathogenesis with large granular lymphocyte leukemia

Abstract

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8⁺ T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

Figure 1.

Felty syndrome patients harbor STAT3 mutations. (A) STAT3 mutations (presented as protein level amino acid changes) identified in study patients. Mutation variant-allele frequencies (VAFs) in the sample with the highest VAF are shown in the table. All samples and mutations detected are shown in Online Supplementary Table S1. (B) Schematic of sequencing results of all six patients with STAT3 mutations. Each patient is presented separately, and the presence of a mutation is indicated by −/+. Presented in smaller font are the data on the VAF and the sample type that was available for sequencing from the time-points. DNA sample types in A-B: Bone marrow (BM) cells, cultured cells from the BM (originally obtained for chromosome analysis); BM MNC: bone marrow mononuclear cells; PWB: peripheral whole blood; PB CD8⁺: peripheral blood CD8⁺ cells.

Figure 2.

Sequencing of sorted, expanded CD8⁺ T-cell populations. The expanded CD8⁺ T-cell populations that were detected by the initial Vβ flow cytometry screen were sorted via flow cytometry and sequenced with Amplicon sequencing (n=2; patients 4 and 6). (A) Gating strategy for Vβ analysis. The subpopulation of interest was included in subsequent gates and other populations were excluded. (B) The expanded CD8⁺ clones in patients 6 and 4. The expanded clones expressing the Vβ in question, as well as all other CD8⁺ cells were collected and sequenced. (C) The summarized sequencing results of sorted cells. The small number of mutation-positive cells present in the CD8⁺Vβ 3neg cells of patient 6 are likely due to sorting impurities. APC: allophycocyanin; FSC: forward scatter; FITC: fluorescein isothiocyanate; VAF: variant-allele frequencies.

Figure 3.

Felty syndrome patients show STAT3 phosphorylation in bone marrow. Representative immunohistochemical analysis of phospho-Tyr705-STAT3 (red) and CD57 (green) in bone marrow tissue samples of Felty syndrome (n=7) and control samples (n=14) counterstained with hematoxylin. All images presented in the figure are 40× magnified unless otherwise specified. Felty syndrome patients show more phosphorylated STAT3 than controls, but the amount of phosphorylation was not related to the STAT3 mutation VAFs. Patient numbers and mutation status are presented in the figure.

Figure 4.

Felty syndrome patients have a similar cytokine profile as that of LGL leukemia. The heatmap compares the plasma cytokine profiles of healthy controls, RA patients, Felty syndrome patients, and LGL leukemia patients. Data was analyzed as NPX units, and were normalized to allow for coloring on the same scale for the heatmap. Bright blue represents smaller protein concentration, while bright yellow represents higher protein concentration. The heatmap shows all cytokines for which the Q-value (false discovery rate) was less than 0.1. RA: rheumatoid arthritis; LGL: large granular lymphocyte.