STAT3 mutation impacts biological and clinical features of T-LGL leukemia

Abstract

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features. Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8±) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France). Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8± T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia.

Keywords: STAT3 mutation; fas ligand; immunophenotype; large granular lymphocyte leukemia; neutropenia.

Figure 1. Representative Sanger sequences for each STAT3 mutation found

By Sanger sequencing, STAT3 mutations were observed in 36 out of 101 T-LGL leukemia patients. Two more cases were found by ARMS-PCR. Upper each graph the cases and their incidence (%) among STAT3 mutated patients (n = 38) are indicated. Y640F and D661Y accounted for the most frequent mutations found.

Figure 2. Immunophenotype distribution analysis evaluated in STAT3 mutated patients as compared with STAT3 wild type patients within CD8+ T-LGL leukemia

The graphs represent the incidence (%) of each LGL immunophenotype in the group of patients with STAT3 mutations (pie chart on the left) and in the group wild type for STAT3 gene (pie chart on the right). Mutated patients mostly belong to CD16+/CD56- phenotype (37/41, 90.2%), representing only 13.3% (4/30) of wild type patients’ group.

Figure 3. Neutropenia evaluation in the patients subdivided according to their immunophenotypes

(A) Histogram of the percentages of patients with neutropenia (ANC < 1,500 mm³). Neutropenia incidence resulted 90.2% in CD16+/CD56- subset (n = 41), 3.7% in the other immunophenotypic subsets of CD8+ T-LGL leukemia (n = 27) and 3% in CD4+ T-LGL leukemia (n = 33), the difference is highly statistically significant (****P < 0.0001, χ² = 49.5 and χ² = 55.7, respectively, using χ² test). (B) Dot plot indicating ANC level of each patient. The mean of ANC ± SEM in CD16+/CD56- subset (795.46 ± 80.03 mm³) is lower than in the other immunophenotypic subsets of CD8+ T-LGL leukemia (2,855.11 ± 224.54 mm³) and in CD4+ T-LGL leukemia (2,635.76 ± 193.81 mm³; *P < 0.05, using one-way Anova and Tukey’s multiple comparison test).

Figure 4. Western blot analysis of LGLs’ extracts for phosphorylated STAT3, total STAT3 and Actin

(A) Representative cases for the patients subdivided according to their different immunophenotypes are reported. (B) Representative cases for STAT3 mutated patients subdivided into samples where mutation was revealed only by ARMS-PCR (mutated clone < 25% of the entire clone) and those with mutations revealed by Sanger Sequencing. Actin expression is shown as gel loading control.

Figure 5. Fas ligand expression

Dot plots report (A) mRNA transcription levels and (B) plasma levels of secreted Fas ligand of T-LGL leukemia patients subdivided according to their immunophenotypes, CD8+ T-LGL leukemia (distinguished in patients with CD16+/CD56- phenotype and those with all the other phenotypes) and CD4+ T-LGL leukemia. The means and SEM are reported. The expression level observed in the group of CD8+ T-LGL leukemia CD16+/CD56- subset is higher as compared with the two other groups (***P < 0.001, ****P < 0.0001, using one-way Anova and Tukey’s multiple comparison test).

Figure 6. Fas ligand modulation after STAT3 activation/inhibition

Western blot analysis and Real-Time PCR results of patients’ PBMCs after culture in the following different conditions: untreated condition (UT) for 2 hours; with Stattic (15 μM) for 2 hours; stimulated by IL-6 (20 ng/ml) or IL-15 (20 ng/ml) for 1 hour; pretreated for 1 hour with Stattic and then stimulated by IL-6 or IL-15 for 1 hour. (A) STAT3 expression and tyrosine phosphorylation in whole-cell extracts were analyzed. Actin expression is shown as gel loading control. Upper panels show a representative STAT3 wild type sample, lower panels a representative STAT3 mutated sample. Data from two representatives out of six independent experiments are shown. (B) The histogram reports the average fold change of Fas ligand mRNA of patients’ PBMCs. All the values were settled on untreated condition set at 1.0. Data are represented as mean ± SEM of six independent experiments. *P < 0.05 vs. UT; **P < 0.01 vs. IL-6 condition; ***P < 0.001 vs. UT; ****P < 0.0001 vs. IL-15 condition, using one-way Anova and Tukey’s multiple comparison test.