CLONOTYPE AND MUTATIONAL PATTERN IN TCRGD LARGE GRANULAR LYMPHOCYTE LEUKEMIA

Background: T-cell large granular lymphocyte leukemia (T-LGLL) is a rare heterogeneous T-cell neoplasia whose leukemic cells usually express the αβ T-cell receptor (TCR); only a small subset of cases expresses the γδ TCR denoting the TCRγδ LGLL. Currently, among the different LGL diseases, TCRγδ LGLL remains less studied and several clinical and laboratory data already described in TCRαβ-LGLL have not yet been explored in TCRγδ-LGLL. Aims: The aims of this work were 1) to characterize TCRγδ-LGLL defining STAT mutational pattern and CDR3 repertoire diversity/clonal composition (clonotype) and 2) to evaluate correlations among LGL phenotype, mutations, TCR rearrangement and clinical presentations. Methods: In this work 11 patients affected by TCRγδ-LGLL were included. Sanger sequencing was used for mutational analysis on hot-spot regions in the two genes more frequently mutated in LGL disorders, STAT3 and STAT5b. Immunophenotype of LGL clone was defined by flow cytometer analysis. CDR3 repertoire and frequency distribution of TCR gamma gene rearrangements were obtained by Next-Generation Sequencing (NGS). Results: Our results showed that TCRγδ LGLL had a high incidence of STAT mutations, 9 out of 11 patients carrying STAT3 or STAT5b mutations in a mutually exclusive pattern. At variance from CD8+ TCR αβ LGLL and CD4+ TCR αβ LGLL, the first being mostly characterized by STAT3 mutations and the latter by STAT5b, TCRγδ LGLL patients were characterized by both the mutations. Thus, TCRγδ LGLL showed features shared by CD8 and CD4 TCRαβ-LGLL. Consistently, TCRγδ LGLL showed the same correlation between immunophenotype and kind of mutation observed in TCRαβ-LGLL: γδLGL patients with CD16+/CD56- LGL immunophenotype were characterized by STAT3 mutations (as in CD8+ T-LGLL), while γδLGL patients with CD56+ LGL immunophenotype by STAT5b mutations (as in CD4+ T-LGLL). Moreover, we observed that patients with γδLGLs positive for Vδ2 showed usually indolent course, while Vδ1 was linked to a more symptomatic disease (4 out of 5 symptomatic patients were Vδ1+), whereas no correlation was found between mutations and clinical course. By NGS of TCR gamma gene, we observed that all patients were clonal but two, showing a polyclonal pattern borderline with clonality percentage defined by sequencing kit criteria. Interestingly, these two last patients were the only two patients without STAT mutations. As far as the remaining cases are concerned, among STAT3 mutated patients (n=4), 3 were polyclonal and one biclonal, while STAT5b mutated patients (n=5) were more frequently monoclonal (4/5 monoclonal and 1/5 biclonal). In terms of clonal rearrangements, Vg3-Jg1/2, Vg9-JgP and Vg8Jg1/2 were the combination usages most frequently detected. Concerning the clonotype repertoire, CDR3 sequences of the immunodominant clones were present with low frequency in almost all the other γδ patients and two different CDR3 sequences were found shared, each one in two different patients at frequency >10% of the total rearrangements. Summary/Conclusions: Our data indicate that TCRγδ LGLL can be considered at the intersection of the two types of TCRαβ-LGLL, sharing CD8 or CD4 TLGLL mutational features. As already described in TCRαβ-LGLL, also in γδ disease a decreased diversity of TCR repertoire was demonstrated. However, in these γδLGL patients STAT mutations do not correlate with a symptomatic clinical behavior while STAT5b mutations seems to be more frequently linked to monoclonal nature of the LGL lymphoproliferation. Rather, the marker Vδ1 appears to be correlated to symptomatic disease.