Contributions
Abstract: EP611
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
T-cell large granular lymphocyte leukemia (T-LGLL) is a rare lymphoproliferative disease whose etiology is still unknown. T-LGLL includes two subtypes of lymphoproliferation: the most frequent CD8+ T-LGLL (70% of cases) and the less common CD4+ T-LGLL (30% of cases). In the CD4+ variant, the leukemic T-LGLs express the CD4 determinant (either alone or together with CD8), the α/β T-cell receptor (TCR), and display a typical mature cytotoxic and activated/memory T-cell phenotype. Previously, in small series of cases, up to 55% of CD4+ T-LGLL patients have shown to be characterized by STAT5B mutations, at variance from CD8+ T-LGLL cases, who frequently harbor STAT3 mutations and only seldom STAT5B mutations.
Aims
In this study, we collected the largest cohort of CD4+ T-LGLL (n=35) so far described at molecular level in the literature. We evaluated the presence of STAT5B mutations and their correlation with patients’ clinical data. In addition, we characterized the clonal landscape, publicness, and structural similarities of TCRβ repertoire in CD4+ T-LGLL in comparison to healthy TCR repertoires.
Methods
CD4+ T-LGLL patients were enrolled from Hematology Units of 3 Academic Institutions, i.e., Padova (Italy; n=22), Helsinki (Finland; n=6), and Shinshu (Japan; n=7). Buffy coats from healthy controls (n=37) were supplied by the Finnish Red Cross Blood service (Helsinki, Finland). STAT5B deep amplicon sequencing was performed for each patient in LGL or PBMC using Illumina platform on MiSeq instrument. The evaluation of differential STAT5B transcriptional activities was measured through STAT5B luciferase reporter assay. TCRβ sequencing was obtained from genomic DNA (the same DNA used for amplicon sequencing) with Adaptive Biotechnologies’ ImmunoSEQ assay.
Results
We observed 63% (23/35) of the patients harboring STAT5B mutations. In addition to the gain-of-function STAT5B mutations previously described in T-LGLL (N642H, Y665F, Q706L, S715F), we found 6 novel mutations (Q220H, E433K, T628S, P658R, P702A, V712E) located not only in hot spot regions but also in other gene domains. In 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, multiple STAT5B mutations were either coexisting in one clone or in distinct clones. On clinical grounds, patients with STAT5B mutations were distinguishable from STAT5B wild type patients for increased lymphocyte and LGL counts.
The deep sequencing of TCRβ repertoire was obtained on 27 patients. We demonstrated that as well as large LGL expansions, also the non-leukemic TCR repertoires exhibited higher clonality in CD4+ T-LGLL patients than in healthy controls. Considering clones with the size exceeding 5% of the repertoire, we identified 59 different clonotypes and 24% of them were also found among the non-expanded TCR repertoires of other CD4+ T-LGLL patients. In 44% of the patients, the LGL clonal expansion was characterized by TRBV06 (TCRVβ13.1) expression. Nevertheless, structural similarities among LGL clonotypes were infrequent and direct viral antigen matches were not identified.
Conclusion
STAT5B mutations are the hallmark of CD4+ T-LGLL and they associate with higher lymphocyte and LGL counts. Despite the gain-of-function nature of the mutations, patients rarely have any symptoms, and the disease is indolent. TCR repertoire of both leukemic and non-leukemic CD4+ T cells is more clonal than in healthy individuals and TRBV06 is overrepresented hinting towards an initial immune response to an antigen as a likely triggering event of LGL expansion.
Keyword(s): CD4+ T cells, Large granular lymphocytic leukaemia, STAT5, T cell repertoire
Abstract: EP611
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
T-cell large granular lymphocyte leukemia (T-LGLL) is a rare lymphoproliferative disease whose etiology is still unknown. T-LGLL includes two subtypes of lymphoproliferation: the most frequent CD8+ T-LGLL (70% of cases) and the less common CD4+ T-LGLL (30% of cases). In the CD4+ variant, the leukemic T-LGLs express the CD4 determinant (either alone or together with CD8), the α/β T-cell receptor (TCR), and display a typical mature cytotoxic and activated/memory T-cell phenotype. Previously, in small series of cases, up to 55% of CD4+ T-LGLL patients have shown to be characterized by STAT5B mutations, at variance from CD8+ T-LGLL cases, who frequently harbor STAT3 mutations and only seldom STAT5B mutations.
Aims
In this study, we collected the largest cohort of CD4+ T-LGLL (n=35) so far described at molecular level in the literature. We evaluated the presence of STAT5B mutations and their correlation with patients’ clinical data. In addition, we characterized the clonal landscape, publicness, and structural similarities of TCRβ repertoire in CD4+ T-LGLL in comparison to healthy TCR repertoires.
Methods
CD4+ T-LGLL patients were enrolled from Hematology Units of 3 Academic Institutions, i.e., Padova (Italy; n=22), Helsinki (Finland; n=6), and Shinshu (Japan; n=7). Buffy coats from healthy controls (n=37) were supplied by the Finnish Red Cross Blood service (Helsinki, Finland). STAT5B deep amplicon sequencing was performed for each patient in LGL or PBMC using Illumina platform on MiSeq instrument. The evaluation of differential STAT5B transcriptional activities was measured through STAT5B luciferase reporter assay. TCRβ sequencing was obtained from genomic DNA (the same DNA used for amplicon sequencing) with Adaptive Biotechnologies’ ImmunoSEQ assay.
Results
We observed 63% (23/35) of the patients harboring STAT5B mutations. In addition to the gain-of-function STAT5B mutations previously described in T-LGLL (N642H, Y665F, Q706L, S715F), we found 6 novel mutations (Q220H, E433K, T628S, P658R, P702A, V712E) located not only in hot spot regions but also in other gene domains. In 22% (5/23) of STAT5B mutated CD4+ T-LGLL cases, multiple STAT5B mutations were either coexisting in one clone or in distinct clones. On clinical grounds, patients with STAT5B mutations were distinguishable from STAT5B wild type patients for increased lymphocyte and LGL counts.
The deep sequencing of TCRβ repertoire was obtained on 27 patients. We demonstrated that as well as large LGL expansions, also the non-leukemic TCR repertoires exhibited higher clonality in CD4+ T-LGLL patients than in healthy controls. Considering clones with the size exceeding 5% of the repertoire, we identified 59 different clonotypes and 24% of them were also found among the non-expanded TCR repertoires of other CD4+ T-LGLL patients. In 44% of the patients, the LGL clonal expansion was characterized by TRBV06 (TCRVβ13.1) expression. Nevertheless, structural similarities among LGL clonotypes were infrequent and direct viral antigen matches were not identified.
Conclusion
STAT5B mutations are the hallmark of CD4+ T-LGLL and they associate with higher lymphocyte and LGL counts. Despite the gain-of-function nature of the mutations, patients rarely have any symptoms, and the disease is indolent. TCR repertoire of both leukemic and non-leukemic CD4+ T cells is more clonal than in healthy individuals and TRBV06 is overrepresented hinting towards an initial immune response to an antigen as a likely triggering event of LGL expansion.
Keyword(s): CD4+ T cells, Large granular lymphocytic leukaemia, STAT5, T cell repertoire