T-Cell Large Granular Lymphocytic Leukemia with Atypical Immunophenotypes: A Single Center Retrospective Analysis of 17 Cases

Purpose T-cell large granular lymphocytic leukemia (T-LGLL) is characterized by expansion of cytotoxic T cells expressing αβ T cell receptor (TCR), CD2, surface CD3, CD8, CD57 as well as cytotoxic molecules. Atypical immunophenotypes of T-LGLL, including γδ TCR and CD4, reported in a small subset, remains to be well-dened. Methods We retrospectively analyzed immunophotypes and clinicopathologic features of 96 T-LGLL cases. Results We found a total of 17 cases with atypical immunophenotypes including 9 TCRγδ + cases and 8 CD4+ TCRαβ + cases. Pure red cell aplasia was less common in atypical immunophenotypes patients compared to that of typical immunophenotypes [0/17 (0%) vs. 26/79 (32.9%), p=0.005]. STAT3 mutations were also less frequent in atypical immunophenotypes cases, although accompanied with marginal signicance (p=0.086). Patients with atypical showed a similar survival outcome of typical immunophenotypes. needed rare atypical immunophenotypes


Introduction
Large granular lymphocyte (LGL) leukemia is a rare lymphoproliferative disease with three recognized subgroups: T-cell leukemia, chronic natural killer (NK) cell lymphocytosis, and NK cell leukemia (Lamy et al. 2017). Approximately 85% of LGL leukemia cases are of the T-LGL subtype, and less than 10% of cases are described as chronic NK-LGL leukemia.T-cell large granular lymphocytic leukemia (T-LGLL) is a subtype of T-cell neoplasm characterized by persistence of increased large granular cells in the peripheral blood. In addition to the peripheral blood and bone marrow, the leukemia cells of T-LGLL also in ltrate the spleen and liver, leading to splenomegaly and hepatomegaly in some cases. Patients suffering from T- LGLL have a wide variety of clinical presentations, mostly presenting with cytopenia including neutropenia, anemia and thrombocytopenia, and autoimmune diseases, such as rheumatoid arthritis. The typical abnormal lymphocytes in the circulation show moderate to abundant cytoplasm with ne or course azurophilic granules ( Previous studies have proved that there were differences between Asian patients with T-LGLL and western patients with T-LGLL (Kwong et al.2010). For example, there was a signi cantly higher incidence of pure red cell aplasia (PRCA) and a much lower incidence of RA in Asian patients with T-LGLL than that in western patients with T-LGLL (Qiu et al.2013). T-LGLL cases with atypical immunophenotypes have not been systematically investigated. There are no data on the pedigree, clinical and biological characteristics of these atypical immunophenotypic cases. Therefore, in the present study, we retrospectively analyzed the prevalence, the spectrum as well as the clinical and laboratory ndings of cases with atypical immuphenotypes in a cohort of Chinese patients with T-LGLL. We also determined whether there are differences in clinical and laboratory characteristics and clinical results between atypical immunophenotypes cases and typical immunophenotypes cases.

Materials And Methods
Patients with T-LGLL who were diagnosed from 2009 to 2017 were included in this study. The diagnosis of T-LGLL was based on the 2008 World Health Organization (WHO) (Swerdlow et al.2008). Cases with large granular lymphocytes less than 2⋅10 9 /L, when the lymphocytes showed typical morphologic and immunophenotypic features, were also diagnosed as T-LGLL if the clonality of these lymphocytes could be demonstrated. Clinical and laboratory data were then collected for analysis. Appropriate T-LGLL consents were obtained from all donors prior to specimen collection in accordance with the Declaration of Helsinki and approved by the ethics committees of the First A liated Hospital of Nanjing Medical University. Immunophenotyping was performed on peripheral blood or bone marrow specimens.
Antibodies speci c for these following antigens were used including CD3, CD2, CD5, CD7, CD4, CD8, CD16, CD56, CD57, TCR αβ and TCR γδ. These antibodies were incubated with samples from peripheral blood or bone marrow in different combinations. Additionally, the β Mark TCR Vβ Repertoire Kit was utilized for the quanti cation of the TCR Vβ repertoire of the T lymphocytes. This kit contains mixtures of conjugated TCR Vβ antibodies corresponding to a total of 24 different speci cities, which cover approximately 70% of all the normal human TCR Vβ repertoire. The determination of these 24 Vβ expressions was done in 8 tubes, with one tube permitting the detection of 3 different Vβ expressions by using three different antibodies labeled with uorescein isothiocyanate (FITC), phycoerythrin (PE) and FITC plus PE, respectively. Other antibodies to CD3, CD4 and CD8, which were conjugated to different uorophores were added in each tube. After incubation, red blood cells were lysed using Tris-NH4Cl  Seventy-nine patients and 17 patients had typical immunophenotypes and atypical immunophenotypes, respectively. These 79 cases with typical immunophenotypes were CD8+CD4-TCRαβ + . In 17 cases with atypical immunophenotypes, 8 cases were CD4+CD8-TCRαβ + and 9 cases were TCRγδ + . In TCRγδ + cases, 7 cases were both CD4 and CD8 negative while the other two cases were CD4 negative and CD8 positive.
The clinical and laboratory ndings of patients T-LGLL with typical and atypical immunophenotypes were summarized in Table 2. We compared the clinical and laboratory characteristics of patients with atypical immunophenotypes and those with typical immunophenotypes. We found that presence of PRCA was more frequent in patients with typical immunophenotypes than those with atypical immunophenotypes  We then compared the clinical and laboratory features of TCRγδ + T-LGLL with TCRαβ + T-LGLL (Table 3). The frequency of PRCA was slightly lower in patients with TCRγδ + T-LGLL than in those with TCRαβ + T-LGLL [0%(0/9) vs. 30.4%(26/87),p=0.108]. The frequencies of female, STAT3 mutation, fatigue, mouth ulcer, hepatomegaly and arthralgia were lower in TCRγδ + cases than in TCRαβ + cases, although there was no statistical signi cance. There were no differences in other parameters between TCRγδ + cases and TCRαβ + cases. We also explored the distinction between CD4+ cases and CD4-TCRαβ + cases ( Table  4). The prevalence of PRCA was slightly higher in patients with CD4+ T-LGLL than in those with CD4-TCRαβ + T-LGLL [29.9% (26/79) vs. 0% (0/8), p=0.099]. The frequencies of female, anemia, mouth ulcer and STAT3 mutation were also lower in CD4+ T-LGLL than in CD4-TCRαβ + T-LGLL, although this was not statistically signi cant. No differences in other clinical or laboratory features between patients with CD4+ T-LGLL and patients with CD4-T-LGLL. Table 3 Comparison of baseline characteristics between cases with TCRγδ + T-LGLL and TCRαβ + T-LGLL. LGLL, large granular leukemia; TCR, T cell receptor. Table 4 Comparison of baseline characteristics between cases with CD4 + T-LGLL and CD4 − TCRαβ + T-LGLL. LGLL, large granular leukemia; TCR, T cell receptor.

Discussion
In the present study, we analyzed the clinical pathologic characteristics of 96 cases of T-LGLL. The frequency of PRCA in our cohort was signi cantly higher than that in the patients with T-LGLL from western countries. The incidence of rheumatoid arthritis is relatively low. The above ndings were consistent with the study by Kwong et al., in which showed that Asian patients with T-LGLL were more likely to PRCA and much less likely to have RA (Kwong et al.2010).
By analyzing the immunophenotypes of these cases, we identi ed 17 T-LGLL cases with atypical immunophenotypes, including TCRγδ + phenotype and CD4 expression. The frequency of TCRγδ + LGLL (9/96) in our cohort was similar to that in other studies (Sandberg et al.2006

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TLGLLsupplementtables1.docx