High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαβ-Large Granular Lymphocytic Leukemia

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-LGL ranged between 0.36 and 571 cells/μL (3.2–91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/μL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ cells ranged between 0 and 25 TRBC1+ or TRBC1− cells/μL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1− cells/μL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Cyclophosphamide Induces Durable Molecular and Clinical Responses in Patients with Relapsed T-Large Granular Lymphocytic Leukemia (T-LGLL)

T-cell large granular lymphocytic leukemia (T-LGLL) is a clonal proliferation of cytotoxic T-lymphocytes that can result in severe cytopenias. The management of T-LGLL is immune-suppressive therapy, with methotrexate (MTX), cyclophosphamide (Cy), and cyclosporine (CsA) serving as primary frontline agents. While MTX has been the main front line agent to treat T-LGLL, overall response rates (ORR) are less than 40%, with complete response (CR) rates of only 5%. Data from the ECOG5998 (E5998) prospective trial and French cohort studies suggest an improved response with Cy in the 2 nd line setting. Anecdotal evidence suggests that Cy may eradicate the T-LGLL clone, producing complete molecular remission (CMR), which has not been observed with MTX or CsA. The degree to which a CMR can be attained and the lengths of such remissions with Cy remains unknown, particularly in the relapsed setting. We evaluated patients treated with Cy, to assess the duration of response and degree of CMR. We retrospectively evaluated patients treated for T-LGLL with oral Cy. Diagnosis of was based on 2016 World Health Organization Criteria. Patients needed a CD3+ CD8+ population on flow cytometry ≥500 cells/mm 3 and a positive monoclonal T-cell receptor (TCR) by PCR or restriction of TCR-Vbeta on flow cytometry. TCR-Vbeta rearrangement was deemed positive if one or more clone was detected in ≥10% of events. Disease response was defined by the E5998 study criteria. CMR was defined as CR by E5998 criteria and clearance of the TCR PCR gene rearrangement or TCR-VBeta flow cytometry. Time to response (TTR) was measured as time from start of Cy until partial response (PR) or CR, with patients who failed to respond being censored at the end of Cy treatment. Leukemia-free survival (LFS) in patients responding to Cy was measured as time from start of Cy until progression. Patients without progression were censored at last follow up. TTR and LFS were compared across variables using Kaplan-Meier curves with median survival and 95% confidence intervals. A total of 25 patients, with a mean duration of Cy treatment of 8 months, and median follow up time of 19 months, were included in this analysis. Patients were started on 50 mg daily for 2 weeks and then increased to 100 mg if tolerated. Three patients (12%) were treated with Cy as 1 st line, 14 (56%) as 2 nd line, 5 (20%) as 3 rd line, and 3 (12%) as 4 th line. Of the 3 patients that received Cy as 1 st line, none had a response. All refractory patients received MTX prior to Cy. Of the 22 refractory patients, 14 (64%) had a response (6 CR, 8 PR), 7 patients had no response, and 1 could not be determined due to development of multiple myeloma. Of the 6 patients that attained a CR, 50% had a CMR. The median TTR (CR or PR) was 6 months (95% CI: 4-7) while median time to PR was 9 months and median time to CR was 7 months. The median time to CMR was 11 months. In patients that achieved a response, median follow up time was 27 months, with a median LFS of 24 months. Median LFS for those who attained a PR was 20 months, while the median LFS for those who attained a CR was not reached as none progressed (Figure). The median follow-up for patients with a CMR was 17 months with LFS having not been reached due to no progression. There was no significant impact of age, gender, or presence of rheumatoid arthritis (RA) on LFS. Males (48%) had a shorter TTR compared to females (52%) (5 vs 7 months; p=0.05). Patients with RA (28%) also trended towards a shorter TTR (p=0.07). Herein, we demonstrate that patients treated for relapsed T-LGLL with Cy can attain durable remissions, with a prolonged response. Of particular interest is that no patients who attained a CR have relapsed, showing that durable remission is achievable, while no patients that received Cy as 1 st line had a response. Further, in patients that achieved a CR, 50% achieved a CMR which has not been previously demonstrated in the relapsed setting. While limited in cohort size, and additional follow up needed, these data suggest that Cy can produce long term remissions in patients with relapsed T-LGLL and induce CMR. While we demonstrate that CMR is attainable in the relapsed setting, the impact of this on long term disease control compared to clinical CR is unclear. Therefore, we recommend that CMR be used as an endpoint in future studies, particularly prospective trials, to evaluate response to treatment. These data clearly demonstrate that Cy is effective in the setting of relapsed T-LGLL and can induce long term disease control. Figure 1 Figure 1. Disclosures Brammer: Seattle Genetics: Speakers Bureau; Kymera Therapeutics: Consultancy; Celgene: Research Funding.

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-defined. 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 significance (p=0.086). Conclusion Patients with atypical immunophenotypes showed a similar survival outcome to that of typical T-LGLL immunophenotypes. Additional efforts were needed to better understand the pathogenesis of these rare atypical immunophenotypes T-LGLL cases.