Methotrexate therapy of T-cell large granular lymphocytic leukemia impact of STAT3 mutation

T-cell large granular lymphocytic leukemia (T-LGLL) is a rare haematologic neoplasm. Consequntly, there are no large prospective studies of therapy and no uniform therapy recommendations. We analyzed data from 36 subjects receiving methotrexate alone (N = 27) or with prednisone (N = 9) as initial therapy. 31 subjects responded (86%, 95% confidence interval [CI], 73, 95%) with 8 complete responses and 23 partial responses. Median time-to-response was 3 months (range, 1–5 months). Median response duration was 20 months (range, 2–55 months). β2-microoglobulin (β2-MG) and erythrocyte sedimentation rate (ESR) decreased significantly post-therapy (P < 0.0001). Pure red cell aplasia (PRCA) was present in 18 subjects (50%) of our subjects and responded well to methotrexate. 26 subjects (72%) were tested for STAT3 mutation. 9 with a mutation had a median treatment-free survival of 5 months (range, 0.5–13 months), significantly briefer than that of 17 subjects without a STAT3 mutation (19 months, range, 3–97 months; P = 0.012; log-rank test). Methotrexate with or without prednisone is an effective initial therapy of persons with T-LGLL with wild-type STAT3.

IntroductIon T-cell large granular lymphocytic leukemia (T-LGLL) is a lympho-proliferative neoplasm of cytotoxic T cells [1]. There is no standard therapy but immune suppression is commonly used when therapy is needed. The three most commonly used drugs are methotrexate, cyclosporine and cyclophosphamide. Treatment outcomes are heterogeneous and there is only one comparative study which shows immune suppression is effective and mutational profiling predicts response [2].
T-LGLL can present as failure of hematopoiesis or immune-mediated destruction of one or several cell lines including RBCs, neutrophils, and platelets. T-LGLL associated pure red cell aplasia (PRCA) accounts for a significant portion of secondary PRCA [3]. Because T-LGLL and T-LGLL associated PRCA are rare optimal long-term outcomes after immune suppressive therapy are controversial. We report results using methotrexate as initial therapy of T-LGLL in 36 subjects most of whom also had PRCA. Correlative studies were conducted to determine if biomarkers or genetic analysis could predict therapy response.

results clinical variables
Data on the 36 subjects are displayed in Table 1

laboratory results
Four subjects had anaemia (Hemoglobin < 110 g/L) and decreases in neutrophils (< 1.5 × 10E + 9/L) and platelets (< 100 × 10E + 9/L). 24 had only neutropenia, severe in 1. Thrombocytopenia was present in 7 subjects. Anaemia was present in 34 (Table 1). 18 subjects had PRCA (Table 1). 23 subjects tested had normal cytogenetics. Three subjects had a positive rheumatoid factor test one of whom had rheumatoid arthritis. All subjects had clonal rearrangement of the T-cell receptor.
In the 18 subjects with PRCA there were 6 complete and 11 partial responses with an overall response rate of 94% (95% confidence interval, 73-100%). Median timeto-response was 3 months (range, 1-5 months). Median response duration was 20 months (range, 5-55 months). Response rate in subjects with PRCA was not significantly different than in all subjects.

serum biomarkers
We also assessed dynamic changes of ESR and β 2 -MG in serum of subjects as a marker of therapy response. Baseline levels (mean ± SEM) of serum β 2 -MG and ESR levels were 4.67 ± 2.76 mg/L and 61.2 ± 41.1 mm/h. Post-therapy values at the time of best response were 2.76 ± 1.53 mg/L (P = 0.003) and 20.1 ± 21.0 mm/h (P < 0.001).

Toxicity
Adverse events were uncommon and none required stopping therapy. There were 2 grade-1 toxicities, one an alanine aminotransferase increase and one a total bilirubin increase each in 1 subject. Grade-2 toxicities included oral mucositis (1 subject) and nausea (3 subjects).

dIscussIon
We report a high response rate with methotrexate with or without prednisone as initial therapy of T-LGLL. Subjects with associated PRCA had correspondingly high response rates and long response durations. Others report similar response rate but many more relapses [4][5][6]. Time-to-response of subjects receiving methotrexate and prednisone was briefer subjects receiving methotrexate only but this allocation was not randomized and requires confirmation. No responder relapsed immediately after discontinuing prednisone. Response rates were similar in persons with and without PRCA. www.impactjournals.com/oncotarget LGL, large granular lymphocyte; ANA, anti-nuclear antibody; β2-MG, β2-microglobulin; ESR, erythrocyte sedimentation rate.    [7,8]. We found STAT3 mutations in 9 of 26 subjects (35%). Like some, but in contrast to other reports, we found an association between STAT3 mutation and PRCA [8,9]. We also found differences in time-toresponse and treatment-free survival in subjects with and without STAT3 mutations. Others reported STAT3 Y640F is associated with response to methotrexate [2]. These data suggest screening for STAT3 mutations may be useful.
There are important limitations to our study. Numbers of subjects is small compromising our power to detect differences. Although the series was consecutive we cannot exclude referral biases. Assignment to prednisone was not random. Finally, there were too few subjects to test the association between STAT3 mutation and treatment-free survival. Because of these limitations our conclusions require validation. In summary, methotrexate alone or combined with prednisone is an effective initial therapy of T-LGLL with high response rates and durable responses. Persons with associated STAT3 mutation had worse outcome than those with wild-type STAT3.  3) LGL concentration > 2 × 10E + 9/L; (4) T-cell clonality; and (5) persistence > 6 months. PRCA was defined as reported [10].

separation of mononuclear cells and dnA isolation
Blood or bone marrow samples were collected at diagnosis and mononuclear cells isolated by density gradient centrifugation using Ficoll-Hypaque. Genomic DNA was extracted and purified as described using the QIAamp blood kit (Qiagen, Hilden, Germany). DNA concentration and purity were measured with Eppendorf Biophotometer (Eppendorf, Hamburg, Germany).

Multiplex polymerase chain reaction (PCR) analysis for t-cell receptor gene rearrangement
PCR analysis for T-cell receptor gene rearrangement was performed using genomic DNA extracted from the mononuclear cells and performed as previously described [12].

Analysis of stAt3 mutations
DNA from samples from 26 consenting subjects was tested for mutations in exons 20 and 21. STAT3 amplicon sequencing and data analyses were done as described [11]. treatment Indications for treatment included: (1) absolute neutrophil level (ANC) < 0.5 × 10E + 9/L; (2) recurrent infections independent of ANC; (3) symptomatic or RBCtransfusion-dependent anemia; and/or (4) autoimmune conditions such as PRCA or rheumatoid arthritis [13]. Methotrexate was given orally at a dose of 10 mg/mE + 2 weekly. Oral low-dose folic acid (5 mg/d) was given to prevent mouth ulcers. Prednisone (0.5-1 mg/kg/d) could be given for ≤ 2 months of starting methotrexate.

response criteria
Complete response was defined as normalization of blood cell levels and reduction of LGLs to the normal range (ANC > 1.5 × 10E + 9/L; lymphocytes < 4.0 × 10E + 9/L; hemoglobin concentration >110 g/L; platelets >100 × 10E + 9/L). Partial response was defined when either of the following: ANC increase > 50% and > 0.5 but < 1.5 × 10E + 9/L; haemoglobin concentration increase > 20 g/L and RBC-transfusion-independence but < 110 g/L. No response was defined as a response < partial response > 4 months after starting methotrexate but without disease progression. Progressive disease was defined as worsening of hematologic parameters in subjects previously achieving ≥ partial response. Toxicity was graded using the modified NCI Common Terminology Criteria (CTC, version 4.0). Blood cell levels and differentials and kidney and liver function tests were analyzed monthly. Treatment was continued in responders until relapse, death or withdrawal of consent.
endpoints Time-to-response was defined as the interval from starting methotrexate to a ≥ 50% improvement in blood cell level(s). Response duration was defined as the interval from declaring response to relapse. Treatment-freesurvival was defined as the interval from date of diagnosis to starting methotrexate. Survival was defined as the interval from diagnosis to death or last follow-up.

statistical analyses
Statistical analyses used the SPSS program for Windows (version 16.0). Comparisons of proportions and ranks of variables between groups were performed by chisquared-test, Fisher exact test or Student t-test. Kaplan-Meier survival estimates were constructed and differences compared by log-rank test. An effect was considered statistically significant at P < 0.05. P-values were two sided.