Gregorio Barilà

Angela Grassi

Heejin Cheon

Antonella Teramo

Giulia Calabretto

Jasmanet Chahal

Cristina Vicenzetto

Julia Almeida

Bryna C Shemo

Min Shi

Vanessa Rebecca Gasparini

Noemi Munoz-Garcia

Cédric Pastoret

Hideyuki Nakazawa

Kazuo Oshimi

Lubomir Sokol

Fumihiro Ishida

Thierry Lamy

Alberto Orfao

William G Morice

Thomas P Loughran

Gianpietro Semenzato
email: g.semenzato@unipd.it

Renato Zambello
email: r.zambello@unipd.it

Prof Renato Zambello

Prof. Gianpietro Semenzato

Tγδ LGL LEUKEMIA IDENTIFIES A SUBSET WITH MORE SYMPTOMATIC DISEASE: ANALYSIS OF AN INTERNATIONAL COHORT OF 137 PATIENTS

Keywords: Tγδ Large Granular Lymphocyte Leukemia, Treatment, STATs mutations, Vδ2 status

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Large Granular Lymphocyte Leukemia (LGLL) is a rare and heterogenous chronic lymphoproliferative disorder characterized by the clonal expansion of Large Granular Lymphocytes (LGLs) [START_REF] Barila | T cell large granular lymphocyte leukemia and chronic NK lymphocytosis[END_REF][START_REF] Lamy | LGL leukemia: from pathogenesis to treatment[END_REF] . The etiology of LGLL is unknown but a constitutive activation of JAK/STAT pathway is involved in the pathogenesis of LGL proliferation [START_REF] Epling-Burnette | Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression[END_REF] , further supported by the discovery of somatic STAT3 and STAT5b mutations in approximately 40% of patients [START_REF] Koskela | Somatic STAT3 mutations in large granular lymphocytic leukemia[END_REF][START_REF] Jerez | STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients[END_REF][START_REF] Teramo | STAT3 mutation impacts biological and clinical features of T-LGL leukemia[END_REF][START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Rajala | Discovery of somatic STAT5b mutations in large granular lymphocytic leukemia[END_REF][START_REF] Teramo | Insights Into Genetic Landscape of Large Granular Lymphocyte Leukemia[END_REF] . Among LGLLs, the latest WHO classification recognizes a CD3+ T-LGLL and CD3-NK-LGLL, accounting for 85% and 15% of cases, respectively. Moreover, based on surface T cell receptor expression, Tαβ and Tγδ subsets of LGLL can be identified [START_REF] Alaggio | The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: Lymphoid Neoplasms[END_REF] .

While LGLL incidence ranges between 0.2 to 0.72 cases per 1 million individuals per year [START_REF] Barila | T cell large granular lymphocyte leukemia and chronic NK lymphocytosis[END_REF] , the frequency of Tγδ proliferation is still not well defined, and most information has been collected through small retrospective studies. As compared to the more frequent Tαβ LGLL, Tγδ LGLL has been less investigated. Firstly reported by Oshimi et al. in 1988 in a 60 years old female exposed to the radiation in Nagasaki in 1945 [START_REF] Oshimi | Ti (WT31)-negative, CD3-positive, large granular lymphocyte leukemia with nonspecific cytotoxicity[END_REF] , Tγδ LGLL has been described in a sizable number of patients in 2006 by Sandberg et al. who reported an immunophenotypical analysis of 44 cases [START_REF] Sandberg | TCRgammadelta+ large granular lymphocyte leukemias reflect the spectrum of normal antigen-selected TCRgammadelta+ T-cells[END_REF] . Up to now, only 4 retrospective studies including more than 200 LGLL patients are available [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Bareau | Analysis of a French cohort of patients with large granular lymphocyte leukemia: a report on 229 cases[END_REF][START_REF] Sanikommu | Clinical features and treatment outcomes in large granular lymphocytic leukemia (LGLL)[END_REF][START_REF] Dong | Large granular lymphocytic leukemia -A retrospective study of 319 cases[END_REF] , however few cases of Tγδ LGLL were included and only in the Italian cohort [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] . Consequently, the clinical features of Tγδ LGLL and information on the efficacy of treatments in this LGLL variant are still missing. Furthermore, data on the frequencies of STAT3 and STAT5b mutations are nowadays available in Tαβ LGLL, but still limited and controversial in Tγδ LGLL. The Italian group recently reported 25% and 19% of Tγδ cases mutated in STAT3 and STAT5b genes, respectively [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] , while STAT3 mutations were found in all patients included in a small Japanese Tγδ LGLL cohort [START_REF] Yamane | High frequency of STAT3 gene mutations in T-cell receptor (TCR)gammadelta-type T-cell large granular lymphocytic leukaemia: implications for molecular diagnostics[END_REF] .

With this as a background and lacking large cohorts of Tγδ patients, major referral groups dealing with LGLL were invited to join this collaborative study aimed at better characterizing Tγδ LGLL

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METHODS

Study patients

The study cohort included 137 patients affected by Tγδ LGLL followed from 1997 to 2020 at 8 referral centres across the world (France, Italy, Japan, Spain, USA). All patients met the currently approved WHO (World Health Organization) diagnostic criteria for T-LGLL [START_REF] Lamy | LGL leukemia: from pathogenesis to treatment[END_REF][START_REF] Swerdlow | WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF] . T-LGL clonality was assessed by TCRγ gene rearrangement.

Demographic and clinical features, including presence of cytopenias, concomitant autoimmune/inflammatory diseases, secondary primary malignancies, treatment requirement and response, were collected. Response to treatment was evaluated based on periodical clinical and laboratory examinations after at least 4-6 months of therapy, using the currently accepted response criteria for LGLL [START_REF] Poullot | Chronic natural killer lymphoproliferative disorders: characteristics of an international cohort of 70 patients[END_REF] . The frequency of LGLs positive for the characteristic antigens was assessed by flow cytometry using direct immunofluorescence assays combining up to 6 markers/tube, according to standard operating procedures of individual centers. The investigation for LGL surface markers was performed on whole peripheral blood anti-coagulated with EDTA or ACD and on purified PBMCs. The commercially available FITC-, phycoerythrin (PE)-, PE-Cy5-, PE-Cy7-, APC-and APC-Cy7-conjugated mouse monoclonal antibodies (mAbs) used included: anti-CD3, anti-CD4, anti-CD8, anti-CD16, anti-CD56 and anti-CD57, anti-TCRγδ, anti-KIRs (killer Immunoglobulin-like receptors: CD158a, CD158b, CD158e), anti-NKG2A, anti-NKG2C, anti-Vγ9, anti-Vδ1 and anti-Vδ2 from Becton Dickinson (Sunnyvale, CA, USA). This international Tγδ LGL leukemia cohort was compared with a recently reported equal size Italian Tαβ LGL leukemia cohort [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] .

This study was performed according to the Helsinki Declaration and patients gave their written informed consent prior to inclusion in the study. The protocol and informed consent form were approved by the Padua ethics committee (approval number 4213/AO/17).

Statistical analysis

Patients' demographic, clinical and biological features expressed as categorical variables were compared by Fisher's exact test. Patient's overall survival (OS) was calculated from the date of diagnosis to death by any cause or the last known follow-up visit for censored patients. Survival curves were estimated using the Kaplan-Meier method and compared with respect to the patients' demographic and clinical characteristics with log-rank test. Schoenfeld residual testing was applied to assess the proportional hazards assumption. A univariate Cox proportional hazards regression analysis was employed to evaluate the prognostic relevance of each variable. Results for significant variables were presented as hazard-ratios (HR) and 95% confidence intervals.

To determine the effect of response to first-line treatment on PFS and OS we performed a 6months landmark analysis in treated patients categorized by their response status (at least partial response vs. stable disease or progressive disease) at 6 months after the start of therapy. The 6months landmark time was selected a priori, before the beginning of data analysis, since at least 4-6 months of treatment are recommended before correctly assess the response. For landmark analyses, PFS and OS were recalculated by shifting the time origin to 6 months after the start of therapy and patients who experienced the event of progression or death before this time were excluded from the PFS or OS landmark analyses, respectively.

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A restricted mean survival time (RMST) analysis was also performed to compare the Tγδ and Tαβ

LGLL cohorts. RMST is a robust and clinically interpretable summary measure of the survival time distribution, estimable even under heavy censoring and when the proportional hazards assumption is not satisfied, as an alternative to the hazard ratio approach [START_REF] Royston | Restricted mean survival time: an alternative to the hazard ratio for the design and analysis of randomized trials with a time-to-event outcome[END_REF][START_REF] Uno | Moving beyond the hazard ratio in quantifying the between-group difference in survival analysis[END_REF] . This analysis depends on the truncation time point fixed for the RMST calculation. Four different truncation time points (100, 120, 140 and 160 months) were evaluated for the comparison of Tγδ and Tαβ

LGLL cohorts. P values < 0.05 were considered significant. Statistical analysis was conducted using R version 3.6.2. Not being part of the work-up for the diagnosis of LGLL, bone marrow evaluation, either by flow cytometry or immunohistochemistry, was available for only 40/137 (29.2%) patients, showing variable degree of infiltration with a range from less than 1% to 60% of bone marrow cellularity.

RESULTS

Clinical and Immunophenotypic features of Tγδ LGLL patients

Clinical and biological features of patients under study are summarized in

Neutropenia -absolute neutrophil count (ANC) <1,500/mm 3 -and mild anemia -hemoglobin (Hb) <120g/L-were the main relevant clinical features of the entire cohort, being present in 54.2% (65/120) and 49.6% (59/119) of patients, respectively. Severe neutropenia (ANC<500/mm 3 ) and severe anemia (Hb<90g/L) were observed in 25/120 patients (20.8%) and in 25/119 cases (21%), respectively. Thrombocytopenia (PLTs <100,000/mm 3 ) and splenomegaly were detected in 18/119 (15.1%) and in 31/122 (21.4%) patients, respectively. Forty-nine patients (41.5%) were affected by concurrent autoimmune/inflammatory (AA) diseases, mostly rheumatoid arthritis (16/49), autoimmune hemolytic anemia (5/49) and pure red cell aplasia (5/49). Finally, secondary primary malignancies (SPM) were detected in 17/84 patients (20.2%), either at the time of diagnosis or during the follow-up. Seven SPM were hematological (three marginal zone lymphoma, one chronic lymphocytic leukemia, one myelodysplastic syndrome, one plasma cell dyscrasia and one systemic mastocytosis) and ten non-hematological neoplasms, including three cases of thymoma, three cases of thyroid neoplasms, one lung cancer, one prostatic cancer, one cervical cancer and one skin cancer.

Treatment of Tγδ patients

Overall, more than half (53.7%) of patients required therapy during the natural history of the disease. All these patients were treated according to currently accepted indications [START_REF] Lamy | LGL leukemia: from pathogenesis to treatment[END_REF][START_REF] Lamy | How I treat LGL leukemia[END_REF] 1 and Table 1. Overall response (ORR) and complete response (CR) rates were lower in MTX treated patients (26.9% and 7.7%, respectively) compared to patients who received CyA and CTX (ORR: 53.9% and 40%, respectively, CR: 23.1% and 40%, respectively), although the latter therapies were used in lower numbers of cases, particularly CTX. Four patients treated with MTX discontinued the treatment due to toxicity.

Among patients requiring treatment (N=57), landmark analyses for progression free survival (PFS) and overall survival (OS) were performed according to response status at 6 months since therapy initiation, only in the subsets of patients for whom precise timing of response was available (N=20 for PFS and N=29 for OS). Irrespective from the type of first line treatment, responders (patients reaching at least partial response) after 6 months from the start of therapy were characterized by an increase in PFS with respect to non-responders (HR=6.16, 95% CI: 0.77-50.00; log-rank test p=0.05) (Figure 1, Panel A). Notably, although with a p-value not statistically significant, responders at 6 months showed also longer OS as compared to non-responders (log-rank test p=0.13) (Figure 1, Panel B). These results suggest a possible prognostic role of early response to first-line therapy that should be further addressed in future prospective studies, by systematically collecting response times.

STAT3 and STAT5b mutation analysis

DNA samples of 97 and 94 patients were available for STAT3 and STAT5b mutational analyses, respectively. STAT3 mutations were detected in 37 patients (38.1%), with a prevalence of variants as follows: Y640F was detected in 16 cases (43.2%), D661Y in 9 cases (24.4%), D661V and S614R in 2 cases each (5.4%), and the H410R, Q448E, G618R, E638Q, K658F and N647I variants were found in one case each (2.7%). In the Italian cohort, two patients showed the A662_N663delinsH deletion/insertion and an in-frame insertion, G656_Y657ins, as previously reported [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] . In contrast, STAT5b mutations were found in only 4 patients (4.2%), from whom 3 patients carried the N642H variant and one had the Y665F mutation. Of note, STAT3 and STAT5b mutations were mutually exclusive in Tγδ LGLL patients, never being detected concurrently in the same patient.

From the phenotypic point of view, STAT3 mutated patients were characterized by lower frequency of expression of CD56 (3.8% vs 56.1%, p<0.0001), Vδ2 (0% vs 50%, p=0.0003) and Vγ9 (25% vs 57.1%, p=0.04). In addition, they showed a higher frequency of neutropenia (65.7% vs 40.8%, p=0.0288), severe neutropenia (31.4% vs 12.2%, p=0.0519), anemia (55.9% vs 34.7%, p=0.0726), autoimmune/autoinflammatory disorders (59.4% vs 31.5%, p=0.0139). They more frequently required therapy (67.9% vs 37.5%, p=0.0169) (Table 2). In contrast, no significant differences were found between STAT3 mutated and wild-type Tγδ LGLL patients regarding the frequency of cases with LGL counts >2,000/mm [START_REF] Epling-Burnette | Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression[END_REF] Differently than STAT3 mutated cases, STAT5b mutated patients were mostly asymptomatic, with only one patient experiencing mild neutropenia and splenomegaly.

Vδ pattern of expression analysis

Tγδ cells usually express 5 different Vδ receptor families (from Vδ1 to Vδ5), Vδ2 being generally expressed in blood circulating Tγδ cells, while the other subsets are typically enriched in epithelia, liver, and spleen [START_REF] Lalor | Memory gammadelta T Cells-Newly Appreciated Protagonists in Infection and Immunity[END_REF] . In our cohort, flow cytometric Vδ analysis was available in 51 patients, 17 cases (33.3%) resulted Vδ2 positive while the remaining 34 (66.7%) were Vδ2 negative. Within this latter subset of patients, 16/34 (47.1%) were Vδ1 positive while 18 cases were neither Vδ1 nor Vδ2 positive (Table 3).

Vδ2 positive patients displayed a higher frequency of expression of CD56 (100% vs 9.1%, p<0.0001), KIR (64.3% vs 18.8%, p=0.0236), CD94 (76.5% vs 42.9%, p=0.0351) and NKG2A (71.4% vs 6.2%, p=0.0004), while no significant differences were found (vs Vδ2 negative cases) regarding CD16 and CD57 expression (100% vs 81.8%, p=0.1412 and 100% vs 81.8%, p=0.1412, respectively).

Interestingly, all Vδ2 positive cases showed concomitant Vγ9 expression (100%), while only a small fraction of Vδ2 negative patients was also Vγ9 positive (18.2%, p<0.0001).

From the clinical point of view, Vδ2 positive cases displayed a more indolent LGLL. They rarely presented with symptomatic disease including neutropenia (5.9% vs 65.6%, p<0.0001), severe neutropenia (0% vs 31.2%, p=0.0094), anemia (0% vs 56.2%, p<0.0001), severe anemia (0% vs 34.4%, p=0.0090), splenomegaly (0% vs 26.7%, p=0.0371), and concurrent autoimmune/inflammatory disease (6.2% vs 48.4%, p=0.0039), in the absence of treatment requirement (0% vs 54.5%, p=0.0007). Interestingly, STAT mutations were mutually exclusive in Vδ2 negative and Vδ2 positive patients, all STAT5b mutated cases being Vδ2 positive (p=0.0327), whereas all STAT3 mutated patients were Vδ2 negative (p=0.0003) (Table 3).

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Clinical and biological features of Tγδ vs Tαβ LGLL.

To get further insight into the unique clinical and biological features of Tγδ LGLL, we compared our cohort of patients with a recently published Tαβ LGLL cohort of comparable size 7 (Table 4). No significant differences in gender and age were found between the two disease subtypes (p=0.3906 and p=0.2408, respectively), while Tαβ LGLL cases generally showed higher LGL counts than Tγδ

LGLL cases (LGL count >2,000/mm 3 in 54.3% vs 22% patients, respectively; p<0.0001). By immunophenotype, Tγδ LGLL displayed a significantly higher frequency of expression of CD16 (72.3% vs 45.7%, p<0.0001), CD94 (42.7% vs 14%, p<0.0001), NKG2A (22.2% vs 10.1%, p=0.0355) and CD158a (14.3% vs 4.7%, p=0.0330) together with an increased KIR expression (41.1% vs 27.9%, p=0.0876), while they showed lower frequency of CD56 (31.1% vs 48.1%, p=0.0106) and CD57 expression (78.4% vs 94.6%, p=0.0003). Regarding STAT mutations, no significant differences were found between Tγδ and Tαβ LGLL patients in the frequency of STAT3 (38.1% vs 37.9%, respectively; p=1) and STAT5b mutations (4.8% vs 12.5%, respectively; p=0.1130).

From the clinical point of view, Tγδ LGLL patients more frequently showed symptomatic disease in terms of neutropenia (54.2% vs 38.8%, p=0.0161), anemia (49.6% vs 11.6%, p<0.0001), severe anemia (21% vs 8.5%, p=0.0065), thrombocytopenia (15.1% vs 5.4%, p=0.0187), and concurrent autoimmune/inflammatory diseases (41.5% vs 21.7%, p=0.0009) (Table 4).

The markedly different observation times of Tγδ-LGLL and Tαβ-LGLL patients prevented to use a

Fisher's exact test for these comparisons since this could lead to major bias due to lack of consideration of the time variable. Consequently, for secondary primary malignancies and need for treatment the data and the related p-value were not available.

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Survival analysis

All demographic, clinical and biological features were evaluated for association with overall survival (OS) in Tγδ LGLL patients. After a median follow-up of 48 months, the median OS of our cohort was not reached. Splenomegaly was the only variable significantly associated with a shortened OS (log-rank test p=0.0012), with an HR=0.18 (95% CI: 0.06-0.59) (Figure 2, panel A), while other clinical and biological features of the disease had no significant impact on patient OS, including those previously found to be relevant for Tαβ LGLL patients [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] , i.e. STAT3 and STAT5b mutation status, or the presence of severe neutropenia or anemia (Supplementary Figure 2).

Direct comparison of patients' OS between Tγδ

LGLL and the more common Tαβ LGLL is likely to prove a poorer overall outcome for Tγδ LGLL patients vs. Tαβ LGLL (log-rank test p=0.017) (Figure 2, panel B). This result must be interpreted with caution, since the two cohorts have different median follow-up times (Tγδ LGLL, 4 years vs. Tαβ LGLL, 9 years) and the proportional hazards assumption seems not to be fully satisfied due to the lack of events in the Tγδ cohort from 143 months onwards. Given the rarity of Tγδ LGLL, it was not possible to increase the cohort size, consequently we provided a supplementary analysis using a different measure of the effect that does not require the proportional hazards assumption, i.e. the restricted mean survival time (RMST). This analysis confirms a significant disadvantage in terms of survival of Tγδ LGLL patients with respect to Tαβ LGLL (Supplementary Table 2).

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DISCUSSION

Here we report on the largest cohort of Tγδ LGL leukemia patients described so far in the literature with data collected between 1997 and 2020, as the result of a collaborative study involving 8 LGLL referral centers across the world. For the first time, we evaluated the clinical and biological features of this rare subset of T-LGLL on a large number of patients, screened for STAT3 and STAT5b mutations. Overall, our results showed that Tγδ LGLL represents a variant with higher frequency of symptomatic disease and reduced survival as compared to the most common Tαβ

LGLL subtype, despite a similar frequency of STAT3 and to a less extent of STAT5b mutations.

In the past LGLL was considered a unique chronic and indolent disease, except for a few patients presenting with very aggressive disease [START_REF] Semenzato | The lymphoproliferative disease of granular lymphocytes. A heterogeneous disorder ranging from indolent to aggressive conditions[END_REF] . In recent years, however, a better understanding of this disorder has been achieved pointing out the need for therapy in a significant fraction of LGLL patients [START_REF] Teramo | STAT3 mutation impacts biological and clinical features of T-LGL leukemia[END_REF][START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Barila | Dominant cytotoxic NK cell subset within CLPD-NK patients identifies a more aggressive NK cell proliferation[END_REF] . Data provided in this study further encourage distinguishing Tγδ LGLL from Tαβ LGLL, since Tγδ LGLL patients showed unique clinical and biological features. Even though characterized by lower LGL counts, Tγδ LGLs more frequently express the CD16 and CD94 receptors, while the CD56 adhesion molecule and the CD57 immunosenescence-associated protein are less commonly expressed. Most importantly, Tγδ LGLL patients more frequently displayed symptomatic disease due to anemia (often transfusion dependent) potentially partially explained by an increased frequency of autoimmune hemolytic anemia and pure red cell aplasia [START_REF] Chen | Clinical, morphologic, immunophenotypic, and molecular cytogenetic assessment of CD4-/CD8-gammadelta T-cell large granular lymphocytic leukemia[END_REF] , and concomitant autoimmune diseases. Altogether, this translates into a poorer outcome as compared to the more common Tαβ subtype of LGLL.

Overall these results are not consistent with previously reported data that did not show clear clinical differences between Tγδ LGLL and Tαβ LGLL [START_REF] Bourgault-Rouxel | Clinical spectrum of gammadelta+ T cell LGL leukemia: analysis of 20 cases[END_REF] ; however, the T-LGLL cohort reported by Bourgault-Rouxel et al. included only a small number of Tγδ patients (20 cases) compared to the almost 200 Tαβ reported cases, which limits the robustness of the conclusions raised [START_REF] Bourgault-Rouxel | Clinical spectrum of gammadelta+ T cell LGL leukemia: analysis of 20 cases[END_REF] ACCEPTED MANUSCRIPT / CLEAN COPY limitation to be considered in the explanation of the worst outcome in Tγδ LGLL could be related to a high frequency of late-stage diseases due to the challenging diagnosis. As a matter of fact, in our series Tγδ patients showed lower LGLs count and CD57 expression as compared to the more common Tαβ patients. The high frequency of symptomatic patients herein reported within the Tγδ

LGLL cohort may account for the reduced overall survival in this LGLL subtype. Aside this potential bias in survival analysis, our data point to the recommendation to include the Tγδ immunophenotype in the diagnostic work-up of unexplained cytopenia.

Despite the comparable size, the Tγδ and Tαβ LGLL cohorts we studied are characterized by different median follow-up (48 vs. 108 months, respectively), moreover the Tγδ LGLL cohort, due to its retrospective nature, suffers for the presence of several censored data. These findings lead to certain limitations in the interpretation of results. For this reason, an additional RMST analysis has been provided to mitigate these limitations, confirming a significant survival disadvantage for Tγδ LGLL patients with respect to Tαβ LGLL. In future perspective studies aimed at comparing the two cohorts it could be interesting to carefully plan the data collection in order to analyze variables that may depend on observation time, e.g. SPM or need for treatment, with a more appropriate time-to-event approach, thus minimizing any bias due to different follow-up lengths.

According to retrospective studies including few and heterogenous series of patients [START_REF] Loughran | Treatment of large granular lymphocyte leukemia with oral low-dose methotrexate[END_REF][START_REF] Moignet | Cyclophosphamide as a first-line therapy in LGL leukemia[END_REF][START_REF] Battiwalla | HLA-DR4 predicts haematological response to cyclosporine in T-large granular lymphocyte lymphoproliferative disorders[END_REF] , treatment of LGLL still relies on immunosuppressive therapy, where MTX and CTX are used upfront, while CyA is generally reserved for relapsed/refractory patients [START_REF] Barila | T cell large granular lymphocyte leukemia and chronic NK lymphocytosis[END_REF][START_REF] Lamy | LGL leukemia: from pathogenesis to treatment[END_REF][START_REF] Lamy | How I treat LGL leukemia[END_REF] . To date, only one published prospective trial evaluating the efficacy of immunosuppressive therapy in LGLL is available [START_REF] Loughran | Immunosuppressive therapy of LGL leukemia: prospective multicenter phase II study by the Eastern Cooperative Oncology Group (E5998)[END_REF] , and one prospective and randomized trial comparing MTX and CTX as first line therapy in LGLL is currently ongoing (NCT01976182). However, all these studies do not report on the frequency of Tγδ LGLL analyzed and their specific response to therapy. Unexpectedly, MTX treatment led to unsatisfactory response rates in our series of Tγδ LGLL patients, with ORR being ACCEPTED MANUSCRIPT / CLEAN COPY observed in less than a third of patients, including CR in a very limited number of cases (7.7%). In contrast, first line therapy with CyA turned out to provide higher efficacy, with almost half patients responding of whom 23.1% reached complete response. An association between Tγδ LGLL and Pure Red Cell Aplasia (PRCA) has been widely described and it is also known that PRCA patients benefit from CyA treatment. In our cohort, treatment indication for the CyA cohort was available for 14 cases and 12 patients started therapy due to anemia, in 8 cases transfusion dependent; the remaining 2 cases had concomitant diagnosis of PRCA. It can be argued that PRCA has been underestimated in Tγδ LGLL with anemia/severe anemia, thus explaining the high overall and complete response rates obtained with CyA in this subgroup of patients. Of notice, the choice of the appropriate therapy is of utmost clinical relevance since we demonstrated here that responding patients were also characterized by a prolonged PFS and an improved overall survival.

These data could offer a rationale for investigating CyA in the first line treatment of Tγδ LGLL, e.g. in new prospective trials. Altogether, the above results indicate that, besides the distinction between T-LGLL and NK-LGLL, further dissection of T-LGLL into the Tαβ and Tγδ LGLL disease variants is of clinical relevance due to the poorer outcome and distinct treatment response profile of the latter patients. It is also worth noting that Tγδ LGLL cases did not appear as a homogeneous disease entity. Vδ2 positivity was associated with an immunophenotype characterized by Vγ9, CD56, KIR and CD94/NKG2A expression and, on clinical grounds, by lower frequency of symptomatic disease in terms of neutropenia, anemia, splenomegaly concomitant autoimmune/inflammatory disease and need of treatment compared to Vδ2 negative patients. Furthermore, the Vδ2 expression profile also correlated with the STAT mutational status since all STAT3 mutated cases were Vδ2 negative, while the three STAT5b mutated patients were Vδ2 positive. Interestingly, the two subsets of Tγδ

LGLL defined by the Vδ2 expression profile are likely to identify distinct cells of origin of Tγδ LGLL [START_REF] Lalor | Memory gammadelta T Cells-Newly Appreciated Protagonists in Infection and Immunity[END_REF] . In line with this hypothesis, Vδ2 positive Tγδ LGLL might represent the neoplastic counterpart of blood circulating Tγδ cells, while Vδ2 negative Tγδ LGLL might mostly originate from tissue-derived Tγδ cells, with potential pathogenic implications.

Accumulating evidence indicates that the association between STAT3 mutation and symptomatic disease is already recognized in Tαβ LGLL [START_REF] Teramo | STAT3 mutation impacts biological and clinical features of T-LGL leukemia[END_REF][START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Munoz-Garcia | STAT3 and STAT5B Mutations in T/NK-Cell Chronic Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL): Association with Disease Features[END_REF] , recent data also supporting a reduced survival for STAT3 mutated vs STAT3 wild-type patients [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF] . In contrast, the clinical impact of STAT5b mutations is still matter of debate, this mutation being present in the rare aggressive variants of LGLL 8 as well as in indolent CD4+ T-LGLL [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Andersson | High incidence of activating STAT5B mutations in CD4-positive T-cell large granular lymphocyte leukemia[END_REF] . In the Tγδ LGLL setting, the real incidence of STATs gene mutations is still unknown, being studied up to now only in small cohorts of patients [START_REF] Barila | Stat3 mutations impact on overall survival in large granular lymphocyte leukemia: a single-center experience of 205 patients[END_REF][START_REF] Yamane | High frequency of STAT3 gene mutations in T-cell receptor (TCR)gammadelta-type T-cell large granular lymphocytic leukaemia: implications for molecular diagnostics[END_REF][START_REF] Shi | STAT3 mutation and its clinical and histopathologic correlation in T-cell large granular lymphocytic leukemia[END_REF] . In our study mutations in STAT3 and STAT5b were screened in nearly hundred Tγδ LGLL patients and a frequency of STAT3 mutations was found to be comparable with previously reported data in LGLL 4- 6 . Moreover, we also detected 3 Tγδ LGLL patients harboring STAT5b mutations who displayed an indolent disease as observed in CD4+ Tαβ LGLL. In our cohort, we confirm the association between STAT3 mutation and symptomatic disease, particularly with neutropenia, and increased need for therapy, although we did not observe a reduced overall survival for STAT3 mutated cases. These results support a more aggressive disease behavior of Tγδ LGLL, and particularly for patients that do not show Vδ2 expression, independently from the STAT3 mutational status.

In conclusion, data from this large multicentric cohort of Tγδ LGLL highlight the unique biological and clinical hallmarks of this rare variant of T-LGLL, likely associated with a discrete treatment response profile. Despite the similar frequency of STAT3 and STAT5b, Tγδ LGLL cases in general, and Vδ2 negative Tγδ LGLL patients in particular, showed more symptomatic disease and a poorer outcome compared to Tαβ LGLL. At the same time, Tγδ LGLL patients appear to mostly benefit from cyclosporine as first line therapy. Altogether, these results underly the relevance of a precise characterization and subclassification of LGLL. ACCEPTED MANUSCRIPT / CLEAN COPY

Figure 1 :

 1 Figure 1: Progression free survival and overall survival landmark analysis of Tγδ LGLL treated patients. Kaplan-Meier curves showing 6-month landmark analysis for progression free survival (PFS) (Panel A) and overall survival (OS) (Panel B) of Tγδ LGLL patients achieving at least a partial response to first line therapy (Responders) compared to non-responding patients (Non-responders) at 6 months from the start of therapy. Curves were compared by log-rank test. .

Figure 2 .BFigure 1 :

 21 Figure 2. Overall survival analysis of Tγδ LGLL patients. Panel A: Overall survival analysis of the Tγδ LGLL cohort with respect to presence/absence of splenomegaly. Survival curves were estimated using the Kaplan-Meier method and compared by log-rank test. Panel B: Overall survival comparison between Tαβ and Tγδ cohorts. With a median follow-up of 108 months (Tαβ) and of 48 months (Tγδ), median OS was not reached in both the cohorts. Survival curves were estimated using the Kaplan-Meier method and compared by log-rank test.

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Screening for STAT3 and STAT5b mutations STAT3

  Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022 and STAT5b sequencing was performed by Sanger Sequencing or Next Generation Sequencing according to local practice. For the screening of STAT3 and STAT5b mutations by Sanger Sequencing, we used the set of primers reported by Koskela et al 4 and by Rajala et al 8 , respectively, to amplify the hot spot regions for mutations (exon 19-21 for STAT3 and exons 16-18 for STAT5b).

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  Supplementary Table 1. 9%) patients showing partial CD8 expression, otherwise CD4 was mostly absent, with only 3 cases showing partial expression. CD16 and CD57 were typical LGL markers, and they were expressed on the expanded Tγδ cells at the highest frequency (72.3% and 78.4%, respectively), while CD56 was present in 31.1% of cases. A dominant KIR expression was demonstrated in 23/56 patients (41.1%), with CD158b being the most frequently expressed marker (13/56, 23.2%), followed by CD158a (8/56, 14.3%) and CD158e (5/56, 8.9%). CD94/NKG2 receptor expression was Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022 ACCEPTED MANUSCRIPT / CLEAN COPY found in 32/75 cases (42.7%), 12 patients displaying NKG2A (12/54, 22.2%) and 3 patients showing NKG2C positivity (3/30, 10%).

	Median age at diagnosis was 58.5 years (range, 18-92), with 29.4% of subjects being >65 years old.
	No relevant gender prevalence was clearly demonstrated (male 55.9%, female 44.1%). By
	immunophenotype, all patients showed an expansion of CD3+ TCRγδ+ T-cells, demonstrated to be
	clonal on molecular grounds. Tγδ LGLs usually displayed CD8 positivity (64/105, 61.0%), with 23
	out 105 (21.

  Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022 ACCEPTED MANUSCRIPT / CLEAN COPY 1 0 neutropenia, 14/58 (24.1%) for transfusion dependent anemia and 13/58 (22.4%) for symptomatic anemia, 6/58 (10.4%) cases due to combined severe neutropenia and symptomatic anemia while the remnant 5/58 (8.6%) for symptomatic concomitant autoimmune diseases. In 8 cases (13.8%) the primary diagnosis was settled by hematology centers without experience in LGLL and subsequently the patients were moved to the referral centers. Consequently, a clear treatment indication was not available. Considering first line treatment, most patients (34/57, 59.6%) received methotrexate (MTX), while 26.3% (15/57) were treated with cyclosporine A (CyA) and only 10.5% (6/57) received cyclophosphamide (CTX). The remaining two patients received cladibrine and splenectomy as first line treatment. Response rates and the absolute numbers of cases are reported in Supplementary Figure

. In detail, 8/58 (13.8%) patients started therapy due to severe neutropenia, 4/58 (6.9%) due to symptomatic

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  3%, p=0.7628), splenomegaly(22.9% vs 20.8%, p=1) and SPM (21.4% vs 22.7%, p=1) (Table 2). from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022

	(25% vs 15.2%, p=0.3824), expression of KIRs (20% vs 50%, p=0.1413) and CD94 (38.9% vs 57.1%, p=0.2542), thrombocytopenia (17.6% vs 14.Downloaded

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  . A possible Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022

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Table 1 . Response to first line treatment.

 1 Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022 ACCEPTED MANUSCRIPT / CLEAN COPY Abbreviations. ORR: overall response rate. CR: complete response. N/A: not available. Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021013489/1920279/blood.2021013489.pdf by guest on 16 September 2022

			Response rate to first-line therapy	Treatment interruption
	Treatment	N	ORR (%)	CR (%)	N/A	Toxicity
	Methotrexate	34	7/26 (26.9%) 2/26 (7.7%)	4	4
	Cyclosporine A	15	7/13 (53.9%) 3/13 (23.1%)	2	-
	Cyclophosphamide 6	2/5 (40%)	2/5 (40%)	1	-
	Cladribine	1	1/1 (100%)	0/1 (0%)	-	-
	Splenectomy	1	0/1 (0%)	0/1 (0%)	-	-

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Acknowledgments

The authors would like to thank Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 2017-20216).

Conflict of interest disclosure

The authors declare no competing financial interests.

Authorship Contributions

GB designed the research, analyzed data and wrote the manuscript. AG analyzed data, performed statistical analysis and wrote the manuscript. HJ, AT, GC, JC, CV, BS, VRG, NMG, HN and CP provided patient's samples and patient's data. JA, MS, KO, LS, FI, TL, AO, WGM and TL participated to the analysis of data and critically reviewed and edited the manuscript. GS provided funding, participated to the analysis of data, and critically reviewed and edited the manuscript. RZ designed the study, analyzed data, wrote the manuscript, and supervised the study.

Conflict of interest disclosure

The authors declare no competing financial interests.