Polyclonal Immune Response in T-LGL Leads to Clonal Expansions Preceding Occurrence of STAT3 Mutations Further Solidifying Clonal Dominance
Authors: Hirsch Cassandra M. , Clemente Michael , Chomczynski Peter , Przychodzen Bartlomiej P. , Nagata Yasunobu , Adema Vera , Williams Louis , Visconte Valeria , Lichtin, MD Alan , Mustjoki Satu , Sekeres Mikkael A. , Maciejewski Jaroslaw P. 
Published: October 2019

Abstract
T cell large granular lymphocyte leukemia (T-LGLL) characterized by excessive clonal cytotoxic T cell proliferation, is often accompanied by cytopenias and is thought to result from immune-mediated suppression, either by misguided immune antigenic recognition or mimicry triggered by auto-, tumor or viral antigens. Irrespective of the mode of evolution, T-LGLL progresses via: i) purely reactive clonal outgrowth in the context of a polyclonal immune response or: ii) a transforming event such as a STAT3 mutation (STAT3MT) within the immunodominant clone.
Studies of TCR rearrangement using DNA based NGS of the TCR Vβ complementarity-determining region 3 (TCR VB CDR3) facilitate analyses of T cell clonality, as the CDR3-based "biological barcodes" allow for clonal quantification. We used serial CDR3 clonotyping accompanied by clonal STAT3 mutant burden measurements to recapitulate the pathological cascade in the course of T-LGLL. We asked if a polyclonal/oligoclonal immune response is the primary process that is clonally "imprinted" by STAT3MT, which would likely arise with the most proliferative clone. We were interested in the dynamics of underlying clonal behavior during treatment.
Our initial cohort included 207 well characterized LGL patients. Most presented with anemia (46%), and/or neutropenia (46%). VB expansion was present in 94% of cases, with an average LGL count of 2317k/uL .STAT3MT were found by targeted deep sequencing in 38% of patients in 4 common hotspots: 42% Y640F, 34% D661Y, 11% D661V, and 8% N647I, with an average clonal burden of 28%. Multiple STAT3MT variants were found in some patients. From this cohort, serial samples obtained at presentation and throughout clinical course were obtained from 20 representative cases (10 STAT3MT and 10 STAT3WT), and subjected to analysis of clonal dynamics, including simultaneous deep TCR VB and STAT3 NGS. Patients were sequenced at an average of 4 time points (range 2-8). At least one major clonotype was identified in all patients, and multiple major clonotypes were identified in half.
Analyses of clonal architecture revealed that STAT3 clones arose with VB expanded clones. In all cases, the TCR clonal burden was greater than that of STAT3MT demonstrating for the first time that STAT3MT is not the ancestral event for clonal expansion; rather, it evolves within the pre-expanded immunodominant clone.
More than half of the patients were treated with immunosuppressive therapy (IST) achieving an approximate 40% response rate. Distinct patterns of clonal dynamics were seen following treatment (see Figure). In some patients, both the STAT3 (if present) and the major TCR clone decreased upon successful therapy. A correlation between a specific IST treatment and a clonal burden decrease was not found. In a subset of patients, the clones persisted despite a hematologic response, suggesting the major clonotype was functionally silenced. We also observed a common phenomenon of TCR "clonotype switching", whereby therapy contracts one major clonotype, while another previously "minor" clonotype emerges. These newly expanded clones did not harbor STAT3MT, and most patients with "switching" were resistant to IST therapy. Multiple clonotypes were present at initial sampling in a few patients without STAT3MT and persisted at the same rate in subsequent samplings, precluding identification of a clear immunodominant clonotype. A stable or increasing clonal burden of both STAT3MT and VB CDR3 was seen in IST non-responders.
In sum, STAT3MT were found to be a secondary event to the clonal expansion of the TCR VB clonotype, as a response within an already expanded clonotype and not the initiator of clonal expansion. The dynamics of both the STAT3MT and the TCR VB clonotype can be assessed over disease course and treatment regimens and demonstrate additional clinical ultility when applied to larger prospective clinical trials. The difficulty in finding a direct correlation between response to specific IST and a decrease in TCR VB clonal burden may be due to variable time frames between samplings, heterogeneity of IST regimens and response assessment, or large asymptomatic clonotypes. Prior to this study, the association of remission and elimination of immunodominant clonotypes was unclear. Our results suggest that clonal elimination is not necessary for complete clinical response; rather, the clone can be contracted to a manageable clonal burden or silenced.

Disclosures
Mustjoki: Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.