Ibrutinib Increases the Clonality of TCR Repertoire in Patients with Chronic Lymphocytic Leukemia
Authors: Baptista Maria Joao , Basumallik Neil , Herman Sarah E. M. , Cook Erika M. , Ahn Inhye E. , Wiestner Adrian , Sun Clare C. 
Published: October 2019

Abstract
The T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL) is more clonal than in healthy individuals. Shared TCR clonotypes among patients may reflect the expansion of T-cell clones that recognize common antigens. It is unknown whether these antigens are tumor-derived to which T cells react, or microbial or auto-antigens that promote expansion of both T cells and the leukemic clone. Ibrutinib inhibits Bruton tyrosine kinase and interleukin-2-inducible kinase and has been shown to modulate the T-cell compartment of patients with CLL.
We performed deep sequencing of the TCRβ repertoire (Adaptive Biotechnologies) in 26 patients with CLL prior to treatment with ibrutinib, at the time of response, and either at disease progression or during sustained remission. TCR clonality was calculated as the inverse of Shannon entropy normalized to the number of productive clones in a sample and it accounts for both the number of unique clonotypes and the extent to which one or a few clonotypes dominate the sample repertoire. Clonality values may range between 0 and 1 with values closer to 1 indicating a more oligoclonal repertoire. Nonparametric tests were applied for statistical analyses, and paired analyses were performed to compare metrics across different time points.
The median productive clonality in CLL patients before treatment was .1795, more than double the clonality reported for healthy donors (.0750, provided by Adaptive Biotechnologies). Differences in clonality were studied between subgroups of patients defined by clinical and biologic features. Overall, the TCR repertoire in treatment naïve patients (clonality = .1454, n = 15) was less clonal than in patients with relapsed/refractory CLL (clonality = .2468, n = 11, P = .002). β2-microglobulin ≥ 3.5 mg/L and CD8+ T-cell counts above the normal reference range were also associated with higher clonality (P = .030 and .003, respectively). Further, CD8+ T-cell counts (median 1134 /uL, IQR 670 - 2474) when assessed as a continuous variable, were correlated with clonality (ρ = .620, P = .001).
Next, we evaluated the TCR repertoire at partial (n = 23) or complete (n = 2) response after a median duration of 12 months (range 6-36) on ibrutinib. Clonality increased at the time of response compared to baseline (median increase 33.2%, P = .048) despite a concomitant decrease in CD4+ and CD8+ T-cell counts. In 12 patients who subsequently developed progressive disease, analysis of the TCR repertoire revealed a decrease in clonality (median decrease 5.7%, P = .026) from the time of response to relapse (median 13 months, range 4-32) while CD4+ and CD8+ T-cell counts increased. In contrast, clonality did not significantly change among patients with sustained response to ibrutinib measured 12 months after the first response time point.
Overrepresented clonotypes comprising more than .05% of the TCR repertoire at baseline or at the time of response were selected for further analyses (median of 95 clonotypes per patient). Eighteen out of 25 patients shared at least one clonotype. In total, 20 shared clonotypes were identified and interestingly, their frequencies decreased with ibrutinib treatment (P = .003).
Interrogation of overrepresented clonotypes in public databases identified 46 clonotypes previously reported in viral infections (77%) followed by those associated with autoimmune conditions (13%) and cancer (10%). The most commonly implicated viruses were Epstein Barr virus, cytomegalovirus and influenza. We observed the convergence of multiple clonotypes recognizing the same viral epitopes and 8 clonotypes were shared between at least 2 patients. However, they behaved differently on treatment with an increase in frequency in some patients and decrease in others.
In summary, we show that ibrutinib induces a more clonal TCR repertoire and that it is lost at relapse. Overrepresented clonotypes were generally patient-specific and correlated with CD8+ T cells. These findings suggest that CD8+ T-cell clonotypes recognizing private antigens expand and mediate anti-tumor activity during treatment with ibrutinib.
This research was in part supported by the Intramural Research Program of the NIH, NHLBI.

Disclosures
Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding.