Inactivating BTK mutations in large B‐cell lymphoma in a real‐world cohort: Strong correlation with BCL2 translocation

Abstract Inactivating mutations in Bruton's tyrosine kinase (BTK) in patients with follicular lymphoma (FL) have recently been reported. These mutations were found in BTK inhibitor‐treatment naïve patients. Here, we report the BTK mutation status in a real‐world cohort of patients with non‐Hodgkin lymphoma. We found primary BTK mutations in 7.7% of patients with large B‐cell lymphoma (LBCL) and in 14.1% of patients with FL. All patients with BTK‐mutated LBCL were BCL2 translocation positive, and the correlation between BCL2 translocation and BTK mutation persisted even when patients with known transformation from FL were excluded.

. The introduction of BTK inhibitors as part of the treatment regimen in chronic lymphatic leukemia (CLL), lymphoplasmacytic lymphoma (LPL) and mantle cell lymphoma (MCL) has been very successful [2]. BTK inhibitor therapy has also been tested in DLBCL patients in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy in several clinical trials with varying success [3][4][5][6]. The effect may depend on the subtype and age of the patients, as the BTK inhibitor ibrutinib plus R-CHOP seems to improve event-free survival and overall survival in younger patients (<60 years of age) with the ABC subtype [7].
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. In patients with X-linked hypogammaglobulinemia (XLA), inactivating germline mutations in BTK result in an immunodeficiency characterized by hypogammaglobulinemia and a decreased number of B lymphocytes, which causes early onset of severe bacterial infections in the patients. The mutations causing XLA are scattered throughout the coding regions, with many unique mutations and some recurrent hot spots [8].

PATIENTS AND METHODS
The study included an unselected cohort of 320 non-Hodgkin lymphoma patients subjected to next-generation sequencing (NGS) as BTK mutations were classified using Varsome [10], literature review, and BTKbase [11]. To compare the characteristics of patients with and without BTK mutations, Fisher's exact test was used, and p < 0.05 was considered statistically significant. Data analysis was performed using R statistical software (Foundation for Statistical Computing, Vienna, Austria).

RESULTS AND DISCUSSION
Mutations in the BTK gene were found in 9/117 (7.7%) patients diagnosed with LBCL and in 9/64 (14.1%) patients diagnosed with FL ( active mechanism in some B-cell lymphomas rather than a passenger mutation phenomenon.
Inactivating mutations in BTK have previously been described in patients with FL and transformed DLBCL [12,13]. Two of the mutations found in our LBCL cohort were also reported in patients with FL by Krysiak et al. [12], whereas the rest of the mutations found in our study were not previously described in FL or transformed LBCL These results support that inactivation of BTK could be an active mechanism, even though the mechanism for increased AKT activation by BTK inactivation is still unknown. Interestingly, CXCR4 mutation, which is correlated with decreased sensitivity to BTK inhibition with ibrutinib in patients with LPL, also causes increased AKT activation and subsequent MAPK1/2 signaling [14]. Of note, secondary BTK mutations that confer resistance to BTK-inhibitory therapy in CLL have recently been reported to support continuous AKT activation as well [15].  Table S1.

Inactivating mutations in
ABC subtype is dependent on BTK signaling. However, the absence of inactivating BTK mutations among ABC patients should be confirmed in larger patient cohorts.

ACKNOWLEDGMENTS
Torsten Holm Nielsen gratefully acknowledges support from the LM Byg foundation and the Sejer and Lis Klüwer Persson foundation.

CONFLICT OF INTEREST
The authors declare they have no conflicts of interest.

ETHICS STATEMENT
Data were collected retrospectively from patient files, and the study was approved as a Health Data Registry Study Research Project by the Capital Region of Denmark (journal no.: R-21010067).

FUNDING INFORMATION
The authors received no specific funding for this work.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.