Marginal Zone Lymphoma Is Associated with Dysregulated Peripheral Blood Immunity Which Is Not Changed By Ibrutinib-Venetoclax Treatment
Authors: Davis Joanne Elizabeth , Le Lina HH , Ludford-Menting Mandy , Holzwart Nicholas , Handunnetti Sasanka M , Tam Constantine S. , Anderson Mary Ann , Ritchie David S. , Koldej Rachel 
Published: December 2024

Marginal Zone Lymphoma (MZL) is a non-Hodgkin Lymphoma with known associations with chronic inflammatory disorders and infections. Despite this link between chronic inflammation and MZL, little is known about MZL circulating immunology. In prior studies, we described immunological repair in Mantle Cell Lymphoma (MCL) patients treated with ibrutinib and venetoclax under the AIM study (Davis et al 2020, Blood Advances). In the current study, we utilised serial samples from MZL patients treated on the same clinical trial (AIM; NCT02471391) to analyse the immunology of MZL and the impact of ibrutinib-venetoclax treatment.
PBMC samples from 14 R/R MZL patients were collected at Baseline, week 4, week 16, 12 months and 24 months on study as part of a pre-planned analysis. All patients received 560mg ibrutinib daily with venetoclax from week 4 ramping up to 400mg at week 8. Baseline samples from 1 MZL screen fail patient were included in the baseline immunology analysis only. Immune profile was assessed using a single-tube 29 antibody panel on an Aurora spectral flow cytometer and compared to the immune profile of 10 age matched healthy donors. Results were correlated with centrally determined PET/CT response.
Compared to healthy donors, MZL patients exhibited normal proportions of Monocytes, MDSCs and mDCs, with reduced proportions of pDCs (P&lt;0.01). While there was no difference in total proportions of CD4 or CD8 T cells at baseline, the proportion of central memory (CM) cells were increased in CD4 (P&lt;0.001) and in CD8 (P&lt;0.01) T cells. Furthermore, CD4 Terminal Effector Memory (TEM) cells were decreased in MZL patients (P&lt;0.05), and PD-1 expression was increased on all CD4 T cell subsets with the highest increase seen in CM cells (P&lt;0.0001). PD-1 expression was also increased on Naïve (P&lt;0.001) and CM (P&lt;0.0001) CD8 T cells. Overall, this suggests alterations in T cell homeostasis with T cells accumulating in the CM subset.
NK cells from MZL patients had altered memory subsets with a trend to increased Immature NK cells and decreased Regulatory NK cells (P&lt;0.01) compared to healthy donors. Furthermore, Immature NK cells had increased TIM3 expression (P&lt;0.05) with increased PD-1 expression on Regulatory NK cells (P&lt;0.05), suggesting significant changes in NK cell homeostasis.
Upon ibrutinib-venetoclax treatment, there was rapid clearance of circulating B cells. Surprisingly, in contrast to our prior work in MCL patients where there was significant immunological repair of T cell subsets over time, MZL patient T and NK cell populations were unchanged with treatment. To explore this in further depth, T cell intracellular cytokine production and degranulation was examined in 6 patients and compared to age matched healthy donors. Expression of IFNγ, TNFα and CD107a were increased 2-6-fold in MZL patients (P&lt;0.001), with the proportion of T cells co-expressing IFNγ, TNFα and Il-2 increased 12-fold (P&lt;0.001).
To assess biomarkers of treatment response to ibrutinib-venetoclax, patients were stratified into Complete Responders (CR; n=6) vs non-CR (n=8) according to PET/CT response at week 56. TIM3+ Immature NK cells were increased in CR patients at baseline (P&lt;0.01) which returned to healthy control levels by 12 months, whereas TIM3+ Mature NK cells were increased in non-CR patients at baseline (P&lt;0.05) which returned to healthy control levels by week 4 (P&lt;0.05). In addition, Monocytes, TIM3+ Monocytes and M2 Macrophages increased to week 16 in those achieving CR, Partial Response or Stable Disease, while those that developed Progressive Disease by week 56 had no change in Monocyte proportions between these early timepoints.
To our knowledge, this is the first study to examine peripheral blood immunology in MZL as a single disease group rather than a combined cohort of non-Hodgkin Lymphoma. The data demonstrates the profound immunological changes associated with MZL including dysregulated expression of immune checkpoints across multiple subsets. In contrast to our prior MCL studies, we did not see improvements in T cell dysregulation suggesting the presence of an antigenic driver that persists despite tumour clearance. Rather, ibrutinib-venetoclax treatment response was associated with changes to innate immune populations which are currently being investigated in further detail. Given the lack of T cell repair, immunotherapy protocols utilising ibrutinib may be less effective in MZL.