Bruton´s tyrosine kinase (BTK) inhibitor (BTKi)s block the B-cell receptor (BCR) signaling cascade by binding to the BTK enzyme preventing the proliferation and survival of malignant and normal B cells. During the past decade, the clinical use of BTKis for the treatment of B-cell malignancies has exponentially grown, changing the treatment landscape for chronic lymphocytic leukemia (CLL) in particular. At present, three different covalent BTKis, ibrutinib, acalabrutinib and zanubrutinib, are FDA-approved and many new inhibitors are under development. Despite having remarkable selectivity for BTK, the first-in-class BTKi ibrutinib can also bind, with various affinities, to other kinases. The combined inhibition of BTK (“on-target” effect) and other kinases (“off-target” effect) can have additive or synergistic anti-tumor effects but also induce undesired side effects which might be treatment-limiting. Such “off-target” effects are expected to be more limited for second-generation BTKis. Moreover, the blockade of BCR signaling also indirectly affects the tumor microenvironment in CLL. Treatment with BTKis potentially impacts on both innate and adaptive immunity. Whether this affects infection susceptibility and vaccination efficacy requires further investigation. Here, we summarize the available knowledge on the impact of BTKis on the immune system and discuss the possible clinical implications. Indeed, a deeper knowledge on this topic could guide clinicians in the management and prevention of infections in patients with CLL treated with BTKis.

In lymphomas of B-cell origin, cancer cells orchestrate an inflammatory microenvironment of immune and stromal cells that sustain the tumor cell survival and growth, known as a tumor microenvironment (TME). The features of the TME differ between the different lymphoma types, ranging from extremely inflammatory, such as in Hodgkin lymphoma, to anergic, leading to immune deficiency and susceptibility to infections, such as in chronic lymphocytic leukemia. Understanding the characteristic features of the TME as well as the interactions between cancer and TME cells has given insight into the pathogenesis of most lymphomas and contributed to identify novel therapeutic targets. Here, we summarize the preclinical data that contributed to clarifying the role of the immune cells in the TME of different types of lymphomas of B-cell origin, and explain how the understanding of the biological background has led to new clinical applications. Moreover, we provide an overview of the clinical results of trials that assessed the safety and efficacy of drugs directly targeting TME immune cells in lymphoma patients.

Ibrutinib is a covalently binding inhibitor of the B-cell receptor signaling-mediator Bruton's tyrosine kinase (BTK) with great efficacy in chronic lymphocytic leukemia (CLL). Common side effects like atrial fibrillation (AF), bleeding and infections might be caused by ibrutinib's inhibition of other kinases in non-B cells. Five-year follow-up of plasma biomarkers by proximity extension assay and immune cell numbers by flow cytometry during ibrutinib treatment revealed that 86 of the 265 investigated plasma biomarkers significantly changed during treatment, 74 of which decreased. Among the 12 markers that increased, 6 are associated with cardiovascular diseases and therefore potentially involved in ibrutinib-induced AF. Comparison between healthy donors and X-linked agammaglobulinemia (XLA) patients, who have nonfunctional BTK and essentially lack B cells, showed indicative changes in 53 of the 265 biomarkers while none differed significantly. Hence, neither B cells nor BTK-dependent pathways in other cells seem to influence the levels of the studied plasma biomarkers in healthy donors. Regarding immune cells, the absolute number of T cells, including subsets, decreased, paralleling the decreasing tumor burden. T helper 1 (Th1) cell numbers dropped strongly, while Th2 cells remained relatively stable, causing Th2-skewing. Thus, long-term ibrutinib treatment has a profound impact on the plasma proteome and immune cells in patients with CLL.

Lenalidomide alone or in combination with GM-CSF and low-dose CTX as immune adjuvant to the Apo-DC vaccine elicited tumor-specific T-cell responses in CLL patients. However, unexpected toxicity was observed and caution is suggested in further exploring this drug as immune adjuvant in CLL.

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro , subclinical doses of platinum-based drugs prevented the generation of COX-2 + M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

In classical Hodgkin lymphoma (cHL), the malignant cells represent only a small fraction of the tumor. Yet, they orchestrate a lymphocyte-dominated tumor microenvironment (TME) that supports their survival and growth. The systemic effects of this local immunomodulation are not fully elucidated. Here, we aimed at characterizing circulating lymphocytes and plasma proteins in relation to clinical parameters and treatment effect. Peripheral blood (PB) samples were obtained from 48 consecutive patients at diagnosis and at 2 time points after successful primary treatment. Single-cell suspensions were prepared from lymph node (LN) biopsies obtained for routine diagnostic purposes. Twenty healthy individuals were included as controls. Cells from PB and LN were analyzed by flow cytometry, and plasma proteins by Proximity Extension Assay. We found that the frequencies of T and B cells positively correlated between the LN and the PB compartments. Compared to controls, cHL patients had higher frequencies of proliferating T cells as well as higher expression of programmed death (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-4 in circulating T cells, and lower naive T-cell frequencies. Advanced-stage patients had fewer NK cells with a functionally impaired phenotype. Differences in the immune profile were observed in patients with a high tumor burden and with high inflammation, respectively. Most of these deviations disappeared after standard first-line treatment. Patients who received radiotherapy involving the mediastinum had low T-cell counts for a prolonged period. Our findings suggest that the immunomodulation of lymphocytes in the TME of cHL might affect immune biomarkers in the PB.

Ibrutinib is used continuously in CLL. This phase 1b/2 study interim analysis explored on‐off‐repeat dosing to reduce toxicity. After 12 months, 16/22 patients (73%) remained in first off‐phase irrespective if initial CR/PR or TP53 aberration. Grade 3‐4 infections were reduced from 55% to 5% during a similarly long off‐phase (P < .01). Treg and exhausted T‐cells increased (P = .01). Six patients restarted ibrutinib at early progression and remain drug‐sensitive. Our interim analysis shows a durable off‐phase in most patients, with reduced infections and cost‐saving potential. If toxicity‐driven permanent cessation of ibrutinib will be affected will be explored in the extended study.

In this study, we analyzed the changes occurring in the peripheral blood mononuclear cells (PBMC) during long-term treatment with the Bruton's tyrosine kinase inhibitor ibrutinib in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) patients (pts). PBMC populations were assessed by flow-cytometry including CLL cells, Natural Killer (NK) cells, T-cell memory subsets, helper subpopulations (Ths) and regulatory T cell (Tregs) subsets. Peripheral blood samples were collected before start of treatment and at week (wk) 4, 10, 16, 22 and at 8, 12, 24 and 36 months (mo) of treatment. Eleven pts were included in the study, 8 males and 3 females, with median age 73 years. Nine age- and sex-matched healthy individuals were included as controls. Comparisons between pts and controls were done with the non-parametric Mann-Whitney U test; between follow-up time points and baseline in pts by Wilcoxon rank test for paired comparisons. Simple regression analyses and the Pearson´s rank test were used to estimate the relationship between different cell populations. Eight pts had high-risk and 3 intermediate-risk disease according to the modified Rai staging system; 8/11 had 17p deletion or TP53 mutation. Three pts had mutated IgHV genes, 3 unmutated, 2 had borderline mutational status (97-98% identity), and 3 were not assessed. Median number of previous treatments was 1 (range 1-4) and median lymphocyte count 45 x 109/L (range 3.1-109.5). At 12 mo, overall response rate was 100%. All pts achieved partial response, with 63% fulfilling all complete response criteria (Hallek M et al, Blood 2018), but for minimal residual disease negativity. Eight pts were still on treatment at 24 mo, 7 pts at 36 mo. Treatment discontinuation was due to: death (n=1), allogeneic transplantation (n=1), disease progression (n=1) and toxicity (n=1). In 3 pts, ibrutinib dose was reduced due to toxicity; the remaining pts stayed on full-dose (420 mg/day p.o.) during the whole treatment period. At study entry, eight pts had hypogammaglobulinemia but none had ongoing IgG replacement therapy. Of these, 5 started IgG replacement therapy during treatment. All 11 patients experienced G2 infections. After an initial increase in circulating CLL cells due to redistribution lymphocytosis, a gradual reduction occurred during treatment becoming significant at wk 16 (p<0.005) (Fig. 1A). As we previously reported (Palma M et al, ASH abs 4322, 2017), at study entry CD8+cells were significantly higher compared to controls (p=0.0005). CD8+cells started to decrease at wk 10 (p=0.02) and normalized (i.e. reached values not significantly different from controls) by 12 mo (Fig. 1B). On the other hand, CD4+T cells, at baseline not significantly different from controls, decreased also gradually from wk 16, reaching by 24 mo levels below normal (p=0.02) (Fig.1D). We show here that the reduction of CD8+ cells correlated with that of CLL cells (r=0.45, p< 0.0001) (Fig. 1C) and that the correlation between the number of CD4+ cells and CLL cells was even more significant (r=0.66, p< 0.0001) (Fig.1E). The expression of the immune checkpoints PD-1 and CTLA-4, at baseline aberrantly high on both CD4+ and CD8+ cells and mainly in the antigen-experienced subsets, decreased gradually during treatment. Among the helper subsets, Th1 (CCR6-/ CXCR3+) cells were at baseline higher compared to controls (p=0.0003), while Th2 (CCR6-/ CXCR3-) did not significantly differ (Fig.2A-B). As expected, this resulted in a Th1/Th2 ratio significantly higher than in controls (median 2.6 vs 0.26, p<0.0001) (Fig.2C). During treatment, both Th1 and Th2 cells decreased. Th1 normalized by 12 mo, while Th2 cells decreased below normal levels at 12 mo (p=0.006). However, Th1/Th2 ratio never normalized during 36 mo of treatment. The strongest direct correlation was between the decrease of Th1 cells and CLL cells (r=0.68, p< 0.0001). During treatment, NK cells decreased and also Tregs (CD4+/CD25+/ CCR4+/CD127low), in particular the Ag-experienced non-activated subset (CD45RO+HLA-DR-). In conclusion, the reduction of tumor burden directly correlated with the reduction of all T-cell subsets, but most significantly with those subsets involved in the antitumor immune response, i.e. CD8+cells and, among the CD4+subset, Th1 cells. Whether these changes also compromised the anti-infection immunity is difficult to assess, due to the fact that most of the pts had also underlying hypogammaglobinemia. Disclosures Palma: Takeda Pharma AB: Research Funding. Österborg:Gilead: Consultancy, Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Abbvie: Research Funding. Mellstedt:Kancera AB: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sandoz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria; Astra-Zeneca: Equity Ownership; Novo Nordisk: Equity Ownership; Global Health Sciences: Speakers Bureau.