Immunotherapy in non-Hodgkin lymphoma

Introduction

Cancer immunotherapy has been a focus of research and investigation since the recognition of T-cells as integral to the immune response in the mid-twentieth century (1). Allogeneic stem cell transplantation represents a powerful yet non-specific form of immunotherapy. Its curative potential in most subtypes of advanced non-Hodgkin lymphoma (NHL) strongly supports a role for immunotherapy in these diseases. However, strategies to induce a host anti-tumor immune response without the complexity and toxicity of allogeneic adoptive immunotherapy have been very challenging to develop. The first therapeutic advance in pharmaceutical immune-oncology was the use of recombinant IL-2 in renal cell carcinoma and melanoma, leading to the Food and Drug Administration (FDA) approval of high dose recombinant IL-2 for these diseases in 1992 and 1998, respectively (2,3). Since that time, the ability to generate monoclonal antibodies against tumor antigens and to use tumor antigens in order to educate the immune system through tumor vaccines moved the field closer to a tumor-specific immune response (4,5). Similarly, the discovery of, and ability to grow and expand, tumor infiltrating lymphocytes (TILs) for reinfusion into melanoma patients laid the groundwork for cellular immunotherapy (6,7). Further genetic engineering of autologous T-cells with either engineered T-cell receptors (eTCRs) or chimeric antigen receptors (CARs) directed against tumor antigens has expanded the field of adoptive immunotherapy and the breadth of target diseases (8-10). Finally, the discovery of immune checkpoint molecules like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) and the elucidation of their role in tumor immune evasion has led to the development of inhibitory therapeutic monoclonal antibodies for the treatment of cancer patients which have revolutionized oncology (11-15). Since the FDA approval of high dose IL-2 in the 1990s, the field has seen the approval of a tumor vaccine for prostate cancer (16), two CAR T-cell products targeting CD19 (17,18), and several immune checkpoint blockade inhibitors in a variety of tumors (13-15). Together, these advances have established immune-oncology as a fundamental paradigm in the treatment of tumors across the cancer spectrum. The impact on the treatment of patients with NHL has been variable and heterogeneous among immunotherapy modalities and tumor targets, which reflects the complex and heterogeneous biology of NHL. The most recent and promising avenues of treatment are CAR-T-cells and immune checkpoint blockade. This review will explore their efficacy and limitations in NHL.

CAR T-cells in NHL

The discovery and clinical application of TILs really laid the groundwork for the revolution in engineered cell therapies, which is poised to soon change treatment paradigms in NHL. Following the initial promising results in melanoma, research shifted towards the genetic engineering of autologous T-cells with gene constructs for eTCRs or CARs directed toward tumor antigens. T-cells have been engineered to target dozens of tumor antigens and have been tested in clinical trials in an equally broad number of tumor types. To date, their greatest success has been with anti-CD19 directed CAR T-cells in B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL) and other B-cell NHLs.

Anti-CD19 CAR T-cell efficacy

In 2010, investigators at the National Cancer Institute (NCI) reported the results of the first NHL patient to have been treated with anti-CD19 directed CAR T-cells (19). The CAR construct contained a CD28 costimulatory molecule and would later be developed as axi-cel. This patient with heavily pre-treated follicular lymphoma had a partial response (PR) with subsequent progression at 32 weeks, but CD19+ B-cells were absent from the blood or bone marrow at 36 weeks and CAR T-cells were detected in the blood through 27 weeks following infusion. This provided a proof-of-principle that CAR T-cells could persist and launch a successful immune attack on target cells. Five years later, the same group reported their experience with this construct in 15 patients with B-cell NHL [including DLBCL and primary mediastinal B-cell lymphoma (PMBL)] and chronic lymphocytic leukemia (CLL) (20). The overall response rate (ORR) was 92%. Among seven DLBCL patients, the CR rate was 57% and the ORR was 86%. The ORR in indolent lymphoma and CLL was 100%. Many responses were durable through the 22-month follow-up.

The early success of axi-cel in B-cell NHL, and in DLBCL and PMBL in particular, led to the pivotal phase 2 ZUMA-1 study in patients with refractory high-grade B-cell lymphoma (HGBL), DLBCL, PMBL, and transformed follicular lymphoma (tFL) (18). This multicenter study treated 101 out of 111 enrolled chemotherapy refractory patients with axi-cel. No bridging therapy was allowed and the average manufacturing time from leukapheresis to the delivery of the product was 17 days. There was only one manufacturing failure; the other 9 patients did not receive their cells due to adverse events, disease progression or because they had unmeasurable disease prior to lymphodepleting chemotherapy. Among the treated patients, ORR was 82% (82% in DLBCL/HGBL; 83% in PMBL/tFL). Complete responses were observed in 54% of patients (49% in DLBCL/HGBL; 71% in PMBL/tFL). There was no clear association between response and any baseline patient or disease characteristic, including CD19 positivity. Response was associated higher peak levels of CAR T-cells. As in the early phase study, responses were durable, with 70% of complete responses (CRs) (42% of treated patients overall) maintained after a median of 15.4 months. These results compared favorably with those of a historical cohort of similar patients assembled for purpose of this comparison, with CR rates of 54% (with axi-cel) versus 7% and median overall survival (OS) not reached at >15 months versus a median OS of 6.3 months (21). This study led to the FDA approval of axi-cel for the treatment of relapsed/refractory DLBCL, HGBL, PMBL and tFL following 2+ prior lines of therapy in October 2017.

A second anti-CD19 CAR T-cell construct, tisagenlecleucel (t-cel) was FDA approved for the treatment of relapsed/refractory DLBCL, tFL, and HGBL after two prior lines of systemic therapy in April 2018 based on the results of the pivotal JULIET study by Novartis. This construct contains a 4-1BB rather than a CD28 costimulatory domain, which differentiates it from axi-cel. In preclinical models, CD28 constructs had a more rapid and higher peak CAR T-cell expansion than 4-1BB CAR T-cells (22). T-cel was tested in a pilot study in 28 patients with DLBCL (n=14) or FL (n=14) (23). In this study, bridging chemotherapy was allowed and administered to 10/28 patients. The median time from apheresis to infusion was 39 days. The ORR in DLBCL was 50%, with 43% CRs. In FL, the ORR was 79%, with 71% CRs. At a median follow-up of 28.6 months, 86% of DLBCL and 89% of FL responders (43% and 70% of treated DLBCL and FL patients, respectively) remained in response. Although the sample size was small, response in DLBCL appeared to be independent of cell of origin, high-risk cytogenetics, and disease burden. Among 10 patients with DLBCL and available pre-treatment biopsies, responding patients had lower levels of PD-L1, PD-1, LAG3, and TIM3 in the tumor and tumor microenvironment. CAR T-cells were detectable in almost all patients in response between 6 and 24 months. Only 2 patients in response lost detectability of CAR T-cells early after their infusion. Similarly, lack of B-cell depletion was associated with early disease progression in 2 patients; all patients in CR had B-cell depletion, and in half this was ongoing at 6.7 months.

The follow-up pivotal, multicenter JULIET study screened 160 patients to treat 106; 49 patients did not receive their cells due to declining health or disease progression (n=38) or due to an inability to manufacture cells (n=11). Patients were eligible for this study if they had relapsed or refractory DLBCL/HGBL or tFL following 2 or more prior lines of therapy. Bridging chemotherapy after apheresis was allowed and given to 90% of patients. The FDA reviewed data for 68/92 patients dosed in the US with at least 3-month follow-up (Kymriah package insert). Some patients not included either did not have lymphoma (n=1), or had achieved a CR following bridging chemotherapy (n=8). Among these 68 patients, the ORR was 50%, with a CR rate of 32%. With a median follow-up of approximately 6 months, approximately 30% of patients remain in CR and 7% of patients remain in PR. Durable responses were associated with T-cel persistence, lasting up to 378 days after infusion in some patients.

A third anti-CD19 CAR T-cell product, lisocabtagene maraleucel (liso-cel) is being developed by Juno Therapeutics for the treatment of relapsed/refractory DLBCL/HGBL, PMBL and tFL following 2 or more prior lines of therapy. Like T-cel, this construct has a 4-1BB costimulatory domain. One distinguishing characteristic of liso-cel is that it is engineered to have a defined composition of CD4+ and CD8+ CAR T-cells. The TRANSCEND-NHL study of liso-cel in B-cell NHL initially enrolled a more heterogeneous group of B-cell NHL patients, testing different doses and dosing schedules. The CORE cohort was later defined to include only patients with DLBCL/HGBL and tFL as part of the pivotal study, and included 73 patients (24). Of patients apheresed, 13 did not receive treatment due to progressive disease and/or death, 2 patients hadn’t received product due to an inability to manufacture cells, and 5 patients had withdrawn consent. Twelve patients are not included in the analyses as they received a cellular product that did not meet product specifications. Within the CORE cohort, the ORR was 80%, with a CR rate of 59%. Approximately 80% of CRs (41% of treated patients) remained in response at 6 months. Response was independent of adverse prognostic factors for these diseases, as was seen in the ZUMA-1 and JULIET studies (24).

The success of these 3 CAR T products in relapsed/refractory DLBCL/HGBL, PMBL and tFL has provided the impetus for many ongoing and planned studies in different settings and in different NHL subtypes. For example, axi-cel is currently being evaluated in indolent B-cell NHL (ZUMA-5, NCT03105336) and mantle cell lymphoma (ZUMA-2, NCT02601313). It is also being tested in a randomized trial against standard of care therapy [salvage chemotherapy/autologous stem cell transplant (ASCT)] in the second-line setting for patients with primary refractory, or early relapsing (<12 months) DLBCL, HGBL, and tFL (ZUMA-7, NCT03391466). Similar trials for t-cel and liso-cel are in the pipeline.

Anti-CD19 CAR T-cell toxicity

While anti-CD19 CAR T-cell therapy has had remarkable efficacy and has established a new and effective standard of care for relapsed/refractory aggressive B-NHL, it is associated with unique and important toxicities, most notably cytokine release syndrome (CRS) and neurotoxicity. The incidence of these toxicities varies with target antigen and target disease, and likely with the different constructs. For example, both CRS and neurotoxicity occur at higher frequency in B-ALL than B-NHL. There is also a higher incidence of neurotoxicity with anti-CD19 directed CAR T-cells than those that target other antigens, like BCMA. High tumor burden and pretreatment inflammatory markers may also associate with higher rates and grade of toxicity. Whether there is more high-grade CRS or neurotoxicity with CD28, as compared with 4-1BB, CAR T-cells constructs is not yet clear. Because of theoretical differences in kinetics of CAR T-cell expansion, it has been hypothesized that rates of high-grade CRS and/or neurotoxicity may be increased following treatment with CD28 CAR T-cells. While there have been differences reported across the studies to support this hypothesis, caution may be warranted in comparing across studies. First, the patient numbers are small. Second, the patient populations treated on these studies are different, as was their management prior to and after CAR-T infusion. Patients on the ZUMA-1 study had refractory disease, whereas patients on the JULIET and TRANSCEND studies had either relapsed or refractory disease, which could mean they had decreased tumor bulk or were less sick as their disease had not been growing through their last line of therapy. Bridging therapy was allowed on JULIET and TRANSCEND, and while it is not expected that these patients would have a durable response to such chemotherapy, a transient response was possible, yielding an improved disease burden at the time of their CAR T-cell infusion. In support of this is the fact that eight patients on the JULIET study had a complete response to bridging therapy prior to CAR T-cell infusion.

CRS

The rapid expansion and activation of T-cells upon reinfusion into a patient with an active CD19+ B-cell malignancy is associated with release of pro-inflammatory cytokines like IL-6, IL-15, and INF-g, among others. The result is a clinical syndrome, CRS, of high fevers, fatigue, and malaise that can progress to cause hypotension, capillary leak, respiratory distress, and end-organ dysfunction. The timing, severity, and duration of CRS vary between different anti-CD19 CAR T-cell products, and by baseline patient and disease characteristics, but it typically occurs within 1–5 days of CAR T-cell infusion and lasts 5–8 days, corresponding with CAR T-cell expansion and peak CAR T-cell levels. It has been theorized that the CD28 CAR T-cell constructs may be associated with earlier and more pronounced CRS due to a more rapid CAR T-cell expansion.

The ZUMA-1 study used the Lee criteria (25) for CRS grading, and on this study 13% of patients experienced grade 3 or higher CRS (18). Any grade CRS occurred in 93% of patients. The median time to onset of CRS was 2 days, and the median duration of CRS was 8 days. There were two cases of fatal CRS, one due to hemophagocytic lymphohistiocytosis (HLH) and one due to cardiac arrest, while all other CRS events were fully reversible. At the outset of this study, little was known about the effect that drugs like tocilizumab and/or corticosteroids would have on the activity and efficacy of CAR T-cells and so they were used more sparingly. With data showing that they did not impact efficacy, they were used earlier as the study progressed and rates of grade 3 or higher CRS improved. In all, 43% and 27% of patients received tocilizumab and glucocorticoids, respectively.

The JULIET study used a CRS grading system developed at the University of Pennsylvania (26). In this scale, patients with grade 3 CRS would largely have had grade 2 CRS by Lee Grading. The incidence of grade 3 or higher CRS was 23%, but only 6% required vasopressor support, and the incidence of any grade CRS was 74% (Kymriah package insert). These results were similar to those observed on the single center experience with T-cel in B-cell NHL (23). The median time to CRS was 3 days, and the median duration was 8 days. Tocilizumab and/or steroids were administered to 21% of patients overall. Just over 25% of patients on the JULIET study received CAR T-cells as an outpatient and approximately 75% could remain outpatient for 3 or more days before developing CRS.

Finally, on the TRANSCEND study, the rates of any grade and grade 3 or higher CRS (Lee grading) were quite low, with only 37% of patients in the CORE cohort experiencing CRS of any grade (24). Only 3% of CORE patients had grade 3+ CRS. In the FULL cohort, the median time to onset of CRS was 5 days, with 7% of patients receiving tocilizumab and 10% receiving corticosteroids. Like with the JULIET study, the delay in onset of CRS allows for the potential of outpatient dosing of liso-cel, which is currently being explored.

Neurotoxicity

CAR-T induced neurotoxicity can manifest with a variety of clinical symptoms including confusion, aphasia, tremors, seizures, obtundation and coma. The pathophysiology of this syndrome is less well understood than that of CRS. Nonetheless, recent animal and human studies have described the importance of endothelial injury and increased blood-brain barrier (BBB) permeability, as well as the increase in levels of proinflammatory cytokines and CAR-T-cells in the cerebrospinal fluid (CSF) (27-31). One study in patients treated with anti-CD19 CAR-T demonstrated an association between high-grade neurotoxicity and early onset CRS, as well as with younger age, B-ALL diagnosis, tumor burden, and earlier and higher peak CAR T-cell expansion (29). High-grade neurotoxicity was associated with disseminated intravascular coagulation (DIC), higher angiopoietin (ANG) 2 to ANG1 ratio, and a low ADAMTS13 to vWF ratio consistent with an acquired thrombotic microangiopathy (28,29). Consistent with other studies, high-grade neurotoxicity was associated with increased numbers of CAR T-cells as well as inflammatory cytokines in the CSF. Together these observations suggest a model by which CAR T-cell-induced inflammation can elicit endothelial activation as well as a brain pericyte stress response leading to increased permeability of the BBB and subsequent influx of proinflammatory cytokines and T-cells into the CSF. Fortunately, except in cases of fatal cerebral edema, which is rare, all neurotoxicity observed on the B-cell NHL studies has been completely reversible.

On ZUMA-1, the incidence of neurotoxicity of any grade was 64%, with 28% of patients experiencing grade 3+ neurotoxicity (18). Neurotoxicity generally occurred later than CRS, with a median time to onset of 5 days, and median resolution on day 17 following infusion. Again, severe neurologic toxicity on this study was associated with higher peak levels of IL-2, GM-CSF, and ferritin. On JULIET the incidence of neurologic toxicity was 58%, with 21% of patients experiencing grade 3 or higher neurotoxicity (Kymriah label). These numbers are higher than those seen on the single center experience with T-cel in B-cell NHL, where 39% of patients had neurotoxicity of any grade, and 11% of patients had grade 3 or higher neurotoxicity (23). One patient with FL on this study had grade 5 neurologic toxicity; at autopsy the brain exhibited diffuse gliosis with severe, widespread neuronal loss and white matter degeneration and a macrophage/CD8 T-cell infiltrate. On TRANSCEND, the incidence of neurotoxicity in the CORE cohort was 25%, with 15% experiencing grade 3 or higher neurotoxicity (24). There were no grade 5 neurologic events. The median time to onset of neurotoxicity was 10 days.

Anti-CD19 CAR T-cell resistance

There are two main mechanisms of CAR T-cell resistance that to date have been the focus of investigation, although there will likely be several more. The first is loss of target antigen, and the second is T-cell exhaustion and/or inhibition. Loss of CD19 has been described following anti-CD19 CAR T-cell therapy in both B-ALL and B-NHL. In B-ALL, 10–30% of patients will relapse with CD19 negative disease. Genetic analysis of two of these relapses demonstrated loss of one copy of CD19 and alternative splicing of the other, resulting in loss of exon 2, which had acquired frameshift or missense mutations that would have otherwise led to loss of expression of the second copy of CD19 and likely cell death (32). This truncated CD19 is not detected by CD19 immunostains or antibodies, fails to trigger CAR T-cell killing, and partially rescues the cancer cell from the effects of CD19 loss. Strategies in development to address antigen loss include the development of multivalent CAR T-cells that target more than one tumor antigen (33).

T-cell exhaustion is an alternative mechanism of CAR T-cell resistance that may be relevant across many tumor types. Primary resistance to adoptive immune effector cell transfer can occur in tumors that have an inhibitory tumor microenvironment, or that upregulate ligands for T-cell inhibitory receptors. In addition, upon antigen stimulation T-cells upregulate PD-1 and CTLA-4 on their surface while upon immune attack, tumor cells increase expression of ligands for those receptors. The result is an acquired form of resistance with T-cell exhaustion and immune escape. In mouse models of mesothelioma, PD-1 antibody therapy restored the function of anti-mesothelin directed CAR T-cells (34). There are case reports of the successful use of anti-PD-1 antibodies to patients having progressed following anti-CD19 CAR T-cell therapy in lymphoma; these clinical responses were associated with a rise in CAR T-cell numbers as well as an increase in pro-inflammatory cytokines within 24 hours of anti-PD-1 antibody therapy, and recrudescence of CAR T-cell related toxicities (35). There is now a study of pembrolizumab for the treatment of relapsed or refractory lymphoma following anti-CD19 directed CAR T-cell therapy (NCT02650999), as well as a phase 1/2 study of the PD-L1 antibody atezolizumab with axi-cel for refractory DLBCL (ZUMA-6, NCT02926833). Other strategies to overcome inhibition of CAR T-cells involve further genetic modification of CAR T-cells such that the genes for PD-1 and other inhibitory receptors are knocked out using gene-editing technologies. Alternatively, CAR T-cells are being engineered into “armored CARs”, which have an inducible or constitutively active proinflammatory cytokine gene construct (e.g., IL-12) to improve CAR T-cell activation or persistence.

Finally, the exact composition of the infused CAR T-cell products is variable, even in liso-cel where the CD4:CD8 ratio is controlled. In addition to selecting for certain T-cell subsets, prior therapies may affect both the health of T-cells collected and the proportion of T-cell subtypes at leukapheresis. Drugs like ibrutinib have immunomodulatory effects that may improve CAR T-cell activity through its effect on T-cell subsets. The immunophenotype of effector CAR T-cells may be important, as response in CLL has been associated with a higher proportion of memory-like CD8 T-cells with an IL6/STAT3 like gene signature, whereas resistance was associated with gene signatures associated with differentiation, glycolysis, exhaustion and apoptosis (36). Exploring new targets, new combinations, and new and additional ways of genetically engineering CAR T-cells are the major movements in the field to both improve outcomes in established diseases and to expand the indication for these therapies for new diseases.

Immune checkpoint blockade in B-cell NHL

As discussed above, targeting T-cell checkpoint or co-stimulatory pathways may emerge as an important platform to overcome resistance to CAR T-cell therapy. Its efficacy as a primary treatment strategy for relapsed/refractory B-cell NHL, though, has not been uniformly promising. The more common B-cell and T-cell NHLs largely lack significant overexpression of the PD-1 ligands PD-L1 and PD-L2, and perhaps as a consequence the efficacy of checkpoint inhibition in these diseases has been disappointing. However, certain subtypes of NHL with more frequent perturbations of PD-1 ligands may be more sensitive to PD-1 inhibition.

Immunologic features of the tumor and its microenvironment in NHL

Common subtypes of B-cell NHL, including DLBCL and FL, rarely (<10%) overexpress PD-L1 or PD-L2. Despite this, levels of PD-L1 and PD-L2 expression in these diseases do correlate with a worse outcome with chemoimmunotherapy (37). While the tumor cells themselves may not overexpress PD-L1 and PD-L2, the immunophenotype of the tumor microenvironment in these diseases may lead to tumor immune evasion. There is evidence that immune escape may play a role in DLBCL progression, given the incidence of mutations in the genes for β2-microglobulin (29%) and CD58 (21%) in this disease in addition to aberrations in HLA-1 and CD58 expression in the absence of genetic mutations; this may prevent recognition by, and activation of, CD8+ T-cells and NK cells, respectively (38). The tumor cell in DLBCL may also express chemokines and cytokines that attract and retain M2 macrophages and regulatory and exhausted T-cells within the microenvironment; pre-rituximab, an increased in intratumoral CD68+ macrophages was associated with inferior OS in this disease (39,40). Immune evasion therefore likely represents an important survival mechanism, but may not depend principally on engagement of the PD-1 pathway.

In FL, GEP identified two signatures associated with prognosis in the pre-rituximab era, which correlated with gene expression by tumor infiltrating immune cells (41). A profile including predominantly T-cell markers (immune-response 1) was more favorable, whereas a GEP including genes expressed by macrophages and dendritic cells (immune-response 2) was associated with worse outcomes. The use of rituximab, however, has paradoxically made the immune-response 2 signature a favorable prognostic marker (42). There are other reasons to suspect that FL can be targeted by immunotherapy. FL cells alter the gene expression of CD4+ and CD8+ TILs, resulting in impaired lymphocyte function and motility (43). Further interaction between FL cells and the microenvironment are being elucidated, such as the interactions between FL and follicular helper T (T_FH) cells and their effect on macrophage polarization and lymphoid differentiation (44,45), or the presence of distinct populations of CD4+ TILs differentiated by their level of PD1 and TIM-3 expression (46-48). A high proportion of PD-1+TIM3+ T-cells, representing exhausted T-cells, outside of the lymph node follicle has been associated with a poor prognosis, and inhibition of TIM3 by anti-TIM3 antibodies can restore function of these T-cells (47). Reports of the prognostic value of PD-1 expression on TILs in FL have been conflicting, however, and it may be that the impact on outcome is T-cell subset dependent (49,50). Altogether, these studies highlight the richness complexity of FL’s interaction with the immune system, and hold the tantalizing promise of therapeutic success by disabling key mechanisms of immune escape.

While the above support a role for immune evasion in the pathobiology of DLBCL and FL, they do not specifically support the use of PD-1 blockade. In contrast, a number of NHL histologies have specific biological characteristics that suggest a potential dependence on the PD-1 pathway and vulnerability to PD-1 blockade. The model for this is classical Hodgkin lymphoma (HL), which harbors near-universal abnormalities at 9p24.1, resulting in amplification and overexpression of PD-L1 and PD-L2, as well as JAK2 (51). Gene expression profiling (GEP) highlights the relatedness of this disease to PMBL. Indeed, PMBL frequently overexpresses PD-L1 and PD-L2 as a result of genetic amplification or translocation events involving the PD-1 ligands (52,53). Gray zone lymphoma (GZL) between HL and PMBL is a disease with a distinct methylation profile from PMBL and HL, although it shares some biologic and pathologic similarities with these diseases. Unsurprisingly, it also has frequent alterations at 9p24.1 (54). A genomic analysis of primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL) revealed genetic similarities between these diseases and PMBL (55). His analysis identified recurrent copy number gains at 9p24.1 and translocations involving PD-L1 and PD-L2 in these diseases. These diseases (PMBL, GZL, PCNSL, PTL) share a high frequency of 9p24 abnormalities, which may render them susceptible to PD-1 blockade. In addition, in a separate analysis EBV+ DLBCL tumors were shown to have frequent overexpression of PD-L1, although in this case induced directly by EBV-encoded proteins rather than 9p24 deregulation (56). T-cell/histiocyte-rich large cell lymphoma (TCHRLCL) was also found to overexpress PD-L1 in a majority of patients while nodular lymphocyte-predominant HL was found to have high PD-L1 expression in one study but not another (57,58). A small series of patients with Richter syndrome (RS) with a high response rate to PD-1 blockade sparked interest (59). Interestingly, these lymphomas frequently overexpress PD-1, not PD-L1 (80%) (60). Finally, several T-cell lymphomas and histiocytic tumors overexpress PD-L1, including ALK+ anaplastic large cell lymphoma (ALCL), angioimmunoblastic T-cell lymphoma (AITL), the virally-driven entities NK/T-cell lymphoma and adult T-cell leukemia/lymphoma, and follicular dendritic cell sarcoma (57). PD-L1 expression within tumor cells has been seen in up to 27% of patients with cutaneous T-cell lymphoma (CTCL) and 15% of patients with peripheral T-cell lymphoma (PTCL), but expression in the tumor microenvironment is much higher (73% and 39%, respectively) (61). Certain T-cell lymphoma subtypes, like cutaneous T-cell lymphomas, are associated with a high mutational burden, which has been associated with greater response to checkpoint blockade in other cancer subtypes (62).

Clinical experience of immune checkpoint blockade in NHL

DLBCL

As discussed above, DLBCL not otherwise specified (DLBCL NOS), which represents the most common lymphoma diagnosis worldwide, rarely overexpresses PD-L1 or PD-L2. Initially, the phase 1 results with nivolumab were encouraging with an ORR of 36%, and one durable response (63). Unfortunately, the as yet unpublished results of the CheckMate-139 phase 2 study of nivolumab in DLBCL NOS did not confirm the earlier results, with an ORR of 3% and 10% in patients who were ASCT ineligible or who had relapsed after ASCT, respectively. Median duration of response was 8.3 and 11.4 months in the 2 cohorts respectively and median duration of complete response has not yet been reached (clinicaltrials.gov). This suggests that remissions in DLBCL are rare, but may be durable when they do occur, and support the idea of testing this modality in specific subsets as discussed below.

The use of checkpoint inhibition in DLBCL may be more effective in combination. A phase 1 study tested the combination of PD-1 and CTLA-4 blockade, with preliminarily no indication of improvement in ORR (64). Despite this, other combinations are currently being explored. These include combinations of PD-1 blockade with agonist antibodies against 4-1BB (utomilumab; NCT02951156) or CD27 (varlilumab; NCT03038672), as well as with other immunomodulatory agents like lenalidomide or ibrutinib. Through correlative and biomarker-driven science on these studies, taking advantage of technical improvements in immunologic profiling both in the tumor and in the blood, the immunologic synapse of this disease may soon be better understood, informing rational and more effective combinations and targets.

Follicular lymphoma

Somewhat more is known about the interface between FL and the immune system, and there is evidence that this disease is sensitive to immunologic surveillance as demonstrated by the efficacy of allogeneic stem cell transplant and reports of spontaneous remissions in this disease. A few FL patients were treated on the early phase studies of immunotherapy agents like pidilizumab and ipilimumab and responses were seen (65,66). These patients were included in a phase 1 study of nivolumab, with ORR of 40% and a CR rate of 10% (63). Unfortunately, as in DLBCL these early promising results were not replicated in phase 2. In unpublished results from the CheckMate-140 trial of nivolumab, the ORR was only 4%, with one patient out of 92 achieving a complete response. Among the few responding patients, responses were durable with a median duration of response of 10.4 months, but the median progression-free survival (PFS) was only 2.2 months (clinicaltrials.gov). Combination of PD-1 blocking agents with anti-CD20 antibodies like rituximab has yielded more encouraging results, although they were tested in patients with rituximab-sensitive disease, where the expected benefit of CD20 directed therapy will be higher. Clinical trials of rituximab or obinutuzumab with pembrolizumab or atezolizumab, respectively, have yielded encouraging results with ORRs in the 57–67% range (67,68). Median duration of response to rituximab/pembrolizumab was 14.1 months, and median PFS was 11.4 months. With the R-pembrolizumab combination, the CR rate was 50%, which does suggest a possible synergistic effect of the combination. Furthermore, this study included blood and tumor biomarker analysis; there was no relationship between PD-L1 levels by immunohistochemistry in the tumor and response, but there was a relationship between a CD8 effector gene signature and the interferon-γ score in the blood and response. Rituximab in combination with immunotherapy targeting other T-cell receptors, including the 4-1BB agonist antibody utomilumab, has also been explored. This combination yielded responses in 27% of patients, including responses in one-third of patients with rituximab-refractory disease (69). Numerous combination studies are underway combining PD-1 or PD-L1 blocking agents with histone deacetylase inhibitors, lenalidomide, ibrutinib, chemotherapy, or other immunotherapy agents. As in the case of DLBCL, correlative science involving tumor and blood samples on these trials will inform a rational immunotherapy approach to the treatment of this innately immunosensitive tumor.

T-cell lymphomas

Twenty-three patients with T-cell lymphomas, including CTCL (n=13) and PTCL (n=5), were included on the phase 1 nivolumab trial; ORR was 15% in CTCL patients and 40% in PTCL (63). Two responding patients had responses that were ongoing at 24- and 50-week follow-up, and one patient had a response ongoing at 18 months. Further investigation of pembrolizumab in CTCL and Sezary syndrome resulted in ORR of 55% and 27%, respectively, with durable responses in responding patients and patients achieving disease stabilization (70). The 12-month PFS was 69%. These results are provocative, although given the tremendous biological heterogeneity of T-NHL, larger studies are needed to better understand the role of PD-1 blockade in these diseases. There are also several ongoing studies exploring combinations of PD-1 or PD-L1 with other agents active in T-cell lymphoma, including pralatrexate, romidepsin, azacitidine, and lenalidomide.

PD-L1/PD-L2 overexpressing lymphomas

The ability to selectively target specific lymphoma subtypes with PD-1 blockade based on their biology has already yielded several encouraging results. Based on the known recurrent alterations in PD-L1 and PD-L2 in PMBL, these patients were included in the phase 1 KEYNOTE-013 study of pembrolizumab in patients with hematologic malignancies. Amongst seventeen patients with PMBL, the ORR was 41%, with a CR rate of 12% (71). These responses were durable, with 6/7 responding patients remaining in response at a median follow-up of 11.3 months. These promising results, as well as the early experience of checkpoint inhibition in RS (ORR 44% in 9 patients), led to a phase 2 study of pembrolizumab in PMBL and RS, which treated 53 patients with PMBL (KEYNOTE-170, NCT02576990) (59,72). At last report the response rate in PMBL was 45%, with a CR rate of 11. The median duration of response had not been reached at ~10 months. This led to the FDA approval of pembrolizumab in PMBL after failure of 2+ lines of prior therapy. Results in Richter’s transformation have not yet been reported. In one small case series, PD-1 blockade yielded complete responses in 3 of 3 patients with GZL (73). Based on the identification of recurrent alterations involving PD-L1 and PD-L2 in PCNSL and PTL, 5 patients with PCNSL (n=4) or secondary CNS lymphoma (n=1) were treated off-label with the anti-PD-1 antibody nivolumab (74). The ORR was 100%, with a CR rate of 80%. These responses were durable, ranging from 13+ to 17+ months. Based on these results, nivolumab is being investigated in relapsed/refractory PCNSL and PTL (CheckMate-647, NCT02857426). The EBV-driven NK/T-cell lymphoma also seems to be a good target for PD-1 blockade, with an ORR of 100% in a small study (75). An ongoing multicenter phase 2 study is currently testing pembrolizumab in biologically selected subtypes of lymphoma and hematologic malignancies including EBV+ lymphoma, T-cell/histiocyte rich DLBCL, plasmablastic lymphoma, AITL, and several histiocytic disorders (NCT03316573).

Targeting the innate immune system

Immune checkpoint blockade has mostly targeted PD-1 (and less commonly in NHL CTLA-4) to date, but advances in the understanding of how the innate immune system is regulated identified new potential immunotherapy targets in oncology. The “don’t eat me” molecule CD47, which is overexpressed by a large variety of cancers including DLBCL and FL, binds to a receptor on macrophages and prevents phagocytosis of the cancer cell. Hu5F9 is a first-in-class monoclonal antibody to CD47. It was tested in combination with rituximab in a phase 1 study in relapsed/refractory DLBCL and FL, 90% of which were rituximab-refractory, with promising results (76). ORRs were 40% and 71% in DLBCL and FL, respectively, with CR in up to 1/3 of DLBCL patients and 43% of FL patients. These responses to date have been durable, with 91% of patients remaining in response at a median follow-up of 6 months and one CR ongoing at 14+ months, but the follow-up overall has been short. The phase 2 study of this combination is ongoing (NCT02953509). Targeting the innate, rather than adaptive, immune system may represent an effective strategy in these diseases.

Conclusions

The field of cancer immunotherapy has grown by leaps and bounds since the early days of high dose IL-2 therapy and allogeneic stem cell transplantation. Through an improved understanding of how the immune system is regulated, and new techniques in genetic engineering, we can now target tumors effectively with both antigen-independent and antigen-specific therapies. The most promising immunotherapeutic innovation in NHL, and in B-cell NHL in particular, has been anti-CD19 directed CAR T-cell therapy, with durable responses seen in nearly 40% of patients with advanced aggressive B-cell NHL. This therapy is now being explored in other B-cell NHL subtypes, including indolent lymphomas and mantle cell lymphoma. Emerging information regarding mechanisms of resistance with this therapy has informed strategies to enhance immune activation of effector T-cells, either through their use in combination with immunomodulatory agents or through further genetic modification. While immune checkpoint blockade has been so far disappointing in all-comer trials of DLBCL and FL, the biologically predictable success of PD-1 blockade in HL has allowed similar biological insights to direct PD-1 blockade to apparently vulnerable NHL subtypes, most notably PMBL. Although numerous combination studies are currently underway with both checkpoint blockade and CAR T-cells, a better understanding of the immune synapses and immune microenvironment in each specific NHL subtype will undoubtedly permit a new generation of clinical trials that capitalize on the early success of immunotherapy for the treatment of NHL and, we hope, eventually provide safe and definitive treatments across the NHL spectrum.

Acknowledgments

Funding: P Armand gratefully acknowledges the generous and unwavering support of the Harold and Virginia Lash Foundation.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Astrid Pavlovsky) for the series “Diffuse Large B-Cell Lymphoma” published in Annals of Lymphoma. The article has undergone external peer review.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/aol.2018.12.01). The series “Diffuse Large B-Cell Lymphoma” was commissioned by the editorial office without any funding or sponsorship. CA Jacobson: consultancy for Kite, Novartis, Pfizer, Humanigen, Precision Biosciences, Celgene; P Armand: consultancy for BMS, Merck, Affimed, Pfizer, Infinity; research funding (institutional): BMS, Merck, Roche, Tensha, Affimed, Sequenta, Otsuka, Sigma Tau. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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