PD-1 Expression and Clinical PD-1 Blockade in B-Cell Lymphomas

From www.bloodjournal.org at DUKE MEDICAL LIBRARY on September 21, 2019. For personal use only.

Review Series

THERAPEUTIC ANTIBODIES PD-1 expression and clinical PD-1 blockade in B-cell lymphomas

Zijun Y. Xu-Monette,1 Jianfeng Zhou,2 and Ken H. Young1,3

1Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX; 2Department of Hematology and Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; and 3Graduate School of Biomedical Science, The University of Texas Health Science Center at Houston, Houston, TX

Programmed cell death protein 1 (PD-1) blockade tar- expression on intratumoral T cells, and diffuse large B-cell geting the PD-1 immune checkpoint has demonstrated lymphoma, which variably expresses PD-1 and PD-L1. In unprecedented clinical efﬁcacy in the treatment of primary mediastinal large B-cell lymphoma, which fre- advanced cancers including hematologic malignancies. quently has 9p24.1 alterations, the ORR was 35% in a This article reviews the landscape of PD-1/programmed phase 2 trial of pembrolizumab. In contrast, the ORR with death-ligand 1 (PD-L1) expression and current PD-1 pembrolizumab was 0% in relapsed chronic lymphocytic blockade immunotherapy trials in B-cell lymphomas. leukemia (CLL) and 44% in CLL with Richter trans- Most notably, in relapsed/refractory classical Hodgkin formation in a phase 2 trial. T cells from CLL patients have lymphoma, which frequently has increased PD-11 tumor- elevated PD-1 expression; CLL PD-11 T cells can exhibit inﬁltrating T cells, 9p24.1 genetic alteration, and high a pseudo-exhaustion or a replicative senescence phe- PD-L1 expression, anti-PD-1 monotherapy has demon- notype. PD-1 expression was also found in marginal zone strated remarkable objective response rates (ORRs) of lymphoma but not in mantle cell lymphoma, although 65% to 87% and durable disease control in phase 1/2 currently anti-PD-1 clinical trial data are not available. clinical trials. The median duration of response was 16 Mechanisms and predictive biomarkers for PD-1 block- months in a phase 2 trial. PD-1 blockade has also shown ade immunotherapy, treatment-related adverse events, promise in a phase 1 trial of nivolumab in relapsed/ hyperprogression, and combination therapies are dis- refractory B-cell non-Hodgkin lymphomas, including fol- cussed in the context of B-cell lymphomas. (Blood. 2018; licular lymphoma, which often displays abundant PD-1 131(1):68-83)

Introduction does not have a high mutational burden compared with other cancers24 but has a higher frequency of PDL1/PDL2/JAK2 genetic Programmed cell death protein 1 (PD-1),1 predominantly alterations in Reed-Sternberg cells25 derived from germinal-center expressed on activated T cells, is an important immune Bcells.26,27 PD-1 blockade has also shown clinical activity in several checkpoint receptor. PD-1 transmits inhibitory signals into T cells types of B-cell non-Hodgkin lymphoma (B-NHL) with variable after ligation with PD-1 ligands (PD-Ls), PD-L12,3 or PD-L2,4 on PD-L1 expression. However, the reasons for differential efﬁcacy neoplastic cells and in the tumor microenvironment. In addition – to binding to PD-1, PD-L1 can bind to CD80/B7-15,6 and PD-L2 can in lymphoma patients are not well understood. Because PD-1 1 – bind to RGMb,7 promoting tolerance. However, PD-L1 function blocking antibodies bind only to PD-1 cells and not to PD-1 28 through T-T interaction can be context-dependent,8-11 and other cells, PD-1 expression and its clinical role in lymphoma need costimulatory receptors for PD-L1/PD-L2 may exist.12-15 The inter- to be better understood. This review provides a summary of actions between PD-1 and PD-L expression on T cells, lymphoma current studies on PD-1/PD-L1 expression and PD-1 blockade cells, and antigen-presenting cells as described in the literature in B-cell lymphomas. are summarized in Figure 1.2,4-6,9,16-19 PD-1 expression and clinical roles in In recent years, we have witnessed the great success of im- munotherapies with PD-1 or PD-L1 blockade in many types of B-cell lymphomas cancers, supporting the essential role of PD-1 and PD-L1 in Unlike solid tumors, B-cell lymphoma cells express major his- immune suppression. PD-L1 expression, but not PD-1 expression, tocompatibility complex (MHC) class II and costimulatory CD80/ has been associated with clinical response to PD-1 blockade in CD86 molecules that are functionally active,29 allowing these many clinical trials. The highest response rate to PD-1 blockade lymphoma cells to act as antigen-presenting cells by themselves was achieved in classical Hodgkin lymphoma (cHL),20-23 which (Figure 1).29-35 However, high rates of decreased or absent

PD-1 is also abundantly expressed in follicular lymphoma (FL) but with complicated expression patterns. T cells that inﬁltrate FL CD80 PD-L1 1 1 +/– –/+ tumors, which are skewed toward the CD4 CD45RO pop- T cell ulation, have higher PD-1 expression (mean amount, 287 cells PD-1 PD-L1/L2 T cell – per mm2 in grade 1-2 FL46; mean proportion, 6.21% to 21.8% of 46,47 1 PD-L1 CD80 all cells ; mean level, ;70% to 82% of CD4 T cells) than CD80PD-1 CD28 1 – –/+ peripheral T cells.48 PD-1 T cells are more frequently observed – + PD-1 CD28 ICOS in intrafollicular or perifollicular regions and less frequently – PD-L1 PD-L1 + + observed in interfollicular regions.47,49-51 A study identiﬁed 2 TCR – /X 1 PD-1 different subpopulations of intratumoral PD-1 T cells in FL

TCR biopsy specimens: those residing in the lymph node follicles with MHC bright PD-11 intensity (PD-1high) and those residing in inter- 1 low ? PD PD CD80 MHC follicular regions with dim PD-1 intensity (PD-1 ). The former BCR -L1 -L2 ICOSL 1 1 CD86 PD-L1 CD86 had a follicular helper T cell (TFH) phenotype (CXCR-5 BCL-6 CD80 1 Lymphoma PD-L2 CD4 , secreting interleukin-21 [IL-21] and supporting B-cell B cell Antigen APC growth in vitro), whereas the latter had exhausted phenotypes (TIM-312B41OX402)50 and function (reduced cytokine production and phosphorylation of STAT1/3/4 in response to cytokine Figure 1. Potential interaction involving PD-1/PD-L1 between receptors and stimulation).52 However, another study showed that FL-infiltrating ligands on lymphoma cells, professional antigen-presenting cells (APCs), and high 1 – 1 T cells. PD-1–PD-L1 interactions are highlighted in red. Functional consequences of PD-1 CD4 T cells included TFH and CXCR5 ICOS non-TFH the interaction are denoted by “1” (stimulatory) and “–” (inhibitory) signs. The “1/–“ cells, and both cell types were unresponsive to cytokines (Table signs indicate context-dependent effects of PD-L1–CD80 interaction on T cells. For 2).48 In line with these complex PD-1 expression patterns and PD-1 expression on malignant B cells in some non-Hodgkin B-cell lymphomas, PD-1 T-cell functions, the prognostic impact of PD-1 expression in FL ligands and functions are currently unclear (indicated by “?”). TCR, T-cell receptor. is inconsistent in the literature (Table 1).46,47,49-55 expression of b2M/MHC class I (79% to 83.2%) and MHC class II Compared with FL, the prevalence of PD-11 expression is lower in (46.8% to 67%) have been observed in cHL.36,37 Decreased MHC DLBCL (39.5% to 68.6%).56-59 Studies of PD-1 expression in DLBCL expression occurs less frequently in NHL.38,39 However, 41% to are more recent than those in HL and FL. Our group has analyzed 61% of patients with diffuse large B-cell lymphoma (DLBCL), the PD-1 expression in a large cohort of patients with DLBCL by us- most common aggressive B-NHL, have loss or aberrant ex- ing state-of-the-art fluorescent multiplex immunohistochemistry pression of MHC class I37,39,40 associated with B2M mutations and techniques (unpublished data); Figure 2 shows the spatial ex- deletions (frequency, 29%).40 Eighty percent of patients with pression of PD-1 on T cells and PD-L1 on lymphoma cells and primary testicular lymphoma (PTL) and 50% of patients with antigen-presenting cells in a DLBCL sample. Several studies have primary central nervous system lymphoma (PCNSL) have lost quantified the number of PD-11 TILs in DLBCL samples. Muenst both class I and class II MHC expression.37 Moreover, PD-1 et al46 found mean values of 27 PD-11 cells per mm2 and 1.1% of expression is frequently increased on tumor-infiltrating T cells all cells in 184 DLBCL samples. Kiyasu et al57 observed a range in B-cell lymphomas. of0to1288PD-11 cells per mm2 in 236 patients with DLBCL, and the number was lower in activated B-cell–like (ABC) DLBCL and The clinical role of PD-1 expression is most notable in Hodgkin DLBCL with PD-L1high expression (membranous and/or cytoplasmic lymphoma (HL). One study showed that PD-1 levels were staining in $30% of lymphoma cells). However, in other studies, markedly elevated in tumor-infiltrating T cells (53% to 76%) in PD-11 cell numbers were positively associated with PD-L1 ex- three patients with HL compared with healthy volunteers41; PD-1 pression (but not with the ABC subtype58,60). The amount of PD-11 expression in peripheral blood was also significantly higher in TILs positively correlated with PD-L1 expression in tumor cells patients with HL (nodular sclerosis, n 5 5; mixed cellularity, n 5 5) and/or macrophages in a study of 126 DLBCL patients58 and in than in patients with B-NHL.41 In a study of 189 patients with cHL, ABC-DLBCL in a study of 253 DLBCL patients,61 as well as in the median amount of PD-11 tumor-infiltrating lymphocytes (TILs) histiocytes (but not PD-L1 on tumor cells) in a study of 20 de novo was 27 cells per mm2, and the mean was 269 cells per mm2.42 and 11 transformed (from FL) DLBCL patients (Table 2).62 The Lymphocyte-rich cHL had the highest PD-1 expression (median, presence of or a high amount of PD-11 TILs in DLBCL samples has 203 cells per mm2; mean, 1044 cells per mm2), whereas nodular been associated with favorable, unfavorable, or no prognostic sclerosis cHL had the lowest (median, 16 cells per mm2; mean, 275 effect in different studies (Table 1)56-59,63; increased PD-1 levels cells per mm2). PD-1 rimming around tumor cells was observed (percentage expression) in the peripheral blood of DLBCL patients only in a small subset of cHL (lymphocyte-rich cHL and mixed have been associated with poorer prognosis.64,65 cellularity cHL),42 in contrast with the characteristic PD-11 rosettes in nodular lymphocyte–predominant HL.42-44 However, in another In contrast to FL and DLBCL, chronic lymphocytic leukemia (CLL) cHL study (nodular sclerosis, n 5 93; mixed cellularity, n 5 25; and small lymphocytic lymphoma (SLL) tissues have low numbers of lymphocyte-depleted, n 5 2), intratumoral PD-1 expression was PD-11 TILs (mean, 13 cells per mm2)46; however, the mean expression undetectable in 42% of patients and present at low levels in intensity of PD-1 was higher in CLL/SLL than in reactive lymph node another 42% of patients.45 Despite the difference in the preva- samples.53 Circulating T cells from CLL patients have been shown to lence of PD-1 expression in these studies, increased PD-11 TILs have elevated PD-1 expression levels compared with those from were consistently associated with poor prognosis (overall survival healthy donors (mean, ;45% of CD41 Tcellsand;30% of CD81 [OS]42 and disease-specific survival45) in cHL patients treated with T cells, respectively),66-69 especially effector (CCR7–CD45RA–) conventional therapies (Table 1). memory CD81 T cells in cytomegalovirus (CMV)-seronegative

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Table 1. Prognostic effects of PD-1 expression in tumor-inﬁltrating T cells in patients with B-cell lymphoma in nonimmunotherapy settings

Lymphoma Antibody Prevalence (No. of patients (manufacturer of PD-11 or Reference with follow-up) or source) PD-11/high cutoff PD-1high (%) Prognostic effect

HL 42 cHL (189) AF 1086 (R&D 23 cells per mm2 52 Increased tumor-infiltrating PD-11 T cells Systems, PD-11 infiltrating correlated with poorer OS independently of Wiesbaden- T cells stage of disease. However, in multivariable Nordenstadt) analysis for OS, PD-11 cell count lost its significance as an independent prognostic factor. 45 cHL (122) NAT (Abcam) .15 cells per high- ;18 PD-1 expression was associated with poor power field or 5-year DSS in univariable analysis and poor .0.5% of all OS in multivariable analysis. nucleated cells

FL/CLL 47 FL (132) NAT105 (Abcam) Cutoffs for low and ;76 (PD-11 cells Low proportion of PD-11 cells in FL tissues was high proportion .5%) and ;25 associated with poor OS and DLBCL of PD-11 cells in (PD-11 cells transformation; high proportion of PD-11 the diagnostic .33%) cells in FL tissues correlated with better OS biopsies were and PFS. #5% and .33%, respectively 49 FL (70) (Spanish National The good-outcome group had higher Cancer Research follicular PD-11 cell densities than the Centre) poor-outcome group. 51 FL (91) NAT (Abcam) $35.6 cells per 49 Increased follicular PD-11 cell density was an high-power field independent prognostic factor for poor OS. 46 FL (46); secondary Polyclonal AF1086 168 cells per mm2 36 Increased PD-11 TILs were associated with DLBCL arising (R&D Systems) or 2.8% of visible favorable DSS in combined 55 cases of FL from FL (9); cells and secondary DLBCL arising from FL; CLL (42) the amount of PD-11 TILs decreased from FL grade 1 to grade 3 and to FL with transformation to DLBCL; the amount of PD-11 TILs was not prognostic in CLL. 53 CLL (71); FL (59) Clone MIH4 CLL patients with poorer OS had increased (phycoerythrin); PD-1 expression (IHC mean intensity) on ab52597, NAT T cells in CLL lymph node samples; FL (Abcam) patients with poorer OS had increased PD-1 expression (IHC mean intensity) on interfollicular T cells; transformation to DLBCL was associated with increased PD-1 expression (IHC mean intensity) compared with paired pretransformation FL samples. 55 FL treated with NAT, ab52587 PD-11 cells In .50% of male High percentage of PD-11 cells in FL biopsies R-CHOP (82) (Abcam) .14.4% of all patients was associated with poorer PFS in male nucleated cells patients (n 5 43). 50 FL (58) NAT (Abcam) Diffuse staining 33 The presence of PD-11 cells in FL samples pattern rather with a diffuse staining pattern (mostly not than the quantity in the follicle) was associated with a of cells shorter time to transformation and inferior OS. 52 FL (32) ab52587 (Abcam) .26% for CD41 50 High PD-11 percentage in interfollicular cells; .45% for CD41/CD81 T cells with dim intensity CD81 T cells significantly correlated with reduced OS; CD41 T cells with bright intensity residing in the lymph node follicles were not associated with survival. 54 FL (92); transformed Polyclonal Increased intrafollicular PD-11 cell numbers in FL (52); HPA035981 diagnostic biopsies were associated with nontransformed (Sigma Life transformation; the number of intrafollicular FL (40) Science) PD-11 cells was significantly decreased upon transformation.

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Table 1. (continued)

Lymphoma Antibody Prevalence (No. of patients (manufacturer of PD-11 or Reference with follow-up) or source) PD-11/high cutoff PD-1high (%) Prognostic effect

DLBCL 46 DLBCL (138) Polyclonal AF1086 The amount of PD-11 TILs was not associated (R&D Systems) with survival. 56 DLBCL (70) Median of cell 50 High amounts of PD-11 cells, FOXP31 cells, counts under and CD41 T cells were all associated with high-power ﬁelds improved clinical outcome. 57 DLBCL (236) Clone NAT105 The median 50 Unclear. (Abcam) number of PD-11 TILs 64 DLBCL (46) (eBioscience) 38.7 High PD-1 levels (percentage expression) on T cells in the peripheral blood correlated with low effective rate of chemotherapy. 58 DLBCL (126) Clone MRQ-22 .0 PD-11 cells 68.6 Presence of tumor-inﬁltrating PD-11 cells (Cell Marque) correlated with prolonged PFS (P 5 .005) and OS (P 5 .026). 65 DLBCL (60) Clone MIH4 PD-11 percentage 16.7 High PD-11 percentage expression on CD41 (phycoerythrin) in peripheral T cells in the peripheral blood correlated blood CD41 with poor EFS and OS. T cells $30.25% 59 DLBCL (76) MRQ-22 Presence of 39.5 Presence of PD-11 TILs in DLBCL tissues was (ZSGB-BIO) PD-11 cells associated with prolonged OS. 63 DLBCL (102) (AbD Serotec) PD-11 cells 50 High PD-11 cell numbers were associated with accounting for poor survival. 13.1% of all cells

Anti-PD-1 clone names were not available in references 49, 63, and 64. DSS, disease-specific survival; EFS, event-free survival; IHC, immunohistochemistry; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. patients with early-stage CLL.67 CD81 T cells from these patients DLBCL, and extranodal MZL.43,46,73 One recent small-scale study had reduced proliferation capacity and cytotoxicity activity but using dual-color immunofluorescent staining for PD-1 and CD3 increased cytokine production (pseudo-exhaustion).67 However, showed that PD-1 was mainly expressed on tumor B cells in CLL- in CMV-seropositive CLL patients, the expanded CMV-specific involved tissues,76 in contrast with the CD21/CD41 T-cell pre- CD81 T cells had decreased PD-1 levels and were functionally dominance of PD-1 expression in CLL tissues shown in a previous intact compared with T cells from healthy controls70,71;PD-1levels study.66 Although in vitro studies have shown that PD-1 inhibits inversely correlated with the absolute number of effector T cells B-cell receptor signaling77 and B-cell activation,78 it seemed that and malignant B cells.71 In contrast, another study of patients with the PD-1 expression on CLL cells did not affect B-cell receptor early-stage CLL found that CMV-serostatus was not associated signaling significantly and was not associated with prognosis,68,75 with PD-1 levels on CD81 T cells but inverted CD4:CD8 ratios although it may modulate ZAP-70 function.68 were. Inverted CD4:CD8 ratios were also associated with reduced progression-free survival (PFS), preferential expansion of CD81 Together, these data suggest that PD-1 expression and clinical T cells skewed toward terminally differentiated effector CD81 roles vary across and within B-cell lymphoma entities. However, Tcells(CD81CCR7–CD45RO–), and a T-cell replicative senescence differences may also result from methodologic (eg, assessed by phenotype (CD571CD28–CD27–).69 These disparate data may PD-11 cell numbers, percentage expression, or staining intensities; suggest that PD-1 expression on T cells has a context-dependent by numbers of PD-11 TILs or numbers of PD-11 T cells; by per- role in CLL. PD-1 expression seemed to have limited prognostic centage of all cells or T cells; by tissue expression or peripheral significance in CLL/SLL.46,53 expression; or by immunohistochemistry or flow cytometry) and technical (use of different antibodies and positivity cutoffs) dis- Studies in other types of B-cell lymphomas found numerous parities among studies,79 illustrating the challenge in assessing the PD-11 T cells in marginal zone lymphoma (MZL) but almost no dynamic tumor microenvironment and T-cell responses. Similar PD-11 cells in mantle cell lymphoma (MCL).46,72,73 issues may exist in clinical studies of PD-1 expression as a biomarker for PD-1 blockade therapy. Furthermore, in addition to the PD-1 expression in reactive T cells, weak PD-1 expression on neoplastic B cells was also reported in 92% of patients with CLL/SLL and 7% to 22% of PD-L1 expression and clinical roles in patients with DLBCL or high-grade FL.60,74 PD-1 was also weakly expressed on circulating CLL cells (levels, ;47%) in 91% of B-cell lymphomas patients and increased after CD40/CD40L/IL-4 stimulation in A functional study demonstrated that only after PD-1 ligation vitro.74,75 However, other studies using different PD-1 antibodies can PD-1 block T-cell activation.80 Therefore, we brieflyre- showed that PD-1 was expressed only in rare cases of CLL/SLL, viewed the expression and clinical significance of PD-L1, the

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Table 2. Observations associated with PD-1 expression on T cells in B-cell lymphomas

Reference No. of Patients Antibody (manufacturer) Associations

HL 41 19 (BD Pharmingen) PD-1 levels in the peripheral blood T cells of HL patients tended to be higher in active disease and declined along with treatment; PD-1 and FOXP3 seemed to be alternatively expressed (expressed in different patients). 42 189 AF 1086 (R&D Systems) PD-11 cell density was lower in patients with 9p24.1 gains (near PDL2 locus) and in those with high numbers of FOXP31 Tregs but positively correlated with densities of granzyme B1 and TIA-11 cells. 61 280 760-4448 (Ventana Roche) There was no correlation between 9p24.1 alterations, EBV status, and rosetting of PD-11 T cells in HL.

FL 47 132 NAT105 (Abcam) PD-1 expression in FL tissues correlated with low grade, good performance status, and low LDH; PD-1 expression was alternative to FOXP3 positivity in T cells (expressed in different patients). 46 49 Polyclonal AF1086 (R&D Systems) The amount of PD-11 TILs was higher in FL grade 1 to 2 than in FL grade 3 and further decreased in FL with transformation to DLBCL. 51 91 NAT (Abcam) The number of PD-11 cells in FL tissues correlated with the number of FOXP31 Tregs. 55 82 NAT, abcam52587 (Abcam) PD-1 positivity in FL biopsies was associated with male sex and high serum b2M levels. 48 14 clone EH12.1 High PD-1 expression levels in TILs correlated with decreased cytokine signaling response, including IL-4–induced phosphorylation of STAT6 and IL-10/IL-21–induced phosphorylation of STAT3. 52 32 ab52587 (Abcam) Intratumoral PD-1high T cells: CD41CXCR51BCL-61TIM-32; PD-1low T cells: TIM-31BCL-62CXCR52.

CLL 69 77 (BD Biosciences) CLL patients with inverted CD4:CD8 ratios had signiﬁcantly increased PD-1 levels on CD81 T cells in CLL blood samples.

DLBCL high 56 70 Mouse mAb Number of PD-1 (TFH) cells positively correlated with number of CD41 T cells. 57 236 Clone NAT105 (Abcam) The number of PD-11 TILs was lower in groups of patients with B symptoms, extranodal sites, bulky masses, ABC subtype, and PD-L11 ($30%) DLBCL. 64 46 (eBioscience) Increased blood PD-1 levels were associated with stage III and IV, B symptoms, IPI .2, elevated LDH, and high Ki-67; PD-1 levels decreased after 4 courses of chemotherapy. 61 253 760-4448 (Ventana Roche) Tumor PD-L1 expression levels positively correlated with the amount of PD-11 TILs in ABC-DLBCL. 58 126 Clone MRQ-22 (Cell Marque) The number of PD-11 TILs positively correlated with PD-L1 expression levels on tumor cells and/or macrophages. 62 31 An increased number of PD-11 TILs negatively correlated with tumor Ki-67 and positively correlated with PD-L1 expression in histiocytes but not with PD-L1 on tumor cells. 65 60 Clone MIH4 (phycoerythrin) PD-1 expression in tumor biopsies did not correlate with PD-1 expression in the peripheral blood.

Anti-PD-1 clone names were not available in references 41, 64, and 69. IPI, International Prognostic Index; LDH, lactate dehydrogenase.

primary PD-1 ligand, on malignant B cells and in the tumor mi- number alterations (97% by fluorescent in situ hybridization,84 croenvironment in B-cell lymphoma entities. PD-L2 has lower compared with ;40% by array comprehensive genome hybrid- expression except in HL and primary mediastinal large B-cell ization85) and Epstein-Barr virus (EBV) infection (23% to 32%).83,86 lymphoma (PMBCL).21,81,82 Interestingly, 9p24.1 gains were associated with fewer PD-11 cells in cHL (Table 2).42 Both PDL1/PDL2 gene amplification and PD-L1 Most patients with cHL have strong tumor PD-L1/PD-L2 ex- expression have been associated with poorer prognosis (PFS84 pression (rate, 70% to 87% in those receiving conventional and event-free survival61) in cHL. PD-L1 expression was also found treatment61,83 and 94% to 100% in patients with relapsed or re- on nonmalignant cells (predominantly tissue macrophages83)in fractory [R/R] cHL in anti-PD-1 clinical trials21,23) because of high cHL but seemed to have a less significant role than tumor PD-L1 frequencies of 9p24.1 (locus harboring PDL1/PDL2/JAK2)copy expression.22,23

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B cells53,66; however, other studies showed that PD-L1 expres- + + PD-1 CD3 sion was mainly in the tumor microenvironment (likely histiocytes)76 but not on tumor cells (very low).61,74,76 In circulating CLL cells, PD-L1 levels were increased compared with normal controls in 1 study66 but were not increased in 2 studies.74,75 Whether PD-L1+ the low tumor PD-L1 expression contributes to the pseudo- CD68+ exhaustion of T cells in CLL is unknown.70,71 However, studies + + PD-L1 CD20 showed that the PD-1/PD-L1 axis is functionally active in CLL, as evidenced by the close interaction between PD-11CD41 T cells and PD-L11 CLL cells in the CLL proliferation center, the spike in PD-1 expression on proliferating T cells and in PD-L1 expression on CLL cells after stimulation in vitro, and impaired interferon-g (IFN-g) production by CD81 T cells after treatment with soluble PD-L1, which was reversed by anti-PD-L1 and/or anti-PD-1 an- 66 Colors: PD-1, CD3, PD-L1, CD68, CD20 tibodies in vitro. Like PD-1, PD-L1 expression in CLL/SLL had no prognostic significance in most studies,61,66 but increased expression intensity was associated with poor prognosis in a Figure 2. Representative overlaid image of PD-1 and PD-L1 expression in a de small-scale analysis.53 novo DLBCL sample by fluorescent multiplex immunohistochemistry. Fluores- cent colors show the following: blue for PD-11, green for PD-L11, red for CD31, yellow for CD681, and cyan for CD201. Examples of PD-11 T cells are indicated by the In other B-NHL entities, the rates of PD-L1 expression on neo- red arrows (co-localization of PD-11 and CD31 signals are shown in magenta), a plastic B cells are low: ;5% in FL, ;10% in high-grade MZL, and 1 1 PD-L1 CD68 cell (macrophage/dendritic cell) is indicated by the yellow arrow, and 0% in MCL.47,61,74,87 PD-L1 expression levels were shown to be a PD-L11 lymphoma cell (CD201) is indicated by the white arrow. Original magni- low in FL47,61 but the mean expression intensities were higher in fication 320. FL than in reactive lymph nodes.53 PD-11 TILs in FL may receive inhibitory signals from PD-L1 expressed by a small fraction of In contrast to HL and PMBCL cell lines with high PD-L1 ex- histiocytes48 and regulatory T cells (Tregs).47 In a small number pression, most DLBCL cell lines do not express PD-L1 in vitro of patients with B-NHL, intratumoral CD41CD25– T cells, but not but do express PD-L1 in vivo.25,41,87 PD-L1 was shown to be Tregs, expressed PD-1, which interacted with PD-L1 on Tregs expressed in 26% to 75% of DLBCL samples with PD-L11 cutoff and inhibited proliferation of CD41CD25– T cells.17 Alternative $5%61,87-89 and more frequently in ABC-DLBCL,57,60,87,88 and expression of PD-1 and FOXP3 was also observed in studies tumor PD-L1 expression has been associated with either poor of FL and HL (Table 2).41,42,47 However, in another study, the prognosis or no significant prognostic effect.57,58,61,90 PDL1/ number of PD-11 TILs and FOXP31 Tregs in 91 samples of FL PDL2 amplifications are rare in DLBCL, and 9p24.1 copy gain showed a positive correlation.51 The prognostic significance of was identified in only 6% to 19% of patients with DLBCL.57,88,91 PD-L1 expression on T cells and histiocytes in FL is unclear. Another form of genetic alteration, disruption of the PDL1 39 untranslated region, was identified in 8% of DLBCL samples.92 Collectively, tumor PD-L1 expression has been better charac- PD-L1 was also shown to be expressed in the tumor microen- terized than PD-1 expression in B-cell lymphomas, but PD-L1 vironment of DLBCL (mainly on macrophages), either concur- expression in the tumor microenvironment has not been well rently with tumor PD-L1 expression or alone (prevalence, 30% to defined and probably not separated from tumor PD-L1 ex- 38%), but this has not been correlated with significant prog- pression in some studies. PD-1 and PD-L1 data have consistently nostic effect.57,58,83,87,90 EBV1 (frequency, 3% to 4% in western demonstrated the clinical importance of PD-1 signaling in HL; countries93,94) DLBCL has high PD-L1 expression on malignant accordingly, anti-PD-L antibodies restored the IFN-g production B cells (mediated by latent membrane protein 1, an EBV- function of HL-infiltrating T cells in vitro.41 PD-1/PD-L1 expres- encoded antigen86)andinfiltrating macrophages,83 but the sion is heterogeneous in B-NHL and has been inconsistently clinical role of PD-L1 expression in EBV1 DLBCL is currently associated with prognosis. PD-L1 blockade in DLBCL resulted in unknown. Moreover, a soluble form of PD-L1 was detected in the either enhanced IFN-g secretion by T cells87 or no effect on the plasma but not in the serum of peripheral blood from DLBCL low production of IFN-g in vitro.41 PD-1 blockade restored patients, and its expression levels had no correlation with PD-L1 proliferation and cytokine production of T cells cocultured with expression on tumors. Elevated soluble PD-L1 expression was EBV1 DLBCL cell lines in vitro.99 For FL, disruption of the tumor found to be either associated89,90 or not associated95 with poorer microenvironment by in vitro culture over time could restore the prognosis. cytokine signaling in PD-1high TILs, and an anti-PD-1 monoclonal antibody (mAb) had no effect beyond isotype control.48 In a CLL Remarkably, PMBCL, PCNSL, and PTL have .50% rates of 9p24.1 mouse model, PD-1 blockade only modestly improved T-cell copy gains.82,91 PMBCL also has a high rate of 9p24.1 rearrange- function and was insufficient to restore the antitumor activity of ment (20%) associated with PD-L1/PD-L2 overexpression.96 T cells,100 likely because increased CD200, CD270 (herpesvirus Half of these rearranged PDL1/PDL2 genes in PMBCL were entry mediator; receptor for B- and T-lymphocyte attenuator), fused to the CIIA gene, leading to downregulation of MHC class II PD-L1, and CD276 (B7-H3) expression on CLL cells was also expression,97 which has been linked to incremental decreases in relevant for impaired T-cell metabolism, actin synapse formation, patient survival.98 and T-cell dysfunction.53,100 In contrast, early PD-L1 blockade restored CD81 T-cell function and prevented the development In CLL/SLL lymph node samples compared with reactive lymph of CLL in vivo.101 PD-L1 blockade with an anti-PD-L1 antibody node samples, PD-L1 expression was increased on neoplastic or PD-1 fusion protein in vitro partly restored the proliferation of

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CD41CD25– T cells from the tumor sites of B-NHL (FL, DLBCL, LAG-3,60 or TIM-350,52,118), or other therapeutic agents (such as and MCL).17 These preclinical studies provide a rationale for lenalidomide53) may have synergistic effects in B-cell lympho- targeting the PD-1 immune checkpoint in B-cell lymphomas. mas. Numerous combination therapies with nivolumab or pembrolizumab are being tested in B-cell lymphomas, including combinations with chemotherapy, radiotherapy, rituximab, Therapeutic targeting of PD-1 with PD-1 MEDI-551 (anti-CD19), ipilimumab (anti-CTLA-4), brentuximab blockade in B-cell lymphomas vedotin (anti-CD30 antibody-drug conjugate), blinatumomab (bispecific anti-CD19/CD3 T engager), AFM13 (bispecific PD-1 antibodies currently approved by the US Food and Drug anti-CD30/CD16A), lenalidomide, PI3K inhibitors (idelalisib, Administration (FDA) include nivolumab and pembrolizumab. TGR-1202), Bruton tyrosine kinase inhibitors (ibrutinib, acalabruti- These fully human or humanized immunoglobulin G4 mAbs nib), varlilumab (anti-CD27), urelumab (anti-CD137), G100 (TLR4 block the binding of PD-L1/PD-L2 to PD-1 because of their high agonist), SEA-CD40 (anti-CD40), MK-1454 (STING agonist), BMS- affinity and specificity (Figure 3A). One structure study sug- 986016 (anti-LAG-3), epacadostat (indoleamine 2,3-dioxygenase gested that the mechanisms of antagonism are highly similar 1 inhibitor), L-NMMA (nitric oxide synthase inhibitor), veliparib among these mAbs,102 but another study showed that nivolumab (PARP inhibitor), histone deacetylase inhibitors (entinostat, and pembrolizumab bind to completely different areas of PD-1.103 vorinostat), RRx-001 (epigenetic agent), dendritic cell therapy, LTX- Functional assays demonstrated that treatment with nivolumab 315 (oncolytic peptide), CD19/CD22 chimeric antigen receptor enhanced antigen-specific T-cell responses, including proliferation T cells (AUTO3), EBV-specific T cells (EB-VST cells), mogamulizu- and cytokine production of effector T cells28;noadverseimmune- mab (anti-CCR4), IFNg-1b, DPX-Survivac (immunotherapeutic related events were observed in cynomolgus macaques, although survivin vaccine), and personalized synthetic long peptide vac- nivolumab showed immunoreactivity to endocrine cells in normal cines with polyinosinic:polycytidylic acid (TLR3 agonist). In ad- human tissues.28 dition, other PD-1 antibodies (AMP-514, SHR-1210, PDR001, BJB-A317, JS001, REGN2810, and IBI308), PD-L1 antibodies In multiple clinical trials in B-cell lymphomas, nivolumab, (atezolizumab, durvalumab, avelumab, and CK-301), and oral pembrolizumab, and another mAb, pidilizumab (CureTech), small-molecule CA-170 (PD-L1, PD-L2, and VISTA antagonist) have shown clinical activity (Table 3). However, pidilizumab is no as monotherapy and in combinations are also being tested in longer recognized as an anti-PD-1 mAb; therefore, pidilizumab clinical trials for patients with HL or B-NHL. trials104-106 are not discussed in this review. Across lymphoma fi entities, anti-PD-1 monotherapy showed the greatest ef cacy in Some combination trials have interim results available. Nivolu- 20-23,76,107-115 R/R cHL. In a phase 1 trial of nivolumab, the objective mab and ipilimumab combination therapy had ORRs of 74% in 20 response rate (ORR) was remarkable (87%). In phase 2 trials, HL and 20% in B-NHL in a phase 1 trial.108 Nivolumab combined nivolumab and pembrolizumab had similar ORRs (68% and 69%) with ibrutinib showed clinical activity in a small number of pa- 22,23 and OS rates (99%) at 6 months, leading to accelerated FDA tients with R/R CLL or RT.111 The combination of pembrolizumab approval in 2016 for nivolumab and in 2017 for pembrolizumab. and rituximab showed a high ORR of 80% in relapsed FL.112 High complete response rates (50% to 65%) have been reported for 107 In R/R FL, the ORR was 40% with nivolumab in a phase 1b trial the combination of nivolumab and brentuximab vedotin in and 80% with combined pembrolizumab and rituximab in a patients with R/R HL in 2 phase 1/2 trials.113,114 phase 2 trial (interim results).112 In R/R DLBCL, the ORR with 107 nivolumab was 36% in a phase Ib trial. In R/R PMBCL, the ORR Nivolumab and pembrolizumab showed acceptable safety 109 with pembrolizumab was 41% in a phase 1b trial and 35% in a profiles with low rates of discontinuation resulting from adverse 115 phase 2 trial (interim results). Notably, a retrospective study events, although close monitoring is warranted, especially for showed that all 4 patients (100%) with PCNSL/PTL (R/R PCNSL patients with a history of acute graft-versus-host-disease.119 or PTL with central nervous system relapse) treated with nivo- Serious immune-mediated adverse events included pneumo- lumab had clinical and radiographic responses, and 3 pa- nitis, neutropenia, thrombocytopenia, hepatitis, rash, dyspnea, tients remained free of progression at 131 to 171 months.110 anemia, colitis, and others. The rates of grade 3 to 4 adverse Phase 2 trials for nivolumab or pembrolizumab monotherapy in events were lower for pembrolizumab (6.4% to 16% in cHL and 12% FL, DLBCL, PMBCL, and PCNSL/PTL are ongoing (CheckMate 140, in PMBCL) than for nivolumab (22% to 41% in cHL and 26% in NHL) CheckMate 139, NCT02362997, KEYNOTE-170,115 NCT02779101, with indicated doses, except for the high 60% rate for pem- NCT03255018, and CheckMate 647), and results informing brolizumab in R/R CLL with RT with prior ibrutinib therapy (Table 3). ORRs, OS, and PFS are awaited. In CLL with Richter transformation (RT; DLBCL), the ORR with pembrolizumab was 44% in a phase 2 Recently, a novel aggressive pattern of hyperprogression was trial, and the median OS was 10.7 months with a median follow-up identified in ;9% of patients receiving anti-PD-1/anti-PD-L1 of 11 months. In contrast, the ORR was 0% in relapsed CLL.76 monotherapy, including 1 (14%) of the 7 lymphoma patients These clinical trials were all single-arm trials for nivolumab or who were studied.120,121 Hyperprogression was associated with pembrolizumab monotherapy in R/R settings. Two-arm trials for elderly age but not baseline tumor burden or specific tumor anti-PD-1 monotherapy in R/R settings and 2-arm and/or single- type.120 In solid tumors, MDM2/MDM4 amplification and EGFR arm trials for combination therapies in first-line or R/R settings aberrations were associated with increased risk of hyperpro- are ongoing, and results are currently not available. gression.121 Among patients whose PD-L11 status was known, 1 (33%) in 3 hyperprogressors had PD-L1– disease, which was Preclinical studies suggested that combining PD-1 blockade higher than the PD-L1– rate (6% [2 of 32]) observed in non- with costimulatory agonists, immune checkpoint blockade (such hyperprogressors (P 5 .24).120 Although the relevance of PD-L1– as blockade of CTLA-4,116 B7-H3,53,117 CD270,53 CD200R,53,100 PD-11 expression to disease hyperprogression is unknown, it

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Figure 3. The mechanism of action of PD-1 blockade in + − PD-L+ tumors and models of potential functional ef- ABPD-L tumor/APC PD-L tumor/APC fects of anti-PD-1 therapy on PD-1+ T cells (CD4+/CD8+) in patients without PD-L expression (PD-L–), according 1 to the literature. (A) In PD-L tumors, the antigen- PD-L1/2 -

fi B7 speci c T-cell response is inhibited because PD-1 is en- CD80/86 Anti-PD-1 gaged by PD-L1/2. PD-1 blockade with anti-PD-1 antibodies Anti-PD-1 prevents PD-L1/PD-L2 from interacting with PD-1 and restores T-cell function (anti-PD-1 mAb acting as an CD28 antagonist). (B) In PD-L– tumors with PD-11CD41 T-cell PD-1 , IL-2,TNF PD-1 CD28 infiltrate, according to Chemnitz et al80 and Bennett Cytotoxicity PI3K et al,122 PD-1 ligation with PD-1 antibodies inhibits CD41 ITIM P ITIM PD-L1 Kinases IFN- γ T-cell proliferation and cytokine production under sub- ITSM SHP-2 ITSM optimal CD3 and CD28 costimulation conditions (anti- PD-1 mAb acting as a PD-1 agonist), which are caused X– – T by inhibition of TCR downstream signaling mediated by + + + SHP-2 associated with immunoreceptor tyrosine-based CD3 T CD4 T switch motif (ITSM) of PD-1. It is unknown whether this CD3 scenario is relevant for some hyperprogressive diseases Proliferation, IFN- γ , IL-2 after PD-1 blockade therapy. (C) The effect of anti-PD-1 antibodies on PD-11CD81 T cells in PD-L– tumors is unknown. If the TCR signaling is strong with optimal CD28 PD-L− tumor/APC PD-L− tumor/APC costimulation and/or TCR engagement (in which case, CD PD-L– is likely caused by mutations or lack of IFN-g receptor on PD-L– tumors, but not because of lack of IFN-g release), PD-1 ligation with anti-PD-1 antibodies may have B7 fi 1 2,122 no signi cant effect on the high CD8 T cell function. Anti-PD-1 Anti-PD-1 Furthermore, if circulating activated T cells express PD-L1 2 1 1 and the PD-1 PD-L1 axis suppresses PD-1 or PD-L1 , TNF- T-cell function, PD-1 blockade could enhance CD41/CD81 PD-1 γ PD-1 CD28

T-cell function, thereby manifesting clinical activity in Cytotoxicity

– IFN- these PD-L patients. (D) As another possibility for anti- X P PI3K CBL -B – ITIM ITIM PD-1 therapy in PD-L tumors, engagement of PD-1 on PD-L1 ITSM Kinases SHP-2 ITSM CD81 T cells by PD-1 antibodies could inhibit TCR signaling (acting as a PD-1 agonist) similar to ligation with the –

natural ligand PD-L1, mediated by SHP-2 phosphatase T

+ + + activity and TCR internalization/degradation as a result of CD8 T CD8 T increased CBL-b ubiquitin ligase activity.123 ITIM, immunoreceptor tyrosine-inhibitory motif. CD3

has been shown in vitro that under suboptimal conditions, and variable PD-L1 resources (tumor, tumor microenvironment, engagement of PD-1 with anti-PD-1 mAbs inhibited rather peripheral blood), as well as the use of different detecting than enhanced CD41 T-cell activation, just like PD-1 ligation antibodies, staining procedures, and PD-L11 cutoffs. Microsatellite by PD-L1 (Figure 3B).80,122 However, engagement of PD-1 on instability (MSI) arising from deficient mismatch-repair (dMMR), has CD81 Tcells123 by anti-PD-1 mAbs in the PD-L1– setting emerged as another predictive biomarker for PD-1 blockade.133 (Figure 3C-D) was not shown. It is also unclear whether clinical Pembrolizumab has received FDA first tissue/site-agnostic approval use of nivolumab and pembrolizumab can act as PD-1 agonists for the treatment of adult and pediatric patients with MSI-high or instead of antagonists28 in PD-L1– patients. Also unknown is dMMR metastatic solid tumors.134 Nivolumab has been ap- whether PD-L1 interacts more with CD80 or other receptors proved for the treatment of patients (age 12 years or older) with during blockade of PD-1,124 whether PD-L1 and PD-L2 have R/R MSI-high or dMMR metastatic colorectal cancer. positive roles,3,8,10,12-15,125-130 and whether some PD-1 polymor- phisms affect the action of PD-1 blockade in patients (nonblocking Efforts have also been undertaken to identify predictive bio- PD-L1 mAbs enhance the binding of PD-L1 to PD-1131), which could markers in PD-1 blockade trials in B-cell lymphomas (Table 4). be relevant for hyperprogression after PD-1/PD-L1 blockade. The first nivolumab phase 1 trial in cHL suggested that PDL1/ PDL2 copy number alterations and high PD-L1/PD-L2 expression were relevant for the high ORR in cHL. In contrast, Biomarker studies and immune tumor-infiltrating T cells largely expressed low levels of PD-1.20 monitoring in B-cell lymphomas Three subsequent trials of pembrolizumab or nivolumab in cHL Predictive biomarkers for response to PD-1 blockade are being examined PD-L1 expression. High prevalence of PD-L1 ex- eagerly pursued. PD-L1 expression is a widely used biomarker in pression (94% to 100%) was found in 2 pembrolizumab trials, but solid tumors, because effective blocking action requires pre- PD-L1 expression defined as either $1% membrane staining existing PD-L12PD-1 interaction in tumors (Figure 3A), and with moderate or strong intensity or detailed stratification by PD-L1 positivity may indicate ongoing immune response.132 staining intensity, membrane staining, and histiocyte scores However, the predictive value of PD-L1 is inconsistent in clinical seemed not to be predictive.21,23 In the nivolumab phase 2 trial, trials, which may be attributable to the complexity of the immune PD-L1 expression levels were calculated as H scores, multiply- signaling network, dynamic and clustered PD-L1 expression patterns, ing the percentage of PD-L11 malignant cells by the average

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Table 3. Results from clinical trials of PD-1 blockade in B-cell lymphoma

Rates of all AEs Phase; no. of PFS; OS; duration of (%), grade 3-4 Lymphoma Antibody, dose patients ORR (%), CR (%) response AEs (%) Reference

PD-1 blockade in HL R/R cHL Nivolumab, 3 mg/kg every 2 wk Phase 1; 23 87, 17 PFS: 86% at 24 wk; OS: 91% at 78, 22 20 1 y and 83% at 1.5 y; 35% of responders maintained a response for more than 1.5 y R/R cHL that progressed Pembrolizumab, 10 mg/kg Phase 1b; 32 65, 16 PFS: 46% at 52 wk; 70% of 97, 16 21 after BV every 2 wk responses lasted longer than 24 wk R/R cHL that failed to Nivolumab, 3 mg/kg every 2 wk Phase 2; 80 66.3, 9 (68, 13 At 6 mo, PFS: 76.9%; OS: 99, 41 22 respond to AHSCT according to 98.7%; at 12 mo, median and BV ICML 2017 PFS: 10.0 mo; per ICML 2017 update) update, with a median follow-up of 23 mo, the median duration of response was 16 mo R/R cHL, progressed after Pembrolizumab, 200 mg once Phase 2; 210 69, 22.4 At 6 mo, PFS: 72.4%; OS: Treatment-related 23 ASCT and/or BV every 3 wk, (median no. of 99.5%; 75.6% of patients had AEs: 63% treatment cycles: 13) a response for $6mo (according to ICML 2017); immune- mediated AEs: 28.6; infusion- related reactions: 6.4

PD-1 blockade in B-NHL R/R NHL and MM Nivolumab, 1-3 mg/kg every Phase 1b; FL, 10; FL, 40, 10; DLBCL, Duration of response: All AEs: 63 (for 107 2wk DLBCL, 11; other 36, 18; other B- 6.0-81.6 wk B-NHL: 71, 26) B-NHL, 10; T- NHL, 0, 0; T-NHL, NHL, 23; MM, 27 17, 0; MM, 4, 4* R/R PMBCL Pembrolizumab, 10 mg/kg Phase 1b; 18 41, 11.8 With a median follow-up of 61, 11.8 109 every 2 wk or 200 mg every 11.3 mo, median duration of 3wkforupto2y response and OS were not reached; in 2 of the 7 patients who responded, the duration of response was 20.51 and 22.41 months R/R CLL with RT and Pembrolizumab, 200 mg every Phase 2; 25 RT (transformed Median OS: 10.7 mo for R/R 100, 60 76 relapsed CLL 3wkforupto2y (transformed DLBCL), 44, 11; CLL with RT after a median DLBCL, 9; relapsed CLL, 0, 0 follow-up time of 11 mo, not relapsed CLL, 16) reached among patients with prior ibrutinib therapy R/R PCNSL and PTL Nivolumab, 3 mg/kg R/R PCNSL, 4; PTL 100, 80 3 patients remained free of 60, 20 110 intravenously every 2 wk (in with CNS progression at 131 to 1 patient with rituximab- relapse, 1 171 months refractory PCNSL, rituximab was continued for 3 doses after beginning treatment with nivolumab)

Abstracts in chronological order R/R hematologic Nivolumab, 3 mg/kg 1 Phase 1; HL, 31; FL, HL, 74, 19; B-NHL, With a median follow-up of Grade $ 3: 29; 108 malignancies ipilimumab, 1 mg/kg every 5; DLBCL, 10; 20, 0; T-NHL, 9, 0; 11.4 mo, median PFS and OS 5 patients (8) 3 wk for 4 doses followed by PMBCL, 1; T-NHL, MM, 0, 0 in: HL: not reached; in discontinued nivolumab monotherapy, 11; MM, 7 B-NHL: PFS 1.5 mo, OS owing to drug- 3 mg/kg every 2 wk 2.9 mo; inT-NHL: PFS related AEs 2.0 mo, OS13.2 mo; in MM: PFS 2.2 mo, OS 7.6 mo R/R CLL/RT Nivolumab, 3 mg/kg 1 Phase 2; R/R CLL, 4; R/R CLL, 75, 0; RT, All immune-related 111 ibrutinib, 420 mg once daily RT, 4; PR CLL after 50, 25; PR CLL, AEs: R/R CLL with ibrutinib, 3 0, 0 RT: 0; PR CLL: 33 Relapsed FL Rituximab 1 pembrolizumab, Phase 2; 30 80, 60 in 15 patients With a median follow-up of 37, 22 in 27 patients 112 200 mg every 3 wk for up to at the interim 7 mo, median PFS, OS, and who had initiated 16 cycles analysis duration of response were therapy not reached R/R HL Nivolumab, 3 mg/kg 1 BV, Phase 1; 19 89, 50 in 18 PFS at 6 mo: 91% Grade 3-5: ;26 113 1.2-1.8 mg/kg every 3 wk evaluated patients for 16 cycles R/R HL BV 1 nivolumab Phase 1/2; 62 85, 64 72, 7 114 R/R PMBCL Pembrolizumab, 200 mg Phase 2; 33 35, 13 58, 18 115 intravenously every 3 wk

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Table 3. (continued)

Rates of all AEs Phase; no. of PFS; OS; duration of (%), grade 3-4 Lymphoma Antibody, dose patients ORR (%), CR (%) response AEs (%) Reference

Pidilizumab trials Advanced stage NHL, HL, Pidilizumab, 0.2-6 mg/kg for Phase 1; FL, 1; Only the FL patient Responding patients: All AEs: 61 104 CLL, or MM one cycle DLBCL, 2; CLL, had a CR; ORR, remained alive at 60 wk 3; HL, 1 CR for B-NHL: 14, 14 DLBCL after AHSCT Pidilizumab, 1.5 mg/kg every Phase 2; 66 ORR, 51 (35 for 35 At 16 mo, PFS: 72%; OS: 85% 96, 32 105 (excluding patients with 42 d for 3 cycles 30-90 d (including patients who had progression and active after transplantation 4 PMBCL and 13 measurable autoimmune disease) transformed disease after indolent B-NHL) AHSCT) Relapsed FL Pidilizumab, 3 mg/kg every Phase 2; 32 66, 52 Median PFS: 18.8 mo 94, 0 106 4 wk for 4-12 infusions 1 rituximab, 375 mg/m2 17 d after the ﬁrst infusion of pidilizumab

Auto-HSCT, autologous hematopoietic stem cell transplantation; AE, adverse effect; CNS, central nervous system; CR, complete remission; ICML, International Conference on Malignant Lymphoma; MM, multiple myeloma; PR, partial remission. *Nivolumab therapy after radiotherapy. intensity of positive staining; PD-L1 expression on malignant expression in the presence of PD-L1 in turn inhibit T-cell Reed-Sternberg cells, but not PD-L1 on infiltrating normal cells, function, including IFN-g production.136 Low PD-L1 expres- was associated with treatment response.22 Moreover, the sion could also result from low IFN-g production by PD-1high magnitude of 9p24.1 gains correlated with H scores, and am- CD81 T cells under suboptimal costiumulation conditions or a plification showed association with treatment response. How- fixed dysfunctional state.137 The implication of these un- ever, 50% of patients with low PD-L1 H scores in the first quartile published data for PD-1 blockade biomarkers is unknown. and 83% of patients with 9p24.1 polysomy also achieved partial remission.22 The predictive values of PD-L2 expression and JAK2/STAT1 signaling, which are increased because of PDL1/ PDL2/JAK2 co-amplification, and other signaling pathways (such Concluding remarks as AP-183 and MEK/ERK/MAPK signaling135 which upregulate In this article, we reviewed the studies on PD-1/PD-L1 expression PD-L1 expression) in cHL are unknown. and therapeutic targeting in B-cell lymphomas and discussed variable roles of PD-1/PD-L1 signaling in different lymphoma In B-NHL (except for PMBCL), PDL1/PDL2 genetic alterations entities. In phase 2 trials, the efficacy of anti-PD-1 monotherapy seemed not to be associated with PD-L1 expression or ORR was remarkable in cHL, moderate in PMBCL (comparable to that to nivolumab.107 In CLL with RT, baseline PD-L1 expression (mainly in melanoma) and CLL with RT, and lacking in CLL without RT; on histiocytes) was associated with response to pembrolizumab in combination therapies have shown encouraging results in FL and a phase 2 study. PD-1 expression, which was found mainly on CLL cHL. Anti-PD-1 monotherapy also showed promise in DLBCL in a B cells, was not statistically associated with treatment response76; phase 1 study. PCNSL and PTL, which frequently have 9p24.1 whether PD-1 expression on CLL cells and PD-1 expression on genetic alterations, may also be suitable for anti-PD-1 immu- T cells have differential associations with treatment response was notherapy according to a retrospective analysis. not determined. T-cell infiltration, 9p24.1 alterations, EBV positivity, and MSI status were not associated with clinical response in In cHL, 9p24.1 amplification and tumor PD-L1 H scores showed CLL with RT.76 In FL, baseline PD-L1 expression in T cells and significant but not absolute correlation with ORR. In CLL with monocytes in peripheral blood samples correlated with response RT, PD-L1 expression in histiocytes correlated with ORR in a to pidilizumab (the antibody specificity has been questioned) and small-scale analysis. However, PD-L1 expression may not be rituximab combination therapy.106 thesoleefficacy determinant. cHL also has an inflammatory microenvironment, strong CD80/CD86 and PD-L2 expression, Data on predictive biomarkers in DLBCL are currently unavail- and increased JAK/STAT signaling in Reed-Sternberg cells.29 able. Because knowledge of PD-128 and PD-L1 (Figure 380,122,123) CD28 is strongly or moderately expressed on HL-infiltrating expression in a large number of pretreatment samples may be T cells but only weakly expressed on DLBCL-infiltrating helpful for understanding responses, we examined PD-L1 and Tcells.30,138,139 CLL cells express multiple inhibitory ligands53 PD-1 expression in EBV2 DLBCL samples and performed cor- but have no or low CD80/CD86 expression.29,32,34 In addition, relative studies (preliminary data). We found that tumor PD-L1 immunologic synapse formation and T-cell metabolism are im- expression compared with PD-1 expression had a much stronger paired in CLL,53,100 and subsets of CD81 T cells are associated with correlation with CD81 T-cell infiltration, whereas the correlation a replicative senescence phenotype.69 Improved understanding of PD-L1 with PD-1 expression showed oscillation-like patterns of these mechanisms will lead to the development of novel (Figure 4), explaining the contradictory observations in previous predictive markers and combination strategies. Vigorous bio- studies.57,58,61 The mechanisms underlying these correlations marker and immune monitoring studies are underway, and we may include that activated T cells express PD-1 and produce can envision that further benefit will be brought to more patients IFN-g, which induces PD-L1, whereas high levels of PD-1 with B-cell lymphoma.

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Table 4. Biomarker studies for PD-1 blockade and pidilizumab therapy in B-cell lymphoma

Drug and Reference lymphoma type Phase ORR (%) CR (%) Biomarker/immune monitoring

PD-1 blockade in HL 20 Nivolumab in R/R 1 87 17 All 10 tumors examined had PDL1/PDL2 copy cHL number alterations and PD-L1, PD-L2, and STAT3 expression; TILs largely expressed low levels of PD-1. 21 Pembrolizumab in 1b 65 16 High prevalence (94%) of PD-L1 positivity in R/R cHL that lymphoma samples; treatment increased progressed after T- and NK-cell numbers in the peripheral blood brentuximab associated with IFN-g, TCR, and expanded vedotin immune signaling gene signatures; however, all these changes were not predictive for treatment response. 22 Nivolumab in R/R 2 66.3 9 PD-L1 expression on malignant Reed-Sternberg cHL that failed to cells, but not PD-L1 expression on inﬁltrating respond to auto- normal cells, was associated with response. HSCT and brentuximab vedotin 23 Pembrolizumab in 2 69 22.4 High prevalence of PD-L1 positivity in R/R cHL that lymphoma samples evaluated by staining progressed after intensity, membrane staining, and histiocyte auto-HSCT and/ score; 1 PD-L12 patient had progression; or brentuximab clinical activity was also seen in patients with vedotin low PD-L1 expression.

PD-1 blockade in B-NHL 107 Nivolumab in R/R 1b FL, 40; DLBCL, FL, 10; DLBCL, PDL1/PDL2 genetic alterations were rare and NHL and MM 36; other NHL, 18; other seemed not to correlate with ORR. 0; MM, 4 NHL, 0; MM, 4 76 Pembrolizumab in 2 44 for CLL with 11 for CLL with Increased PD-L1 expression in the pretreatment R/R CLL with RT RT; 0 for RT; 0 for tumor specimens (mostly from the tumor and relapsed relapsed CLL relapsed CLL microenvironment) was associated with CLL response; increased PD-1 expression had a trend of association with response but it was not statistically signiﬁcant; MSI status, EBV1, and baseline T cells in tumor specimens or peripheral blood were not associated with responses; none of the tested patients had 9p24 copy number gain or ampliﬁcation.

Pidilizumab studies 104 Advanced NHL, 1 55.5 11 During treatment, no changes in CD691 HL, CLL, and MM lymphocytes and levels of IFN-g or tumor necrosis factor-a were observed; a signiﬁcant decrease in CD81 lymphocytes was noted in patients who received 3 mg/kg. 105 CR/PR DLBCL after 2 51 Treatment resulted in increases in absolute auto-HSCT numbers of circulating memory T cells and (including 4 PD-L1-bearing helper T cells and monocytes. patients with PMBCL) 106 Relapsed FL 2 66 52 Baseline PD-L1 (but not PD-1 or PD-L2) expression in peripheral blood CD41, CD81, and CD141 cells correlated with ORR but not PFS; gene signatures at baseline indicating T-cell activation and effector T-cell (but not Treg) function in pretreatment tumor biopsies, as well as baseline signatures indicating function of effector T cells (PD-1intCXCR5int or low low PD-1 CXCR5 ) rather than TFH cells (PD-1highCXCR5high), predicted increased PFS; increased T-cell activation gene expression signatures after treatment with pidilizumab correlated with prolonged PFS.

CR, complete remission; int, intermediate; MM, multiple myeloma; TCR, T-cell receptor.

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A P = 0.0008 P = 0.0032 100 100

80 80 (%) (%) + +

60 60 CD4 CD8 + +

CD3 40 CD3 40 + +

20 20 PD-1 PD-1

0 0 PD-L1– PD-L1+ PD-L1– PD-L1+ CD20+ CD20+ CD20+ CD20+ n = 20 n = 376 n = 20 n = 376 B P = 0.0051 P = 0.0066 100 100

80 80 (%) (%) + +

60 60 CD4 CD8 + + CD3 CD3 40 40 + +

20 20 PD-1 PD-1

0 0 PD-L1low PD-L1high PD-L1low PD-L1high CD20+ CD20+ CD20+ CD20+ n = 345 n = 51 n = 345 n = 51 C

100 100

80 80 (%) (%) + + 60 60 CD8 CD4 + + 40 40 CD3 CD3 + + 20 20 PD-1 PD-1

0 PD-L1+CD20+ 0 PD-L1+CD20+ (%) (%) [0-1] (1-10] (10-30] (30-50] >50 [0-1] (1-10] (10-30] (30-50] >50 n=20 n=185 n=140 n=33 n=18 n=20 n=185 n=140 n=33 n=18

2500 5000

2000 4000 (#) (#) + + 1500 3000 CD4 CD8 + + 1000 2000 CD3 CD3 + + 500 1000 PD-1 PD-1 0 0 + + + + -500 PD-L1 CD20 PD-L1 CD20 (%) (%) [0-1] (1-10] (10-20] (20-30] (30-50] >50 [0-1] (1-10] (10-20] (20-30] (30-50] >50 n=20 n=185 n=101 n=39 n=33 n=18 n=20 n=185 n=101 n=39 n=33 n=18 D E 5000

4000 4000 (#) (#) + + 3000 CD3 CD3

2000 + + 2000 PD-1 PD-1 1000 0 + + PD-L1 CD68 0 (%) [0-10] (10-30] (30-40] (40-70] >70 0 1000 2000 3000 4000 n=31 n=105 n=89 n=159 n=12 PD-L1+ (#)

Figure 4. Correlation between PD-1 and PD-L1 expression in DLBCL. (A) At a low cutoff for PD-L1 positivity on CD201 lymphoma cells, the PD-L11 group (95% of the cohort) had a significantly higher mean level of PD-1 expression than the PD-L1– group. (B) At a 30% cutoff for PD-L1high on CD201 lymphoma cells (according to Kiyasu et al57), the PD-L1high group (12.9% of the cohort) had a significantly lower mean level of PD-1 expression than the PD-L1low group. (C) PD-L1 levels on lymphoma cells were further stratified and correlated to PD-11 levels (%) and absolute PD-11 T cell numbers in the tumor microenvironment. (D) PD-L1 levels (%) on CD681 cells were stratified and correlated to absolute PD-11 T-cell numbers in the same tissues. (E) Dot plot for total PD-L11 and PD-11 cell counts in DLBCL tissues. Each dot represents the data for 1 patient.

PD-1 EXPRESSION AND BLOCKADE IN B-CELL LYMPHOMAS blood® 4 JANUARY 2018 | VOLUME 131, NUMBER 1 79 From www.bloodjournal.org at DUKE MEDICAL LIBRARY on September 21, 2019. For personal use only.

Acknowledgments Authorship This work was supported by National Institutes of Health, National Cancer Contribution: All authors conceived of and designed the study and wrote Institute grants R01CA138688, R01CA187415, and 1RC1CA146299 (K.H.Y.). and approved the article. This work was also partially funded by National Cancer Institute and National Institutes of Health grants P50CA136411 and P50CA142509, Conﬂict-of-interest disclosure: The authors declare no competing ﬁ- and by MD Anderson Cancer Center Support Grant CA016672. Z.Y.X.-M. nancial interests. and K.H.Y. are supported by The University of Texas MD Anderson Cancer Center Institutional Research and Development Fund, Gun- Correspondence: Ken H. Young, The University of Texas MD Anderson dersen Lutheran Medical Foundation Award, Hagemeister Lymphoma Cancer Center, Department of Hematopathology, 1515 Holcombe Blvd, Foundation Award, and the University Cancer Foundation via the Sister Houston, TX 77030-4009; e-mail: [email protected]. Institution Network Fund at The University of Texas MD Anderson Cancer Center. J.Z. is supported in part by the Key Program of Na- tional Natural Science Funds of China (81230052 and 81630006). K.H.Y. also receives research support from Roche Molecular System, Gilead Footnotes Sciences, Seattle Genetics, Dai Sanyo, Adaptive Biotechnology, Submitted 5 July 2017; accepted 28 October 2017. Prepublished online Incyte Pharmaceutical, HTG Molecular Diagnostics and Perfectgen as Blood First Edition paper, 8 November 2017; DOI 10.1182/blood- Diagnostics. 2017-07-740993.

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PD-1 EXPRESSION AND BLOCKADE IN B-CELL LYMPHOMAS blood® 4 JANUARY 2018 | VOLUME 131, NUMBER 1 83 From www.bloodjournal.org at DUKE MEDICAL LIBRARY on September 21, 2019. For personal use only.

2018 131: 68-83 doi:10.1182/blood-2017-07-740993 originally published online November 8, 2017

PD-1 expression and clinical PD-1 blockade in B-cell lymphomas

Zijun Y. Xu-Monette, Jianfeng Zhou and Ken H. Young

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