PD-L1 Expression In Lymphoma Research Articles

Precision in PD-L1 Assessment in Diffuse Large B Cell Lymphoma: Greater Biological Insight Using in Situ Hybridization Approach

PD-L1 expression in diffuse large B-cell lymphoma (DLBCL) has been associated with the activated B-cell (ABC) type, gains/ amplifications at the PD-L1/ 9p24 locus, and worse clinical outcome in the setting of conventional (R-CHOP) chemotherapy. Most of the prior studies have evaluated PD-L1 expression by immunohistochemistry (IHC), utilizing a variety of antibody clones and with various cut-offs for determining positivity. IHC methods for evaluating PD-L1 expression are inherently suboptimal due to the differing sensitivities/ specificities of the various clones and staining platforms and the lack of well-established interpretive criteria. Moreover, quantifying levels of PD-L1 expression by percent of positive cells and/or staining intensity are difficult and highly subjective. Alternative, highly sensitive and quantitative approaches for studying PD-L1 expression are available (e.g. gene expression profiling, flow cytometry), but these methods lack the ability to distinguish PD-L1 expression in lymphoma cells from those of the surrounding immune microenvironment.RNA-based, in situ hybridization on formalin-fixed tissues is a sensitive, specific and more quantitative approach for determining levels of expression on morphologically preserved tissue. We analyzed 69 of DLBCL cases by RNAScope in situ hybridization (Advanced Cell Diagnostics) on an automated Leica platform. PD-L1 expression was quantitated in lymphoma cells using established scoring guidelines from 0 to 4 , where 0 corresponds to no RNA expression detected or only one hybridization signal and 4 corresponds to 10 or more signals per cell. From 69 cases of DLBCLs studied 45% had the score of 0, 17% score of 1, 23% score of 2, 6% score of 3 and 9% score of 4. Results were correlated with IHC (using a variety of commercial antibodies), cell of origin classification and gains/ losses at the 9p24 locus (PD-L1 locus).We observed that PD-L1 expression quantified by RNAScope highly correlated with IHC (p<0.0001, Chi-square test) method. In the lower range of PD-L1 expression, RNAscope had higher sensitivity and dynamic range with 23 cases demonstrating no PD-L1 expression using IHC (score of 0) revealing low levels of amplification using RNAscope: with 15 cases at score 0, 7 cases at score 1 and 1 case at score 2. We also observed that high RNAScope scores categorized as high (3,4) were strongly correlated with non-GCB type and also with samples showing gain of 9p24 (p=0.0061 Chi-square test, p=0.0002, Chi-square test respectively). Interestingly, PD-L1 expression based protein detection using IHC correlated with EBV infection (p=0.008 Chi-square test), while RNAscope score demonstrated no significant correlation (p=0.6 Chi-square test). Comparison of IHC scores and RNAscope scores in EBV positive cases demonstrated that PD-L1 overexpression was not linked to 9p24 amplification, thus suggesting a different biological mechanism of PD-L1 overexpression in EBV-associated cases.These findings shed additional light into potential mechanism of PD-L1 overexpression in DLBCLs and offer a complimentary and highly sensitive practical approach of assessing PD-L1 overexpressing tumors in FFPE tissue. DisclosuresNo relevant conflicts of interest to declare.

MYC inhibition increases PD-L1 expression induced by IFN-γ in hepatocellular carcinoma cells

The effectiveness of immunotherapy targeting the immune checkpoint PD-L1/PD-1 pathway highlights importance of elucidating the regulatory mechanisms of PD-L1 expression in cancer cells. Previous studies demonstrate that oncogene MYC up-regulates PD-L1 expression in lymphomas. In the present study, we investigated the regulatory role of MYC in the PD-L1 expression induced by IFN-γ in HCC cells. Unexpectedly, knockdown of MYC expression using siRNA assay increased the inducible expression of PD-L1 both at mRNA and protein levels. Mechanistically, the inhibition of MYC elevated expression of STAT1, a critical component of IFN-γ signaling pathway, leading to the elevation of PD-L1 expression in HCC cells exposed to IFN-γ. These results suggest that MYC may down-regulate PD-L1 expression in the context of HCC. This study implicates that a combination therapy targeting MYC function and PD-L1/PD-1 pathway might be effective for treatment of HCC.

Evaluation of the diagnostic and prognostic value of PDL1 expression in Hodgkin and B-cell lymphomas

Activation of the programmed death 1 (PD1)/PD1 ligand (PDL1) pathway is important for tumor cells to escape from immune control. The clinical efficacy of therapeutic modulation of the PD1-PDL1 pathway has been recently shown in classical Hodgkin lymphoma (cHL), but little is known about the frequency and diagnostic and prognostic importance of PDL1 expression in lymphomas. The available anti-PDL1 antibody clones E1L3N and SP142 were compared, and a large cohort of Hodgkin lymphomas (n=280) and B-cell lymphomas (n=619) was examined for PDL1 using E1L3N. The results were correlated with the expression of other phenotypic markers, interphase fluorescence in situ hybridization data of the 9p24.1 region (PDL1 locus), and the clinical outcome. PDL1 was expressed on more than 5% of tumor cells in 70% of cHL, 54% of nodular lymphocyte-predominant Hodgkin lymphoma, and 35% of primary mediastinal B-cell lymphomas; in the latter, PDL1 expression correlated with PDL1 gains (ρ=0.573). PDL1 was expressed in 31% of primary diffuse large B-cell lymphomas (DLBCLs), whereas most other entities did not express PDL1. In cHL, expression of PDL1 correlated with increased numbers of granzyme+ T cells (ρ=0.251) and CD68+ macrophages (ρ=0.221) but with decreased numbers of FoxP3+ T cells (ρ=0.145). In activated B-cell-like DLBCL, PDL1 positively correlated with PD1+ T cells, whereas an inverse correlation with FoxP3+ T cells was seen in the germinal center B-cell-like DLBCL. PDL1 expression can be diagnostically valuable in some gray zones around DLBCL and cHL; it identifies an "immune escape" cluster of cHL and activated B-cell-like DLBCL with increased granzyme+ and PD1+ T cells and macrophages and decreased regulatory T cells.

PD-L1 and PD1 Expression in Lymphomas

BackgroundBinding of PD-1 with ligands (e.g. PD-L1) expressed on tumor cells or native antigen-presenting cells results in down-regulation of effector T cell function and represents a potent mechanism of immune evasion across a number of solid tumors and lymphomas especially classical Hodgkin lymphoma (CHL) and aggressive virus-associated B-cell lymphomas. Several biomarkers (different antibodies, gene amplifications and mutations) are being investigated for identifying patients who may benefit from PD-1 checkpoint blockade therapies. It is well known that tumors which strongly express PD-L1 have a high clinical response rate to PD1 blockade. We aimed at identifying the expression of PD-L1 and PD1 in a diverse group of lymphomas. There are several antibodies used to detect PD-L1 expression, however the concordance between these antibodies is not known. We have previously tested concordance of 2 anti-PD-L1 antibodies in histiocytoses [1], but data in lymphomas are lacking. In the current study, we measured PD-L1 expression in lymphoma biopsy samples using 3 antibodies to identify if there was concordance in expression patterns of PD-L1 in various subtypes of lymphomas.MethodsFormalin fixed paraffin embedded tissues from 37 patients with lymphomas of B- and T-cell lineages (both EBV-positive and negative) were investigated for the expression of PD-1 (MRQ-22 antibody) and PD-L1 (MAB1561, SP142 and SP263) using automated immunohistochemical methods. Expression of PD-L1 on 5% or more lymphoma cells was considered positive.ResultsNeoplastic cells expression of PD-L1 was identified in 13/37 cases (35% of all cases). The strongest (3+/>50% cells) and consistent (4/4 cases) expression was observed in Hodgkin RS cells of CHL (2/2 nodular sclerosis and 2/2 lymphocyte depleted). Other B-cell lineage lymphomas positive for expression of PD-L1 included diffuse large B-cell lymphomas (5/9 DLBCL) and lymphomatoid granulomatosis (1/1 LYG). Small lymphocytic lymphoma (n=3), splenic marginal zone lymphoma (n=2) and follicular lymphomas (n=8) were all negative. One of four mantle cell lymphomas (MCL) was borderline (5% of cells) positive. One NK/T-cell lymphoma was strongly (>50% cells) positive; four peripheral T-cell lymphomas (PTCL) and one T cell lymphoblastic lymphoma were negative. PD-L1 expression was observed in both EBV- positive (2/4) and negative (2/3) malignancies. Concordance between any 2 anti-PD-L1 antibodies varied between 86 and 97% (Table 1) with all 3 Abs agreeing in 82%. PD-1 was expressed on malignant cells in 3 cases (2 PTCL and 1 DLBCL); reactive PD-1+ T-lymphocytes were absent in LD CHL and variably present in all other lymphomas.ConclusionsOver-expression of PD-L1was detected in classic HD, diffuse large B cell lymphoma, NK/T cell lymphoma and lymphomatoid granulomatosis. A high concordance in detection of PD-L1 between three antibodies indicates little need for companion diagnostic kits, but correlation between clinical responses and level of PDL1 and PD1 expression in lymphoma should be established in future clinical trials.Table 1Concordance between the PD-L1 antibodies.Antibodies (n)MAB1561SP142SP263MAB1561 (23)--20/23 (86%)SP142 (37)----34/35 (97%)SP263 (35)19/22 (86%)----

Update on checkpoint blockade therapy for lymphoma.

Although cancer cells express antigens recognizable to the immune system, tumors employ a number of diverse mechanisms aimed at subverting the host anti-tumor immune response. Tumor immune evasion pathways have been most thoroughly studied in solid tumors. However, emerging data has demonstrated that malignancies of hematopoietic origin are also able to co-opt their local environment in order to escape immune attack. Activated T cells upregulate negative costimulatory receptors, such as programmed death-1 (PD-1) and cytotoxic lymphocyte antigen-4 (CTLA-4). Engagement of PD-1 or CTLA-4 with ligands expressed on tumor cells or professional antigen presenting cells results in down-regulation of effector T cell function and represents a potent mechanism of immune evasion across a number of human cancers. Antibodies which block PD-1 / PD-L1 interactions have demonstrated remarkable activity in a number of solid tumor subtypes. Interestingly, recent data have demonstrated that in select subtypes of Hodgkin (HL) and non-Hodgkin lymphoma (NHL), the PD-1 ligands are over-expressed due to a genetic amplification of the loci encoding them. Other mechanisms of PD-L1 over-expression in lymphoma have also been elucidated. Reports from early-phase clinical trials of PD-1 blockade have demonstrated remarkable effectiveness in HL, and also appear active against some NHLs. We review the mechanisms of PD-L1 expression in lymphoma and also the early results of anti-PD-1 therapy in this disease.

Abstract IA6: Immune escape mechanisms in lymphoid malignancies.

Abstract Certain lymphoid malignancies utilize potent mechanisms to evade host anti-tumor immune responses including the AP-1-dependent overexpression of Galectin-1 (Gal1) and the genetic amplification and overexpression of the PD-1 ligands. Gal1 recognizes specific cell surface glycans (Gal1 β1 GlcNAc [N-acetyllactosamine] units on the branches of N- or O-linked glycans) on receptors such as CD45, CD43 and CD7 and induces the apoptosis of select T cell subsets – Th1, Th17 and cytotoxic T cells. Th2 cells and regulatory T (Treg) cells have different patterns of sialylation of cell surface glycoproteins, lack Gal1 ligands and resist Gal1-induced death. We recently found that malignant Reed-Sternberg (RS) cells in classical Hodgkin Lymphoma (cHL) and EBV-transformed malignant B cells in post-transplant lymphoproliferative disorders (PTLD) overexpress the secreted carbohydrate-binding lectin, Galectin-1 (Gal1). Gal1 overexpression by Hodgkin RS cells and EBV-transformed B cells promotes the immunosuppressive Th2/Treg-predominant, Th1/cytotoxic T cell-deficient micro-environment in cHL and PTLD. In both of these lymphomas with known constitutive activation of AP-1, Gal1 expression is driven, in large part, by an AP-1–dependent Gal1 enhancer. The AP-1-dependent expression of Gal1 is a useful diagnostic marker and circulating Gal1 levels have prognostic value. In addition, neutralizing Gal1 antibodies have promising immunomodulatory activity in in vitro assays and tumor models. Engagement of PD-1 receptors triggers T-cell “exhaustion” and the progressive loss of effector T-cell function and proliferative capacity. In certain cancers, the tumor cell expression of PD-1 ligands inhibits T-cell activation and promotes the apoptosis of tumor-specific T cells. We recently integrated high-resolution copy number data with transcriptional profiles and identified the PD-1 ligands, PD-L1 and PD-L2, as key targets of the 9p24.1 amplification in cHL and a related lymphoid malignancy, primary mediastinal large B-cell lymphoma (MLBCL). PD-1 ligand gene amplification was associated with increased protein expression in both lymphoid malignancies. In cHLs and MLBCLs, the extended 9p24.1 amplification region also included the JAK2 locus. JAK2 amplification increased JAK2 protein expression and activity and specifically induced PD-1 ligand transcription. Chemical inhibition of JAK2 decreased PD-1 ligand expression. These findings defined 9p24.1 amplification as a disease-specific structural alteration that increased both the gene dosage of PD-1 ligands and their induction via JAK2. Like Gal1, PD-L1 includes an AP-1-dependent enhancer which augments PD-L1 expression in lymphomas with constitutive activation of AP-1. In addition, Epstein-Barr virus (EBV) induces PD-L1 expression via AP-1 and EBV+ cHLs with diploid 9p24.1 and the majority of EBV+ PTLDs express detectable PD-L1. Therefore, AP-1- and EBV-driven malignancies with normal 9p24.1 copy numbers, including cHLs and most PTLDs, may also be amenable to PD-1 blockade. Taken together, these studies identify the PD-1 axis and Gal1 as promising therapeutic targets in certain lymphoid malignancies and define genetic bases for increased expression of these immunoregulatory molecules. For these reasons, clinical evaluation of PD-1 blockade alone and in association with JAK2 inhibition is planned and assessment of antibody-mediated Gal1 blockade is underway. Citation Format: Margaret A. Shipp. Immune escape mechanisms in lymphoid malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA6.