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Supplementary Table from Clinical Impact of Immune Cells and Their Spatial Interactions in Diffuse Large B-Cell Lymphoma Microenvironment

Supplementary Figure from Clinical Impact of Immune Cells and Their Spatial Interactions in Diffuse Large B-Cell Lymphoma Microenvironment

Purpose:Tumor-infiltrating immune cells have prognostic significance and are attractive therapeutic targets. Yet, the clinical significance of their spatial organization and phenotype in diffuse large B-cell lymphoma (DLBCL) is unclear.Experimental Design:We characterized T cells, macrophages, and their spatial interactions by multiplex IHC (mIHC) in 178 patients with DLBCL and correlated the data with patient demographics and survival. We validated the findings on gene expression data from two external DLBCL cohorts comprising 633 patients.Results:Macrophage and T-cell contents divided the samples into T cell–inflamed (60%) and noninflamed (40%) subgroups. The T cell–inflamed lymphoma microenvironment (LME) was also rich in other immune cells, defining immune hot phenotype, which did not as such correlate with outcome. However, when we divided the patients according to T-cell and macrophage contents, LME characterized by high T-cell/low macrophage content or a corresponding gene signature was associated with superior survival [5-year overall survival (OS): 92.3% vs. 74.4%, P = 0.036; 5-year progression-free survival (PFS): 92.6% vs. 69.8%, P = 0.012]. High proportion of PD-L1- and TIM3-expressing CD163− macrophages in the T cell–inflamed LME defined a group of patients with poor outcome [OS: HR = 3.22, 95% confidence interval (CI), 1.63–6.37, Padj = 0.011; PFS: HR = 2.76, 95% CI, 1.44–5.28, Padj = 0.016]. Furthermore, PD-L1 and PD-1 were enriched on macrophages interacting with T cells.Conclusions:Our data demonstrate that the interplay between macrophages and T cells in the DLBCL LME is immune checkpoint dependent and clinically meaningful.

Supplementary Figures 1-2, Materials, Tables 1-4 and Table 7 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Supplementary Table 5 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Supplementary Table 6 from Systematic Analysis of MicroRNAs Targeting the Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is expressed in all stages of prostate cancer progression, including in castration-resistant tumors. Eliminating AR function continues to represent a focus of therapeutic investigation, but AR regulatory mechanisms remain poorly understood. To systematically characterize mechanisms involving microRNAs (miRNAs), we conducted a gain-of function screen of 1129 miRNA molecules in a panel of human prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. In this way, we defined 71 unique miRNAs that influenced the level of AR in human prostate cancer cells. RNA sequencing data revealed that the 3′UTR of AR (and other genes) is much longer than currently used in miRNA target prediction programs. Our own analyses predicted that most of the miRNA regulation of AR would target an extended 6 kb 3′UTR. 3′UTR-binding assays validated 13 miRNAs that are able to regulate this long AR 3′UTR (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371–3p, miR-421, miR-449a, miR-449b, miR-634, miR-654–5p, and miR-9). Fifteen AR downregulating miRNAs decreased androgen-induced proliferation of prostate cancer cells. In particular, analysis of clinical prostate cancers confirmed a negative correlation of miR-34a and miR-34c expression with AR levels. Our findings establish that miRNAs interacting with the long 3′UTR of the AR gene are important regulators of AR protein levels, with implications for developing new therapeutic strategies to inhibit AR function and androgen-dependent cell growth. Cancer Res; 71(5); 1956–67. ©2011 AACR.

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Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. We integrated over 8,000 transcriptomes and 2,000 samples with multilevel genomics of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival, and validated key findings experimentally. Infiltration of cytotoxic lymphocytes was associated with TP53 and myelodysplasia-related changes in acute myeloid leukemia, and activated B cell-like phenotype and interferon-gamma response in lymphoma. CIITA methylation regulating antigen presentation, cancer type-specific immune checkpoints, such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity and predicted survival. Our study provides a resource linking immunology with cancer subtypes and genomics in hematological malignancies.

The tumor microenvironment (TME) and limited immune surveillance play important roles in lymphoma pathogenesis. Here we aimed to characterize immunological profiles of diffuse large B-cell lymphoma (DLBCL) and predict the outcome in response to immunochemotherapy. We profiled the expression of 730 immune-related genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize T-cell phenotypes, including cytotoxic T cells (CD8, Granzyme B, OX40, Ki67), T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188 patients. We observed a high degree of heterogeneity at the transcriptome level. Correlation matrix analysis identified gene expression signatures with highly correlating genes, the main cluster containing genes for cytolytic factors, immune checkpoint molecules, T cells and macrophages, together named a TME immune cell signature. Immunophenotyping of the distinct cell subsets revealed that a high proportion of immune checkpoint positive T cells translated to unfavorable survival. Together, our results demonstrate that the immunological profile of DLBCL TME is heterogeneous and clinically meaningful. This highlights the potential impact of T-cell immune checkpoint in regulating survival and resistance to immunochemotherapy. (Registered at clinicaltrials.gov identifiers: NCT01502982 and NCT01325194.)

Emerging evidence indicates a major impact for the tumor microenvironment (TME) and immune escape in the pathogenesis and clinical course of classical Hodgkin lymphoma (cHL). We used gene expression profiling (n = 88), CIBERSORT, and multiplex immunohistochemistry (n = 131) to characterize the immunoprofile of cHL TME and correlated the findings with survival. Gene expression analysis divided tumors into subgroups with T cell-inflamed and -noninflamed TME. Several macrophage-related genes were upregulated in samples with the non–T cell-inflamed TME, and based on the immune cell proportions, the samples clustered according to the content of T cells and macrophages. A cluster with high proportions of checkpoint protein (programmed cell death protein 1, PD-1 ligands, indoleamine 2,3 dioxygenase 1, lymphocyte-activation gene 3, and T-cell immunoglobulin and mucin domain containing protein 3) positive immune cells translated to unfavorable overall survival (OS) (5-year OS 76% vs 96%; P = .010) and remained an independent prognostic factor for OS in multivariable analysis (HR, 4.34; 95% CI, 1.05-17.91; P = .043). cHL samples with high proportions of checkpoint proteins overexpressed genes coding for cytolytic factors, proposing paradoxically that they were immunologically active. This checkpoint molecule gene signature translated to inferior survival in a validation cohort of 290 diagnostic cHL samples (P < .001) and in an expansion cohort of 84 cHL relapse samples (P = .048). Our findings demonstrate the impact of T cell- and macrophage-mediated checkpoint system on the survival of patients with cHL.

Primary testicular lymphoma is a rare lymphoma entity, yet it is the most common testicular malignancy among elderly men. The majority of the cases represent non-germinal center B-cell-like (non-GCB) diffuse large B-cell lymphoma (DLBCL) with aggressive clinical behavior and a relatively high relapse rate. Due to the rareness of the disease, no randomized clinical trials have been conducted and the currently recognized standard of care is based on retrospective analyses and few phase II trials. During recent years, the tumor microenvironment (TME) and tumor-related immunity have been the focus of many tumor biology studies, and the emergence of targeted therapies and checkpoint inhibitors has significantly modulated the field of cancer therapies. Testicular DLBCL (T-DLBCL) is presented in an immune-privileged site of the testis, and the roles of NF-κB pathway signaling, 9p24.1 aberrations, and tumor-infiltrating immune cells, especially immune checkpoint expressing lymphocytes and macrophages, seem to be unique compared to other lymphoma entities. Preliminary data on the use of immune checkpoint inhibitors in the treatment of T-DLBCL are promising and more studies are ongoing.

Objectives Testicular diffuse large B-cell lymphoma (T-DLBCL) is a rare and aggressive extranodal lymphoma. We have previously shown that high content of tumor-infiltrating lymphocytes (TILs) and PD-1 expressing TILs associate with better survival in T-DLBCL. In this study, we have further characterized distinct TIL subtypes and their proportions in association with patient demographics and survival. Methods We used multiplex immunohistochemistry to characterize TIL phenotypes, including cytotoxic T-cells (CTLs; CD8(+), OX40(+), Granzyme B+, Ki-67(+), LAG-3(+), TIM-3(+), PD-1(+)), CD4(+)T-cells (CD3(+), CD4(+), TIM-3(+), LAG-3(+)), regulatory T-cells (Tregs; CD3(+), CD4(+), FoxP3(+)), and T helper 1 cells (Th1; CD3(+), CD4(+), T-bet(+)) in 79 T-DLBCLs, and correlated the findings with patient demographics and outcome. Results We observed a substantial variation in TIL phenotypes between the patients. The most prominent CD8(+)TILs were Ki-67(+)and TIM-3(+)CTLs, whereas the most prominent CD4(+)TILs were FoxP3(+)Tregs. Despite the overall favorable prognostic impact of high TIL content, we found a subpopulation of T-bet(+)FoxP3(+)Tregs that had a significant adverse impact on survival. Lower content of CTLs with activated or exhausted phenotypes correlated with aggressive clinical features. Conclusions Our results demonstrate significant variation in TIL phenotypes and emphasize the adverse prognostic impact of Tregs in T-DLBCL.

Different miRNA profiling protocols and technologies introduce differences in the resulting quantitative expression profiles. These include differences in the presence (and measurability) of certain miRNAs. We present and examine a method based on quantile normalization, Adjusted Quantile Normalization (AQuN), to combine miRNA expression data from multiple studies in breast cancer into a single joint dataset for integrative analysis. By pooling multiple datasets, we obtain increased statistical power, surfacing patterns that do not emerge as statistically significant when separately analyzing these datasets. To merge several datasets, as we do here, one needs to overcome both technical and batch differences between these datasets. We compare several approaches for merging and jointly analyzing miRNA datasets. We investigate the statistical confidence for known results and highlight potential new findings that resulted from the joint analysis using AQuN. In particular, we detect several miRNAs to be differentially expressed in estrogen receptor (ER) positive versus ER negative samples. In addition, we identify new potential biomarkers and therapeutic targets for both clinical groups. As a specific example, using the AQuN-derived dataset we detect hsa-miR-193b-5p to have a statistically significant over-expression in the ER positive group, a phenomenon that was not previously reported. Furthermore, as demonstrated by functional assays in breast cancer cell lines, overexpression of hsa-miR-193b-5p in breast cancer cell lines resulted in decreased cell viability in addition to inducing apoptosis. Together, these observations suggest a novel functional role for this miRNA in breast cancer. Packages implementing AQuN are provided for Python and Matlab: https://github.com/YakhiniGroup/PyAQN., Author summary This work demonstrates a practical approach to the joint-analysis of multiple miRNA expression profiling datasets acquired with different measurement technologies. The use of different platforms in miRNA profiling can lead to major differences in results. In particular, some miRNA species are less amenable to detection and quantification by certain platforms or designs. Our approach, termed AQuN, combines quantile normalization with special attention to missing entities, to normalize miRNA expression across datasets, technologies, designs and platforms. As we show, our proposed approach uncovers patterns of interest that would not have emerged as statistically significant when analyzing the datasets individually or with other standard-practice normalization methods. Amongst our findings, we noted a previously undocumented miRNA that is significantly over-expressed in samples from estrogen-receptor positive breast cancer patients as compared to samples from estrogen-receptor negative patients. We further investigated this miRNA, hsa-miR-193b-5p, and experimentally show, in cell lines, that its expression level impacts the viability of tumor cells. AQuN is available to the community in the form of Python and Matlab packages. The joint-processed data is also made available for further investigation.

Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68+, median 32%, M2 type CD163+, median 22%), whereas the proportion of TAMs expressing IDO-1 is lower (CD68+, median 5.5%, CD163+, median 1.4%). A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68+), or from CD163+ TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1+ and IDO-1+ TAMs remain independent prognostic factors for freedom from treatment failure (PD-L1+CD68+/CD68+, HR = 2.63, 95% CI 1.17&ndash, 5.88, p = 0.019, IDO-1+CD68+/CD68+, HR = 2.48, 95% CI 1.03&ndash, 5.95, p = 0.042). In contrast, proportions of PD-L1+ tumor cells, all TAMs or PD-L1&minus, and IDO-1&minus, TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL.

Primary testicular lymphoma is a rare and aggressive lymphoid malignancy, most often representing diffuse large B-cell lymphoma histologically. Tumor-associated macrophages and tumor-infiltrating lymphocytes have been associated with survival in diffuse large B-cell lymphoma, but their prognostic impact in primary testicular lymphoma is unknown. Here, we aimed to identify macrophages, their immunophenotypes and association with lymphocytes, and translate the findings into survival of patients with primary testicular lymphoma. We collected clinical data and tumor tissue from 74 primary testicular lymphoma patients, and used multiplex immunohistochemistry and digital image analysis to examine macrophage markers (CD68, CD163, and c-Maf), T-cell markers (CD3, CD4, and CD8), B-cell marker (CD20), and three checkpoint molecules (PD-L1, PD-L2, and PD-1). We demonstrate that a large proportion of macrophages (median 41%, range 0.08-99%) and lymphoma cells (median 34%, range 0.1-100%) express PD-L1. The quantity of PD-L1+CD68+ macrophages correlates positively with the amount of PD-1+ lymphocytes, and a high proportion of either PD-L1+CD68+ macrophages or PD-1+CD4+ and PD-1+CD8+ T-cells translates into favorable survival. In contrast, the number of PD-L1+ lymphoma cells or PD-L1- macrophages do not associate with outcome. In multivariate analyses with IPI, PD-L1+CD68+ macrophage and PD-1+ lymphocyte contents remain as independent prognostic factors for survival. In conclusion, high PD-L1+CD68+ macrophage and PD-1+ lymphocyte contents predict favorable survival in patients with primary testicular lymphoma. The findings implicate that the tumor microenvironment and PD-1. PD-L1 pathway have a significant role in regulating treatment outcome. They also bring new insights to the targeted therapy of primary testicular lymphoma.

Background The epigenome plays a key role in cancer heterogeneity and drug resistance. Hence, a number of epigenetic inhibitors have been developed and tested in cancers. The major focus of most studies so far has been on the cytotoxic effect of these compounds, and only few have investigated the ability to revert the resistant phenotype in cancer cells. Hence, there is a need for a systematic methodology to unravel the mechanisms behind epigenetic sensitization. Results We have developed a high-throughput protocol to screen non-simultaneous drug combinations, and used it to investigate the reprogramming potential of epigenetic inhibitors. We demonstrated the effectiveness of our protocol by screening 60 epigenetic compounds on diffuse large B-cell lymphoma (DLBCL) cells. We identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with doxorubicin and rituximab. These two classes of epigenetic inhibitors achieved sensitization by disrupting DNA repair, cell cycle, and apoptotic signaling. The data used to perform these analyses are easily browsable through our Results Explorer. Additionally, we showed that these inhibitors achieve sensitization at lower doses than those required to induce cytotoxicity. Conclusions Our drug screening approach provides a systematic framework to test non-simultaneous drug combinations. This methodology identified HDAC and HMT inhibitors as successful sensitizing compounds in treatment-resistant DLBCL. Further investigation into the mechanisms behind successful epigenetic sensitization highlighted DNA repair, cell cycle, and apoptosis as the most dysregulated pathways. Altogether, our method adds supporting evidence in the use of epigenetic inhibitors as sensitizing agents in clinical settings.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease whose personalized clinical management requires robust molecular stratification. Here, we show that somatic hypermutation (SHM) patterns constitute a marker for DLBCL molecular classification. The activity of SHM mutational processes delineated the cell of origin (COO) in DLBCL. Expression of the herein identified 36 SHM target genes stratified DLBCL into four novel SHM subtypes. In a meta-analysis of patients with DLBCL treated with immunochemotherapy, the SHM subtypes were significantly associated with overall survival (1642 patients) and progression-free survival (795 patients). Multivariate analysis of survival indicated that the prognostic impact of the SHM subtypes is independent from the COO classification and the International Prognostic Index. Furthermore, the SHM subtypes had a distinct clinical outcome within each of the COO subtypes, and strikingly, even within unclassified DLBCL. The genetic landscape of the four SHM subtypes indicated unique associations with driver alterations and oncogenic signaling in DLBCL, which suggests a possibility for therapeutic exploitation. These findings provide a biologically driven classification system in DLBCL with potential clinical applications.

Primary therapy for diffuse large B-cell lymphoma (DLBCL) is an immunochemotherapy regimen comprising rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). While R-CHOP cures 60% of the patients with DLBCL, those who do not respond or relapse have dismal prognosis, and effective treatment strategies are needed. Due to the plastic nature of the epigenome and its fundamental role in regulating cell identity, we hypothesized that reprogramming the epigenome can overcome resistance to R-CHOP. We developed a novel drug screening protocol to identify epigenetic modifiers that sensitize DLBCL cell lines to immunochemotherapy. Of the herein tested 60 epigenetic compounds, we identified several histone deacetylase (HDAC) and histone methyltransferase (HMT) inhibitors that acted synergistically with immunochemotherapy. We show that sensitization through HDAC and HMT inhibitors is achieved by dysregulating homologous recombination, a central DNA repair pathway, as well as by disrupting the cell cycle and affecting the apoptotic pathway. Epigenetic inhibitors are well-tolerated, which together with our findings support their use in combination with immunochemotherapy in patients with primary refractory and relapsed DLBCL.

Primary testicular lymphoma is a rare lymphoid malignancy, most often, histologically, representing diffuse large B-cell lymphoma. The tumor microenvironment and limited immune surveillance have a major impact on diffuse large B-cell lymphoma pathogenesis and survival, but the impact on primary testicular lymphoma is unknown. Here, the purpose of the study was to characterize the tumor microenvironment in primary testicular lymphoma, and associate the findings with outcome. We profiled the expression of 730 immune response genes in 60 primary testicular lymphomas utilizing the Nanostring platform, and used multiplex immunohistochemistry to characterize the immune cell phenotypes in the tumor tissue. We identified a gene signature enriched for T-lymphocyte markers differentially expressed between the patients. Low expression of the signature predicted poor outcome independently of the International Prognostic Index (progression-free survival: HR=2.810, 95%CI: 1.228-6.431, P=0.014; overall survival: HR=3.267, 95%CI: 1.406-7.590, P=0.006). The T-lymphocyte signature was associated with outcome also in an independent diffuse large B-cell lymphoma cohort (n=96). Multiplex immunohistochemistry revealed that poor survival of primary testicular lymphoma patients correlated with low percentage of CD3+CD4+ and CD3+CD8+ tumor-infiltrating lymphocytes (P

Background: Aggressive non-Hodgkin lymphoma (NHL) relapsing after standard first line chemotherapy represents an unmet clinical need. Currently, a phase 1/2 study with the combination of pixantrone, etoposide, bendamustine, and in CD20 positive tumors, rituximab, in patients with relapsed aggressive NHL of B- or T- cell phenotype (the PREBEN study) is ongoing. Here our aim was to molecularly characterize samples from the PREBEN trial and find clinical correlates for predicting treatment response. Methods: The profiling cohort consisted of 21 patients with pre-treatment RNA samples and clinical data. Nanostring PanCancer Pathways and PanCancer Immune profiling panels (altogether 1348 genes) were utilized for the gene expression analyses. The findings from gene expression analyses were correlated with clinical parameters. Results: Fourteen patients had diffuse large B-cell lymphoma (DLBCL), whereas seven had peripheral T-cell lymphoma (PTCL). In general, the expression of DNA replication genes distinguished DLBCL from PTCL. Additionally, gene expression analyses identified genes having differential expression based on the response to the treatment. Supervised hierarchical clustering of the ten most differentially expressed genes could separate the responding (n=4) and non-responding (n=10) DLBCL patients into two distinct subgroups (Fig. 1A). Similarly, the responding (n=3) and non-responding (n=4) PTCL patients could be separated into distinct subgroups by supervised clustering with the ten most differentially expressed genes (Fig. 1B). Conclusion: Molecular profiles of aggressive NHL are heterogeneous and may be utilized for predicting the treatment response. More detailed molecular analyses are currently ongoing. Disclosures Jørgensen: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. d'Amore:Servier: Research Funding. Leppa:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Research Funding; Bayer: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: combination of bendamustine and pixantrone for relapsed NHL

SUMMARYUnderstanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. Here, we integrated over 8,000 transcriptomes and over 1,000 samples with multilevel genomic data of hematological cancers to investigate how immunological features are linked to cancer subtypes, genetic and epigenetic alterations, and patient survival. Infiltration of cytotoxic immune cells was associated with distinct microenvironmental responses and driver alterations in different cancers, such asTP53in acute myeloid leukemia andDTX1in diffuse large B cell lymphoma. Epigenetic modification ofCIITAregulating antigen presentation, cancer type-specific immune checkpoints such as VISTA in myeloid malignancies, and variation in cancer antigen expression further contributed to immune heterogeneity. Prognostic models highlighted the significance of immunological properties in predicting survival. Our study represents the most comprehensive effort to date to link immunology with cancer subtypes and genomics in hematological malignancies, providing a resource to guide future studies and immunotherapy development.

Additional file 1. Supplementary file containing additional methods and results, as well as the user guide for the Results Explorer.

Primary testicular lymphoma is a rare and aggressive lymphoid malignancy, most often representing diffuse large B-cell lymphoma histologically. Tumor-associated macrophages and tumor-infiltrating lymphocytes have been associated with survival in diffuse large B-cell lymphoma, but their prognostic impact in primary testicular lymphoma is unknown. Here, we aimed to identify macrophages, their immunophenotypes and association with lymphocytes, and translate the findings into survival of patients with primary testicular lymphoma. We collected clinical data and tumor tissue from 74 primary testicular lymphoma patients, and used multiplex immunohistochemistry and digital image analysis to examine macrophage markers (CD68, CD163, and c-Maf), T-cell markers (CD3, CD4, and CD8), B-cell marker (CD20), and three checkpoint molecules (PD-L1, PD-L2, and PD-1). We demonstrate that a large proportion of macrophages (median 41%, range 0.08–99%) and lymphoma cells (median 34%, range 0.1–100%) express PD-L1. The quantity of PD-L1+ CD68+ macrophages correlates positively with the amount of PD-1+ lymphocytes, and a high proportion of either PD-L1+ CD68+ macrophages or PD-1+ CD4+ and PD-1+ CD8+ T cells translates into favorable survival. In contrast, the number of PD-L1+lymphoma cells or PD-L1− macrophages do not associate with outcome. In multivariate analyses with IPI, PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents remain as independent prognostic factors for survival. In conclusion, high PD-L1+ CD68+ macrophage and PD-1+ lymphocyte contents predict favorable survival in patients with primary testicular lymphoma. The findings implicate that the tumor microenvironment and PD-1 – PD-L1 pathway have a significant role in regulating treatment outcome. They also bring new insights to the targeted thera py of primary testicular lymphoma.