Single-cell transcriptomic analysis reveals that the APP-CD74 axis promotes immunosuppression and progression of testicular tumors.

Testicular tumors represent the most common malignancy among young men. Nevertheless, the pathogenesis and molecular underpinning of testicular tumors remain largely elusive. We aimed to delineate the intricate intra-tumoral heterogeneity and the network of intercellular communication within the tumor microenvironment. A total of 40,760 single-cell transcriptomes were analyzed, encompassing samples from six individuals with seminomas, two patients with mixed germ cell tumors, one patient with a Leydig cell tumor, and three healthy donors. Five distinct malignant subclusters were identified in the constructed landscape. Among them, malignant 1 and 3 subclusters were associated with a more immunosuppressive state and displayed worse disease-free survival. Further analysis identified that APP-CD74 interactions were significantly strengthened between malignant 1 and 3 subclusters and 14 types of immune subpopulations. In addition, we established an aberrant spermatogenesis trajectory and delineated the global gene alterations of somatic cells in seminoma testes. Sertoli cells were identified as the somatic cell type that differed the most from healthy donors to seminoma testes. Cellular communication between spermatogonial stem cells and Sertoli cells is disturbed in seminoma testes. Our study delineates the intra-tumoral heterogeneity and the tumor immune microenvironment in testicular tumors, offering novel insights for targeted therapy. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

T cells in testicular germ cell tumors: new evidence of fundamental contributions by rare subsets.

BACKGROUND: Immune cell infiltration is heterogeneous but common in testicular germ cell tumors (TGCT) and pre-invasive germ cell neoplasia in situ (GCNIS). Tumor-infiltrating T cells including regulatory T (Treg) and follicular helper T (Tfh) cells are found in other cancer entities, but their contributions to TGCT are unknown. METHODS: Human testis specimens from independent patient cohorts were analyzed using immunohistochemistry, flow cytometry and single-cell RNA sequencing (scRNA-seq) with special emphasis on delineating T cell subtypes. RESULTS: Profound changes in immune cell composition within TGCT, shifting from macrophages in normal testes to T cells plus B and dendritic cells in TGCT, were documented. In most samples (96%), the CD4+ T cell frequency exceeded that of CD8+ cells, with decreasing numbers from central to peripheral tumor areas, and to tumor-free, contralateral testes. T cells including Treg and Tfh were most abundant in seminoma compared to mixed tumors and embryonal carcinoma. CONCLUSION: Despite considerable heterogeneity between patients, T cell subtypes form a key part of the TGCT microenvironment. The novel finding of rare Treg and Tfh cells in human testis suggests their involvement in TGCT pathobiology, with implications for understanding tumor progression, to assess patients' prognosis, and as putative targets for personalized immunotherapy.

Leucine Zipper 4 Autoantibody: A Novel Germ Cell Tumor and Paraneoplastic Biomarker.

OBJECTIVE: This study was undertaken to describe a novel biomarker of germ cell tumor and associated paraneoplastic neurological syndrome (PNS). METHODS: Archival sera from patients with germ cell tumor-associated PNS were evaluated. We identified a common autoantigen in a human testicular cancer cell line (TCam-2) by Western blot and mass spectrometry. Its identity was confirmed by recombinant-protein Western blot, enzyme-linked immunosorbent assay (ELISA), and cell-based assay. Autoantibody specificity was confirmed by analyzing assorted control sera/cerebrospinal fluid. RESULTS: Leucine zipper 4 (LUZP4)-immunoglobulin G (IgG) was detected in 28 patients' sera, 26 of whom (93%) were men. The median age at neurological symptom onset was 45 years (range = 28-84). Median titer (ELISA) was 1:300 (1:50 to >1:6,400, normal value < 1:50). Coexistent kelchlike protein 11-IgG was identified in 18 cases (64%). The most common presenting phenotype was rhombencephalitis (17/28, 61%). Other presentations included limbic encephalitis (n = 5, 18%), seizures and/or encephalitis (n = 2, 7%), and motor neuronopathy/polyradiculopathy (n = 4, 14%). The most common malignancy among cancer-evaluated PNS patients was seminoma (21/27, 78%). Nine of the 21 seminomas detected by whole-body fluorodeoxyglucose positron emission tomography scan (43%) were extratesticular. Both female patients had ovarian teratoma. Regressed testicular germ cell tumors were found in 4 patients. Exposure of T-cell-dendritic-cell cocultures from chronic immunosuppression-naïve LUZP4-IgG-seropositive patients to recombinant LUZP4 protein evoked a marked increase in CD69 expression on both CD4+ and CD8+ T cells when compared to vehicle-exposed and healthy control cultures. INTERPRETATION: LUZP4-IgG represents a novel serological biomarker of PNS and has high predictive value for germ cell tumors. The demonstrated antigen-specific T-cell responses support a CD8+ T-cell-mediated cytotoxic paraneoplastic and antitumor potential. ANN NEUROL 2021;89:1001-1010.

Identification of novel genes in testicular cancer microenvironment based on ESTIMATE algorithm-derived immune scores.

Testicular cancer is the most common solid malignancy among young men. We downloaded data of testicular cancer patients from The Cancer Genome Atlas database to find novel genes in the testicular cancer microenviroment based on ESTIMATE algorithm-derived immune scores. A total of 156 cases of testicular cancer were included in this study and 165 cases of normal testicular tissues were used. We divided the testicular cancer patients into high- and low-score groups based on their immune scores. We identified 1,226 differentially expressed genes (fold change > 2, false discovery rate < 0.05), including 688 downregulated genes and 538 upregulated genes, between these two groups. The top Gene Ontology terms were involved in the immune response-regulating cell surface receptor signaling pathway, immune response-activating cell surface receptor signaling pathway, external side of the plasma membrane, and receptor ligand activity. By performing the Kyoto Encyclopedia of Genes and Genomes analysis, we demonstrated that cAMP signaling pathway was highly enriched among these differentially expressed genes. High expression of LINC01564, LINC02208, ODAM, RNA5SP111, and RNU6-196P were found to be associated with poor overall survival. The expression of genes was further validated by the Human Protein Atlas and only ALB and IFNG were demonstrated to be differentially expressed between testis tissue and testicular cancer tissue.

High frequency of inactivating tetraspanin C D37 mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

Paraneoplastic Progressive Downbeat Nystagmus, Ataxia and Sensorineural Hearing Loss due to the ANTI-Kelch-11 Protein Antibody.

ABSTRACT: A 45-year-old man with a history of testicular seminoma treated 8 years earlier presented with chronic progressive truncal and limb ataxia, progressive sensorineural hearing loss, and episodic vertigo. Eye movement and neuro-otology examinations showed localizing abnormalities to the bilateral cerebellar flocculus, vermis, and bilateral cerebellar hemispheres. Audiometric testing showed bilateral symmetric sensorineural hearing loss. There was a normal MRI of the brain. Cerebrospinal fluid (CSF) showed modest lymphocytic pleocytosis, and there was an elevated serum choriogonadotrophic hormone. An abdominal CT scan showed a solitary, large retroperitoneal lymph node, and histopathologic examination of the node biopsy showed granulomatous inflammation without microorganisms; eventually, immunohistochemical markers confirmed the diagnosis of metastatic seminoma. Although normal neuroimaging and inflammatory CSF reaction suggested a paraneoplastic etiology, the initial paraneoplastic antibody testing was negative. Subsequent investigation identified a positive kelch-11 protein antibody, thus confirming the paraneoplastic connection between the metastatic seminoma and the subacute neurologic-cochleovestibular syndrome.

Are Changes in the Percentage of Specific Leukocyte Subpopulations Associated with Endogenous DNA Damage Levels in Testicular Cancer Patients?

Chemoresistance of germ cell tumors (GCTs) represents an intensively studied property of GCTs that is the result of a complicated multifactorial process. One of the driving factors in this process is the tumor microenvironment (TME). Intensive crosstalk between the DNA damage/DNA repair pathways and the TME has already been reported. This study aimed at evaluating the interplay between the immune TME and endogenous DNA damage levels in GCT patients. A cocultivation system consisting of peripheral blood mononuclear cells (PBMCs) from healthy donors and GCT cell lines was used in an in vitro study. The patient cohort included 74 chemotherapy-naïve GCT patients. Endogenous DNA damage levels were measured by comet assay. Immunophenotyping of leukocyte subpopulations was performed using flow cytometry. Statistical analysis included data assessing immunophenotypes, DNA damage levels and clinicopathological characteristics of enrolled patients. The DNA damage level in PBMCs cocultivated with cisplatin (CDDP)-resistant GCT cell lines was significantly higher than in PBMCs cocultivated with their sensitive counterparts. In GCT patients, endogenous DNA damage levels above the cutoff value were independently associated with increased percentages of natural killer cells, CD16-positive dendritic cells and regulatory T cells. The crosstalk between the endogenous DNA damage level and specific changes in the immune TME reflected in the blood of GCT patients was revealed. The obtained data contribute to a deeper understanding of ongoing interactions in the TME of GCTs.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.

Immunosenescence profile and expression of the aging biomarker (p16INK4a) in testicular cancer survivors treated with chemotherapy.

BACKGROUND: Cytotoxic chemotherapy can cure advanced germ cell tumors. Nevertheless, cancer treatment may induce cellular senescence and accelerate molecular aging. The aging process implies an increase of cells expressing p16INK4a and changes in lymphocyte subpopulations. Our aim was to study the potential induction of premature immunosenescence in testicular cancer survivors (TCS) exposed to chemotherapy. METHODS: Case-control exploratory study of TCS treated with chemotherapy (≥3 BEP cycles, disease-free ≥3 months) compared with age matched healthy controls. Peripheral blood mononuclear cells were isolated, and lymphocyte subpopulations were analyzed by flow cytometry. CDKN2A/p16INK4a expression in T cells was measured using qPCR. The percentage of lymphocyte subpopulations and the CDKN2A/p16INK4a expression in TCS were compared with the control group using the Wilcoxon signed-rank test. RESULTS: We included 16 cases and 16 controls. The median age was 27 years (minimum 24, maximum 54) and the median time on surveillance was 26.5 months (minimum 3, maximum192). TCS had a lower percentage of total T cells and CD4+ T cells in total lymphocytes. Among the CD4+ T lymphocytes, TCS had less naïve CD4+ and increased memory CD4+ cells. Within the CD8+ T lymphocytes, TCS exhibited a decrease in the percentage of naïve cells and an increase in CD8 + CD45RA + CD57+ cells. TCS also exhibited decreased memory CD19+ B cells compared to the controls. The relative expression of CDKN2A/p16INK4a in T cells was increased in TCS (mean 1.54; 95% CI of the mean: 1.074-2.005; p = 0.048). CONCLUSION: In this exploratory study, TCS showed increased expression of CDKN2A/p16INK4a and a lymphocyte phenotype that has been associated with immunosenescence. Further studies are warranted to define the clinical implications of these alterations in TCS.

Cancer Stem Cell Niche and Immune-Active Tumor Microenvironment in Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) represent the most common neoplasia among young men. Management of TGCTs is an excellent example of curative outcomes in clinical oncology. The unique sensitivity to cisplatin-based chemotherapy regimens has led to establishing TGCTs as a "model of cancer cure." However, mechanisms and factors underlying pervasive growth of TGCTs are still poorly understood. It is suggested that unique cancer stem cell (CSC) niche exists in the testicular tumor microenvironment. CSC niche potentially contributes to the progression of germ cell tumors. Furthermore, rich infiltration of TGCTs with immune cells indicates involvement of immune system in biology of this cancer type. This review summarizes current knowledge regarding specific cancer microenvironment in TGCTs and discusses the role of cancer stem cells as well as immune mechanisms in these tumors.

Immunotherapy: last bullet in platinum refractory germ cell testicular cancer.

Testicular germ cell tumors are chemosensitive with very high cure-rates even in the metastatic setting. However, patients with platinum-refractory and relapsing tumors after autologous stem cell transplant have very poor outcomes despite salvage treatments, and with no effective alternative therapies. Immunotherapy, notably with PD-1 inhibitors, has proven to be very effective in treating various solid tumors. This review summarizes the experience with anti-PD-1 agents (pembrolizumab, nivolumab) in the treatment of testicular germ cell tumor relapsing after multiple lines of treatment, and exposes future trials evaluating newer checkpoint inhibitors in this setting.

May High Levels of Systemic Immune-Inflammation Index and Hematologic Inflammation Markers Suggest a Further Stage in Testicular Tumours?

INTRODUCTION: Due to variety of treatment alternatives for testicular tumours, parameters other than existing staging criteria are also needed. Most studies have revealed the correlation between cancer and inflammation. In this study, we aimed to investigate the value of preoperative inflammatory markers between early-stage testicular tumours and patients with advanced-stage, their relationship with tumour pathology and their importance in predicting stage. To calculate the differences between inflammatory markers, stage 1 tumours localized to the testis and advanced-stage tumours spread beyond the testis were classified into 2 groups according to tumour pathology. MATERIALS AND METHODS: The data of 112 patients undergoing inguinal orchiectomy in between 2008 and 2018 were recorded retrospectively. Neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR) and systemic immune-inflammation index (SII) were calculated by using the numbers of blood cell counts based systemic markers of inflammation. The differences between markers of inflammation were calculated by dividing tumours into 2 groups including early-stage and advanced- stage testicle tumours. RESULTS: According to the results of preoperative inflammatory markers in predicting the stage; in the seminoma group, the difference between the median NLR (2.37 vs. 4.39, p = 0.012), LMR (3.80 vs. 2.40, p = 0.018) and SII (612 vs. 1,127, p = 0.009) of stage 1 and advanced stage were statistically significant, while in the non-seminoma group, only the difference between median PLR (99 vs. 154, p = 0.002) of stage 1 and advanced stage was statistically significant. Sensitivity and specificity of predicting advanced stage according to cut-off values of markers were 69 and 75% in NLR (3.21), 83 and 75% in LMR, and 59 and 75% in SII in the seminoma group; on the other hand, in the non-seminoma group, the sensitivity, and specificity of predicting the advanced stage of PLR cut-off (104) were 71 and 88% respectively. CONCLUSIONS: The clinical use of inflammatory biomarkers in testicular tumours may represent an important step in understanding germ cell tumours biology and in supporting staging criteria and prognostic criteria.

Malignant leydig cell tumor in a 91-year-old man: Case report.

Testicle tumors are a rare entity among men population, accounting for only 1-1.5% of all can-cers among men. The stromal tumors of the sexual cord correspond just 4% of all testicular cancers. Only 10% of them are malignant. The major representative of the sex cord-stromal tumors is the Leydig cell tumor, corresponding to 75 to 80% of the total. It has bimodal age incidence, involving children and adults between 30 and 60 years. We report the caso of a 91-year-old man with malignant Leydig cell tumor, presenting increase of the volume of scrotum, local pain and hyperemia. The are few cases in the literature, only 1 with pacient above 85 years old.

Systemic immune-inflammation index in germ-cell tumours: search for a biological prognostic biomarker.

This corrects the article DOI: 10.1038/bjc.2017.85.

Systemic immune-inflammation index in germ-cell tumours.

BACKGROUND: We evaluated systemic immune-inflammation index (SII) and its association with patient outcome in germ-cell tumours (GCTs). METHODS: Two independent cohorts of patients were analysed; the discovery set (n=171) from a single institution and the validation set (n=181) previously included in a study evaluating PD-L1 in GCTs. The SII was calculated using platelet (P), neutrophil (N) and lymphocyte (L) counts before chemotherapy and correlated with survival using regression analyses and Kaplan-Meier method. RESULTS: In the discovery cohort, the SII was associated with poor risk clinical features. Patients with low SII had significantly longer progression-free survival (HR=0.22, 95% CI 0.12-0.41, P<0.001) and overall survival (OS) (HR=0.16, 95% CI 0.08-0.32, P<0.001) compared to high SII. This index was independent of International Germ Cell Cancer Collaborative Group criteria in multivariable Cox regression analysis for OS and was validated in an independent cohort. When combining PD-L1 expression on tumour infiltrating lymphocytes (TILs) and SII, we identified three distinctive prognostic groups. CONCLUSIONS: High SII was associated with poor outcome in GCTs. Combination of PD-L1 positive TILs and SII could further refine prognosis in GCTs.

Systemic inflammatory markers have independent prognostic value in patients with metastatic testicular germ cell tumours undergoing first-line chemotherapy.

BACKGROUND: The prognostic utility of systemic inflammatory markers has so far not been investigated in patients with metastatic testicular germ cell tumours (GCTs). METHODS: International Germ Cell Cancer Cooperative Group (IGCCCG) risk groups and blood-based systemic inflammatory markers (haemoglobin, leukocytes, platelets (P), neutrophils (N), lymphocytes (L), C-reactive protein (CRP) and albumin) of 146 patients undergoing first-line chemotherapy for GCT were retrieved. In addition, N to L ratio (NLR), P to L ratio and the systemic immune-inflammation index (SII=N × P/L) were calculated. The prognostic ability of these markers for overall survival (OS) were assessed using regression analyses and Kaplan-Meier curves with log-rank tests. RESULTS: In univariate Cox regression, low haemoglobin and albumin as well as high leukocytes, N, NLR, SII and CRP were associated with a shorter OS. In multivariable Cox regression analyses, high leukocyte (hazard ratio (HR) 1.274 (95% confidence interval (CI) 1.057-1.535); P=0.011) and N count (1.470 (1.092-1.980); P=0.011), higher NLR (84.5 (2.2-3193.4); P=0.017) and SII (12.15 (1.17-126.26); P=0.037) remained independent prognostic predictors for OS besides the IGCCCG risk groups. CONCLUSIONS: Systemic inflammatory markers might have prognostic utility for patients with metastatic GCT. The planned IGCCCG update could be an opportunity to test these markers in a larger data set.

Spontaneous CD4+ and CD8+ T-cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients.

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune-privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T-cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg-specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8+ and CD4+ T-cell responses against MAGE-A family antigens were present in 44% (20/45) of patients' samples assayed by ex vivo IFN-γ ELISPOT. The presence of MAGE-specific CD8+ T cells was further determined following short-term in vitro expansion through the use of pMHC-I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE-specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg-specific T-cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T-cell pool following treatment. Spontaneous T-cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype.

Managing seminomatous and nonseminomatous germ cell tumors.

PURPOSE OF REVIEW: In the present review, we summarize the recent developments in the management of germ cell tumors (GCTs). RECENT FINDINGS: Treatment-related acute and late-onset toxicity remains a key challenge in the management of GCTs, with recent evidence showing that the adverse health outcomes of etoposide and cisplatin for four cycles in comparison to bleomycin, etoposide, and cisplatin for three cycles appear to be similar. Recent data showed that multidisciplinary clinic approach and management in experienced academic centers were associated with improved overall survival in GCT patients. There are currently multiple conventional-dose chemotherapy options for salvage therapy in patients with refractory or recurrent disease. In addition, more efficacious high-dose chemotherapy regimens continue to be developed. The role of salvage conventional-dose chemotherapy versus high-dose chemotherapy is currently being investigated prospectively. Recent reports suggested that brentuximab vedotin could be a potential salvage option for cluster of differentiation 30 positive refractory GCTs. On the other hand the results of the first phase II clinical trial investigating pembrolizumab in refractory GCTs were disappointing showing no clinical activity.Finally, deep exploration of the immune profile of GCTs using immunohistochemistry and gene expression profiling has identified that advanced GCT stage was associated with decreased T-cell and Natural killer-cell signatures, whereas T regulatory, neutrophil, mast cell, and macrophage signatures increased with advanced stage. Even though these results indicated that activated T-cell infiltration correlated with seminoma histology and good prognosis, and could be used in the future as a biomarker, this approach needs to be validated in a large cohort. SUMMARY: Remaining challenges to be addressed include minimizing therapeutic toxicity, and improving outcomes in patients with refractory/recurrent GCTs.

PD-L1+ tumor-associated macrophages and PD-1+ tumor-infiltrating lymphocytes predict survival in primary testicular lymphoma.

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.