Unraveling relatlimab (RELA)-specific biology: Biomarker analyses in patients (pts) with metastatic non-small cell lung cancer (mNSCLC) treated with 1L nivolumab (NIVO) + RELA high-dose (HD) and platinum-doublet chemotherapy (PDCT).

BackgroundThe phase 2/3 RELATIVITY-047 clinical trial (NCT03470922) in patients with previously untreated metastatic or unresectable melanoma met its primary endpoint of improved progression-free survival with a combination of nivolumab plus...

e18014 Background: Immunotherapy has revolutionized the landscape of systemic treatment of recurrent or metastatic (R/M HNSCC). However only a small percentage of patients achieve long-term benefit regarding overall response and overall survival from the current immunotherapy. Indeed it has already been demonstrated that HNSCC microenviroment is distinctly immunosuppressive due to the high concentration of several biomarkers such as regulatory T cells (Treg cells) and lymphocyte activation gene 3 (LAG3) . The aim of our study is to investigate if the expression of peculiar biomarkers can affect the therapeutic efficacy of anti-PD-1. Methods: Data from 35 patients with combined positive score (CPS) positive R/M HNSCC receiving first line immunotherapy or immunotherapy in association with chemotherapy, from March 2021 to November 2021 were prospectively reviewed. Treg cells, stromal and intratumoral, and LAG3 expression by cancer cells and CPS expression were evaluated on pre-treatment biopsies. We defined an immune suppressive profile (ISP) as a concomitant presence both of LAG3 and Treg cells. We evaluated the associations between early progression (EP), defines as progression occurred within 3 months, progression-free survival (PFS), overall survival (OS), objective response rate (ORR) and the expression of LAG3 and with the ISP. Results: Twenty-four patients were male (69%), 11 female (31%) and median age was 67 years. Baseline ECOG PS, evaluated before the start of treatment, was 0, 1, and 2 in 14 (40%), 14 (40 %) and 7 (20%) patients, respectively. The primary tumor site was the oral cavity in 19 patients (54 %), the larynx in 13 patients (37 %), hypopharynx in 2 patients (6 %) and sinus in the remaining 1 (3%). Thirteen (37%) patients received pembrolizumab alone while pembrolizumab in association with chemotherapy (carboplatin or cisplatin and 5-fluorouracil) were administered intravenously to 22 (63%) patients. Twenty-eight (80%) patients presented LAG3 expression, whereas Treg cells was reported in the stroma and the tumour in 31 (89%) and 29 (83%) patients respectively. Twenty-three (66%) patients presented both expression of LAG3 and presence of Treg cells. We showed both a statistically significant association between LAG3 expression and EP disease ( p = 0.005) and between ISP and EP ( p = 0.023). We didn’t observed associations between PFS, OS, ORR and LAG3 and Treg status. Conclusions: Our findings showed that in CPS positive R/M HNSCC patients the contemporary expression of Treg cells and LAG3 could better select the patients primary resistant to anti-PD-1. The results of our study identify a novel immunological patients’ profile.

LAG-3: another brake to release in breast cancer?

The immune checkpoint molecule lymphocyte activation gene 3 (LAG-3) is currently being investigated as a possible target for immunotherapy in triple-negative breast cancer (TNBC), frequently as an addition to treatment with programmed cell death protein 1/programmed death ligand 1 (PD-L1) inhibition. However, expression of LAG-3, the frequency of coexpression with PD-L1, and the prognostic significance of this marker have not been studied extensively in TNBC. For this study, tissue microarrays (TMAs) were constructed from surgical specimens of 514 patients with TNBC. TMAs were stained immunohistochemically for LAG-3 and PD-L1 expression. Tumor-infiltrating lymphocytes (TILs) were evaluated on full glass slides. LAG-3 expression was significantly associated with improved overall survival and relapse-free survival. When adjusted for clinicopathologic factors, each increment of 10 LAG-3-positive intratumoral lymphocytes per TMA core was associated with improved overall survival (hazard ratio=0.93, 95% confidence interval: 0.89-0.97, P=0.002), and recurrence-free survival (hazard ratio=0.91, 95% confidence interval: 0.85-0.97, P=0.002). PD-L1 expression on immune cells and PD-L1 expression evaluated with the combined positive score and TILs were also associated with improved survival in both univariate and multivariate analyses. PD-L1 expression on tumor cells was only associated with improved survival in univariate analysis. LAG-3 expression was associated with both TILs and PD-L1 expression. Coexpression of LAG-3 and PD-L1 did not confer additional survival benefits. In conclusion, LAG-3 expression is associated with improved survival in TNBC. LAG-3 is often coexpressed with PD-L1, confirming that TNBC is likely a suitable candidate for cotreatment with LAG-3 and programmed cell death protein 1/PD-L1 inhibitors. However, coexpression does not confer additional survival benefits.

Esophageal squamous cell carcinoma (ESCC) is a malignant epithelial tumor with a high incidence in East Asia and the Middle East. The outcomes for ESCC patients are usually not optimal due to the recurrence and metastasis. This study is aim to examine the expression and the prognostic value of LAG-3 in ESCC. We applied immunohistochemistry analysis to examine the expression of LAG-3, CD4 and CD8 in 287 ESCC cohorts. Our study demonstrated that the decreased LAG-3 expression was significantly associated with CD4 tumor-infiltrated lymphocytes (TILs) (p=0.000), CD8 TILs (p=0.000), and the advanced clinical stages (p=0.041) by Chi-square analysis. Kaplan-Meier survival analysis revealed that higher LAG-3 expression were positively correlated with a better overall survival (OS) (p=0.010) and better progression free survival (PFS) (p=0.006), especially in the patients at stages T1-2 status (p=0.001, OS; p=0.001, PFS), N0 status (p=0.036, OS; p=0.050, PFS), and early stages (I-II) (p=0.006, OS; p=0.008, PFS). Both high of CD4 TIL /CD8 TIL ratio and LAG-3 expression were correlated with longer OS and PFS. Cox proportional hazards regression analysis showed that LAG-3 is an independent biomarker of survival (HR, 0.724; 95% CI 0.526-0.995; p = 0.047) (p=0.036). Taken together, we found that high expression of LAG-3 was correlated with an improved survival and LAG-3 is an independent predictor of survival, suggesting that LAG-3 may serve as a useful immune marker for the prognosis of ESCC.

Aim: To investigate the expression and clinical value of the immune checkpoint marker LAG-3 in breast cancer patients Background: Lymphocyte-activation gene 3 (LAG-3) is a recently discovered immune checkpoint biomarker that is targeted by agents currently being evaluated in early phase clinical trials. LAG-3 functions as a cell surface receptor expressed following T cell activation and negatively impacts T cell functions. This biomarker has not yet been evaluated in large series of breast cancers with long term treatment and outcome data, in the context of subtype and other immune biomarkers. Methods: Two tissue microarray series (a training set with N=330 and a validation set with N = 2203 patients) were constructed from breast carcinoma primary excision specimens from University of British Columbia hospitals, linked to detailed clinical and pathological data. None of these patients had received neoadjuvant treatment. 4µm sections were stained with an antibody to LAG-3 (clone 17B4) by immunohistochemistry using a Ventana Discovery Ultra automated slide stainer. LAG-3+ stromal and intra-epithelial tumor infiltrating lymphocytes (TILs) were reported as absolute counts per tissue microarray core. Stromal TILs (sTIL) were defined as lymphocytes present in the stroma not in direct contact with tumor nest whereas intra-epithelial TIL (iTIL) were lymphocytes in direct contact with carcinoma cells. All descriptive and survival analyses were conducted using SPSS software. Results: LAG-3+ sTILs were found in 16% of breast cancer cases in both the training set and the validation set; LAG-3+iTILs were present in 14 and 11%, respectively. In both the training set and the validation set, the presence of LAG-3 (iTILs or sTILs) was significantly (p<0.001) associated with high grade tumors, estrogen and progesterone receptor negativity, high Ki67 index and with the HER2+ and basal-like subtypes. In survival analyses of ER negative patients, in both sets patients with LAG-3 T cells (iTILs or sTILs) had a significantly improved disease-specific survival (p<0.05). As with other lymphocyte biomarkers, this association was not observed among ER+ patients. Conclusions: LAG-3+TILs are present in breast cancer and are associated with major risk factors and hormone receptor negative subtypes. ER negative breast cancer patients have a better outcome if they contain LAG-3+ TILs, consistent with published data showing better survival among ER- breast cancer patients with immune infiltrates. More than a quarter of ER negative breast cancers contain TILs expressing LAG3, and may represent the most relevant subset to target with emerging checkpoint inhibitors targeting this T cell surface receptor. Citation Format: Burugu S, Gao D, Nielsen TO. Expression of LAG-3 in breast cancer, and its association with subtype and outcome [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD5-08.

Lymphocyte-activation gene 3 in non-small-cell lung carcinomas: correlations with clinicopathologic features and prognostic significance

We aimed to characterize the relationships of lymphocyte activation gene-3 (LAG-3) expression, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density, and to investigate the joint prognostic impact of these three markers in patients with surgically resected esophageal squamous cell carcinoma (ESCC). Expression of LAG-3, CTLA-4 and the density of CD8+ TILs were evaluated by immunohistochemistry in resected ESCC. The associations between LAG-3 expression and clinicopathologic characteristics, as well as patient prognoses, were analyzed. A total of 183 patients were included. LAG-3 expression was observed in 69 (37.7%) patients. Positive LAG-3 expression was significantly associated with CTLA-4 expression (P=0.004). LAG-3 positivity, CTLA-4 positivity, and low CD8+ TIL densities were significantly associated with worsening recurrence-free survival (RFS) [LAG-3: hazard ratio (HR), 1.72; 95% confidence interval (CI), 1.10-2.89; P=0.019; CTLA-4: HR, 1.69; 95% CI, 1.04-2.73; P=0.033; CD8+: HR, 0.60; 95% CI, 0.38-0.94; P=0.025] and overall survival (OS) (LAG-3: HR, 2.09; 95% CI, 1.24-3.53; P=0.006; CTLA-4: HR, 1.47; 95% CI, 0.86-2.53; P=0.161; CD8+: HR, 0.56; 95% CI, 0.33-0.95; P=0.032). Subgroup analysis revealed that the LAG-3 CTLA-4 CD8+ group had the best RFS (P<0.001) and OS (P<0.001). LAG-3 expression was correlated with CTLA-4 expression on TILs. Positive LAG-3 expression was associated with poor prognoses in ESCC. A combination of LAG-3, CTLA-4 expression and CD8+ TILs density could further stratify patients into different subgroups with distinct prognoses.

BackgroundImmune inhibitory receptors play an important role in chronic infections. However, little is known about their role in hepatitis B virus (HBV) infection. Here, we analyzed the relationship between programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) expression on CD4+ T cells and HBV disease progression.ResultsPD-1 and LAG-3 expression was significantly higher on CD4+ T cells from HBV patients than on those from the HCs. In addition, a significant positive correlation was found between the PD-1 and LAG-3 expression levels and the ALT(alanine aminotransferase) level. CD4+ T cell function was inhibited by high PD-1 and LAG-3 levels, and CD4+ T cells with high PD-1 and LAG-3 expression lost the ability to secrete IFN-γ, IL-2 and TNF-α. Furthermore, blockade of the PD-1 and LAG-3 pathways reversed the damage to CD4+ T cell proliferation and cytokine secretion.ConclusionsCD4+ T cell exhaustion during chronic HBV had high PD-1 and LAG-3 expression and the absence of helper T cell cytokines, including IFN-γ, IL-2 and TNF-α. After blocking PD-L1 and LAG-3, CD4+ T cell function in chronic hepatitis B patients was partially restored.

PurposeImmune modulatory therapies including immune checkpoint inhibitors have so far failed to result in clinically meaningful efficacy in glioma. We aimed to investigate lymphocyte activation gene 3 (LAG-3), an inhibitory receptor on immune cells and target of second-generation immune checkpoint inhibitors, in glioma.Methods97 patients with diffuse glioma (68 with glioblastoma, 29 with WHO grade II-III glioma) were identified from the Neuro-Biobank of the Medical University of Vienna. LAG-3 expression in the inflammatory microenvironment was assessed by immunohistochemistry (monoclonal anti-LAG-3 antibody, clone 17B4) and correlated to CD3+ , CD8+ , CD20+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells.ResultsLAG-3+ TILs could be observed in 10/97 (10.3%) IDH-wildtype samples and in none of the included IDH-mutant glioma samples (p = 0.057). Further, LAG-3+ TILs were only observed in WHO grade IV glioblastoma, while none of the investigated WHO grade II–III glioma presented with LAG-3+ TILs (p = 0.03). No association of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and presence of LAG-3+ TILs was observed (p = 0.726). LAG-3 expression was associated with the presence of CD3+ (p = 0.029), CD8+ (p = 0.001), PD-1+ (p < 0.001) TILs and PD-L1+ tumor cells (p = 0.021), respectively. No association of overall survival with LAG-3+ TIL infiltration was evident (median OS 9.9 vs. 14.2 months, p = 0.95).ConclusionsLAG-3 is only rarely expressed on TILs in IDH-wildtype glioma and associated with active inflammatory milieu as defined by higher TIL density. Immune microenvironment diversity should be considered in the design of future immunotherapy trials in glioma.

BackgroundThe diffuse large B-cell lymphoma (DLBCL) has the highest incidence of all lymphomas worldwide. To investigate the functions of lymphocyte activation gene 3 (LAG-3) and programmed cell death 1 (PD-1) in tissues and peripheral blood of patients with DLBCL, the expression of LAG-3 and PD-1 genes in DLBCL-TCGA were analyzed.MethodsLAG-3 and PD-1 mRNA levels in DLBCL were analyzed using data from The Cancer Genome Atlas (TCGA) database. Utilize the Genotype-Tissue Expression (GTEx) database for assessing the variance in the expression of LAG-3, PD-1, and other associated factors between the tissues of DLBCL patients and healthy individuals. Immunohistochemistry was applied to detect the expression of LAG-3 and PD-1 levels in 137 cases of DLBCL tissues and 20 cases of reactive lymphoid hyperplasia. The prognostic value of LAG-3 and PD-1 were assessed using the Kaplan-Meier curve. The Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) and ssGSEA algorithm were used to explore the immune microenvironment of DLBCL. Additionally, the expression and co-expression of LAG-3 and PD-1 were detected on CD4 and CD8 T cells in peripheral blood samples from 100 cases of DLBCL tissues and 30 cases of healthy individuals using flow cytometry.ResultsAccording to TCGA database, LAG-3 and PD-1 gene expression levels were significantly up-regulated in DLBCL tissues. LAG-3 and PD-1 levels were also strongly positively correlated with those of most infiltrating immune cells. Overall survival of patients with high LAG-3 and PD-1 co-expression was significantly shorter than that of patients with low co-expression. In DLBCL patients, LAG-3 and PD-1 were highly expressed in peripheral blood CD8+ T cells. In addition, LAG-3 was highly expressed in CD4+ T cells, while the expression of PD-1 in CD4+ T cells of DLBCL patients showed no significant difference compared to healthy individuals. Additionally, CD8+ T cells and SU-DHL6/OCI-LY3 from patients with DLBCL were co-cultured in vitro; after addition of LAG-3 and/or PD-1 inhibitors alone, an increased perforin and granzyme B secretion levels by CD8+ T cells were detected, as well as an increase in the overall proportion of tumor cells undergoing apoptosis.ConclusionHigh LAG-3 and PD-1 levels significantly inhibit CD8+ T cell function, resulting in weakened ability to kill tumor cells. Combined LAG-3 and PD-1 blockade can restore CD8+ T cell function and provides a potential avenue for development of personalized cellular immunotherapy for DLBCL.

Lymphocyte activation gene 3 (LAG3) is a member of the immunoglobulin superfamily and has been implicated in the development, growth, and progression of several cancers. However, the biological role of LAG3 has not been investigated in pan-cancer datasets. We sought to perform a comprehensive bioinformatics analysis of pan-cancer datasets to determine the relationship of LAG3 with patient survival prognosis, tumor microenvironment, immunotherapy responsiveness, and mechanisms regulating tumorigenesis. We used TCGA, GTEx, TIMER2, GDSC, CTRP, and TISCH databases and online websites to extract data on LAG3 in a variety of cancers, and analyzed pan-cancer patient datasets to explore not only the correlation between LAG3 expression and clinical stage, diagnosis, and prognosis of cancers, but also the correlation between LAG3 expression, gene variants, methylation status, tumor stemness, and tumor immunity. The biological functions of LAG3 in osteosarcoma cells were determined by in vitro CCK-8, wound healing and transwell assays. Finally, through in vivo experiments, the study preliminarily explored the impact of LAG3 on osteosarcoma and its correlation with immune genes. Pan-cancer analysis showed that LAG3 expression was up-regulated in a variety of cancers, and the expression of LAG3 was closely related to the clinical stage, diagnosis and prognosis of cancers. GO and KEGG enrichment analyses showed that LAG3 was enriched in inflammatory, metabolic, and immune signaling pathways in a variety of cancers. Meanwhile, LAG3 expression not only has an impact on patient immunotherapy prognosis and immunotherapy response, but also has a significant effect on drug sensitivity. In vitro experiments have shown that LAG3 promotes the proliferation, migration and invasion of osteosarcoma cells. In vivo xenotransplantation experiments further confirmed that LAG3 promotes the growth of osteosarcoma, and the expression of LAG3 is positively correlated with CD8, CD19, and CD68. Our study suggests that LAG3 is a promising marker for cancer diagnosis, prognosis, and treatment.

Systemic treatment options for patients with locally advanced or metastatic basal cell carcinoma (BCC) are limited, particularly when tumors are refractory to anti-programmed cell death protein-1 (PD-1). A better understanding...

LAG-3+ tumor infiltrating lymphocytes in breast cancer: clinical correlates and association with PD-1/PD-L1+ tumors

Salivary gland carcinomas (SGCs) are rare and can be subdivided into distinct entities, some of which confer a poor prognosis. As targets for effective systemic therapy are warranted, some studies investigated the role of immune-checkpoint proteins PD-L1 and CTLA-4 in SGC. Our study depicts the expression of lymphocyte activation gene 3 (LAG3) in a test cohort and a larger validation cohort, totaling 139 SGCs. LAG3 is expressed on tumor-infiltrating lymphocytes (TILs), mediates T cell exhaustion and is subject to numerous currently recruiting clinical studies. Overall, one-third of SGCs were infiltrated by LAG3-expressing TILs with a strikingly high concordance between the test cohort and the validation cohort (30% and 28.2%, respectively). In the validation cohort, entity-wise LAG3 expression frequencies were highly variable. The highest rates were observed in salivary duct carcinoma (SDC; 66.7%) and adenocarcinoma not otherwise specified (ANOS; 50.0%). We observed LAG3 expression on effector T cells and in smaller frequencies also on FOXP3− T helper cells and FOXP3+ Tregs. LAG3 expression significantly correlated with advanced nodal metastases, cytotoxic T cell infiltrate and TP53 mutations. In the group of adenoid cystic carcinomas, LAG3 expression was also associated with a shorter event-free survival (EFS). Tumors with TP53 nonsense mutations (TP53 null type) exhibited higher LAG3 frequencies and a shorter EFS compared to TP53 wild type. This is the first report of LAG3 expression in SGC, a promising target for immunotherapy. LAG3 blockage could be distinctly applicable for SDC and ANOS, two SGC types with a particularly poor outcome.