Classifying Cancers Based on T-cell Infiltration and PD-L1.

Introduction: The quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient's survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies. However, a framework to assess the PD-L1 status with a high interobserver reproducibility on tumor cells and different types of immune cells has yet to be established. Methods: To study the impact of PD-L1 expression on the tumor immune microenvironment and patient outcome, a framework for fully automated PD-L1 quantification on tumor cells and immune cells was established and validated. Automated PD-L1 quantification was facilitated by incorporating three different deep learning steps for the analysis of more than 80 different neoplasms from more than 10'000 tumor specimens using a bleach & stain 15-marker multiplex fluorescence immunohistochemistry panel (i.e., PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, CD31). Clinicopathological parameter were available for more than 30 tumor entities and overall survival data were available for 1517 breast cancer specimens. Results: Comparing the automated deep-learning based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p<0.0001) as well as immune cells (p<0.0001) and an accuracy of the automated PD-L1 quantification ranging from 90% to 95.2%. Across all tumor entities, the PD-L1 expression level was significantly higher in distinct macrophage/dendritic cell (DC) subsets (identified by CD68, CD163, CD11c, iNOS; p<000.1) and in macrophages/DCs located in the Stroma (p<0.0001) as compared to intratumoral macrophages/DC subsets. Across all different tumor entities, the PD-L1 expression was highly variable and distinct PD-L1 driven immune phenotypes were identified based on the PD-L1 intensity on both tumor and immune cells, the distance between non-exhausted T-cell subsets (i.e. PD-1 and CTLA-4 expression on CD3+CD8+ cytotoxic T-cells, CD3+CD4+ T-helper cells, CD3+CD4+FOXP3+ regulatory T-cells) and tumor cells as well as macrophage/(DC) subtypes. In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival with an area under receiver operating curves (AUC) of 0.72 (p<0.0001) than the percentage of PD-L1+ tumor cells (AUC: 0.54). In PD-L1 positive as well as negative breast cancers a close spatial relationship between T- cell subsets (CD3+CD4±CD8±FOXP3±PD-1±CTLA-4±) and Macrophage/DC subsets (CD68±CD163±CD11c±iNOS) was found prognostic relevant (p<0.0001). Conclusion: In conclusion, multiplex immunofluorescence PD-L1 assessment provides cutoff-free/continuous PD-L1 data which are superior to the conventional percentage of PD-L1+ tumor cells and of high prognostic relevance. The combined analysis of spatial PD-L1/PD-1 data and more than 20 different immune cell subtypes of the immune tumor microenvironment revealed distinct PD-L1 immune phenotypes. Citation Format: Niclas C. Blessin, Elena Bady, Tim Mandelkow, Cheng Yang, Jonas B. Raedler, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Sarah Minner, Eike Burandt, Doris Höflmayer, Guido Sauter, Katharina Möller, Sören A. Weidemann. Automated cell type specific PD-L1 quantification by artificial intelligence using high throughput bleach & stain 15-marker multiplex fluorescence immunohistochemistry in human cancers [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P068.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

BackgroundThe quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient’s survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies....

Background: Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors.Materials, Methods & Results: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes (P = 0.034). Survival curves demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1+ and PD-L1– (P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade (P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-L1+/TILs-High vs. PD-L1–/TILs-Low (P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS.Discussion: In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT.

Histological subtype is associated with PD-L1 expression and CD8+ T-cell infiltrates in triple-negative breast carcinoma

<div>Abstract<p>Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (<i>n</i> = 120) compared with non-TNBC (<i>n</i> = 716; <i>P</i> < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1<sup>+</sup> tumors had greater CD8<sup>+</sup> T-cell infiltrate than PD-L1<sup>−</sup> tumors (688 cells/mm vs. 263 cells/mm; <i>P</i> < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses. <i>Cancer Immunol Res; 2(4); 361–70. ©2014 AACR</i>.</p></div>

<div>Abstract<p>Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (<i>n</i> = 120) compared with non-TNBC (<i>n</i> = 716; <i>P</i> < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1<sup>+</sup> tumors had greater CD8<sup>+</sup> T-cell infiltrate than PD-L1<sup>−</sup> tumors (688 cells/mm vs. 263 cells/mm; <i>P</i> < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses. <i>Cancer Immunol Res; 2(4); 361–70. ©2014 AACR</i>.</p></div>

Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n = 120) compared with non-TNBC (n = 716; P < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1(+) tumors had greater CD8(+) T-cell infiltrate than PD-L1(-) tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses.

P3.03-012 Tumor-Infiltrating Lymphocytes, PDL-1, BAP-1, VEGFR-2 and IGF-1R Expression in Malignant Pleural Mesothelioma: Topic: Mesothelioma Transitional

Interferon alpha (IFNa) is recognized for its potential in cancer immunotherapy by activating immune responses against tumor cells. However, untargeted IFNa's application is limited by systemic toxicity and non-specific activity, affecting both tumor and healthy tissues and resulting in undesirable side effects. To overcome these challenges, we developed the novel trivalent bispecific antibody-cytokine fusion protein RO7694744 designed to provide a specific activation of IFNa on programmed cell death ligand 1 (PD-L1) positive cells, such as tumor cells and immune cells. One antibody arm binds to human PD-L1 with high affinity, whereas the other arm can bind mutually exclusive either to human PD-L1 (with lower affinity) or to IFNa2a, which is fused to the antibody via a peptide linker. Both PD-L1 binders are non-blocking. This antibody design, called DutaFlip, protects the IFNa2a moiety from interacting with IFNa receptors (IFNAR1/2) until it is activated by binding to PD-L1 on the target cells. Our research explored the PD-L1 selective activity of RO7694744 in multiple preclinical models. Using reporter cell assays, human whole blood cell assays, and various tumor cell lines with different PD-L1 expression levels, we demonstrated that IFNa activity depends on PD-L1 expression. Cells with elevated PD-L1 expression showed significantly higher activation compared to those with low PD-L1 levels. In vivo studies further validated these findings. In MDA-MB-231 tumor-bearing mice, RO7694744 treatment resulted in significant tumor growth inhibition (TGI), accompanied by intratumoral immune cell activation, and demonstrated a synergistic effect when combined with checkpoint inhibitor (CPI) treatment. To ensure translational relevance, we confirmed these in vivo observations in patient-derived tumor explants that mimic the human tumor environment. The explant data supported our previous findings, demonstrating PD-L1 specificity and dependency of RO7694744. Our preclinical findings illustrate that RO7694744 selectively targets and activates PD-L1 expressing tumor and immune cells. Clinical experience is needed to confirm whether this targeted strategy can enhance therapeutic efficacy while minimizing adverse effects, thus addressing the key challenge of untargeted IFNa in cancer immunotherapy. Citation Format: Moritz Rapp, Harald Duerr, Gabriel Schnetzler, Davide Bertoldo, Jack Bates, Laura Kruempelmann, Alexandra Baumgartner, Marion Lichtenauer, Sandra Bunte, Kerstin Paetzold, Martina Antoniolli, Malika Ait-Goughoulte, Yvonnne Kienast, Johannes Sam, Steffen Dettling, Martin Lechmann, Robert Mader, Daniela Willend, Barbara Leutgeb, Anja Irmisch, Claudia Mueller, Metodi Donev, Andreas Roller, Nino Gochitashvili, Kerstin Meier, Yan Li, Meher Majety, Marina Bacac, Christian Klein, Pablo Umana. Advancing cancer immunotherapy: PD-L1-targeted IFNa2a fusion protein RO7694744 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3416.

BackgroundRecent advances in immunotherapy have shown promising results in triple-negative breast cancer (TNBC). Studies regarding PDL1/PD1 co-inhibitory pathway demonstrate that PDL1 positive subgroup of TNBCs had better prognosis and increased...

Background The programmed death ligand 1 (PD-L1) has recently emerged as a target immunotherapy in Triple-negative breast cancer (TNBC). However, the tumor-intrinsic role of PD-L1 and its pathway still needs to be fully clarified. Recently, a close association between CD73, PD-L1, cancer cell steaminess and epithelial-to-mesenchymal transition phenotypes is emerging. In addition, we have previously demonstrated that miR-320a and miR-145 target PD-L1, whereas miR-30 family is known to target CD73. In this study, we aimed at investigating the role of these miRNAs as prognostic and predictive dynamic biomarkers for immunotherapy in TNBC. Additionally, we investigated whether PD-L1 exerts cellular autonomic functions in TNBC beyond its role in the immune checkpoint. Results CD73 and PD-L1 expression was assessed in a panel of breast cancer lines and the non-malignant breast cells MCF-10. We showed a significantly higher CD73 and PD-L1 expression in MDA-MB-231 (TNBC, Basal B) than in MCF-10. Conversely, in MCF-7 cells (HR +, Luminal A), CD73 and PD-L1 expression is lower than in MCF-10. Furthermore, we showed that low PDL1 expression is correlated with high levels of miR-320a and miR-145, and CD73 expression is inversely related to miR-30 expression. These putative biomarkers were also validated in two patients with different PD-L1 status. From the analysis of the data obtained by real-time qRT-PCR, we found high plasma level of CD73 and low plasma level of miR-320a, miR-145 and miR-30 in the patient with PD-L1 positive BC. On contrary, in the patient with PD-L1-negative BC, we found low plasma level of CD73 and high plasma level of miR-320a, miR-145 and miR-30. Furthermore, to explore whether PD-L1 could also have an intrinsic role in tumor development and invasiveness, we used stably MDA–MB-231 PD-L1-silenced cells. We found that cell growth, colony formation, migration rate and the ability to form spheres are consistently reduced upon shRNA-mediated PDL1 silencing. In particular, we observed a reduction in the number, diameter and volume of the 3D spheres compared to the control cells. To further demonstrate the role of PD-L1, we treated the PDL1-high expression cells MDA-MB-231, PD-L1 silenced MDA-MB-231 clones, and PD-L1 low expression cells MCF-7 with Durvalumab, an anti-PD-L1. Interestingly, we observed that in PD-L1 silenced clones and MCF-7 cells, the sphere-forming ability was increased in presence of Durvalumab treatment. On the contrary, in MDA-MB-231, Durvalumab inhibited the sphere-forming ability. Conclusion Our data confirm that PD-L1 and CD73 are targets of miR-320a/miR-145 and miR-30 respectively. These could be potential predictive dynamic biomarkers for chemotherapy, ICIs and anti-CD73 therapeutic approach. Future validations of these biomarkers in an extensive series of TNBC patients are needed to support their use in clinical practice. Here, we further characterized the cellular autonomic role of PD-L1 in breast cancer and showed a differential role of basal PD-L1 expression in PD-L1 checkpoint inhibitors treatment efficacy. This suggests a potential role in monitoring PD-L1 expression indirect biomarkers (i.e. miR-320a, miR-145 and CD73) during ICIs treatment.

Expression of programmed death ligand 1 (PD-L1) by cutaneous squamous cell carcinomas has been shown to correlate with advanced disease and risk of metastasis. Lymphocyte activation gene 3 (LAG-3, or CD223) is an inhibitory receptor that interacts with the PD-L1 axis, which has been shown to be a marker of immune exhaustion and a potential immunotherapy target. However, the role of LAG-3 in cutaneous squamous cell carcinoma has not been established. We reviewed 18 cases of locally advanced and/or metastatic cutaneous squamous cell carcinomas and assessed for PD-L1 expression, presence or absence of tumor-infiltrating lymphocytes (TILs), and expression of LAG-3 by TILs. PD-L1 expression was present in 11 of 13 locally advanced primary tumors and 5 of 5 metastases. TILs were present in all 18 tumors, of which 14 cases were positive for LAG-3 expression. In positive cases, LAG-3 was expressed on average by 32% of TILs. Advanced cutaneous squamous cell carcinomas frequently express PD-L1 and are associated with an inflammatory response. LAG-3 expression by TILs was identified in a majority of cases. Our findings suggest that LAG-3 positive tumor-associated inflammatory cells may play a role in the development of advanced disease and offer another potential target for drug therapy.

In this issue of Neuro-Oncology, Berghoff et al present the results of their investigations into the expression of programmed death-ligand 1 (PD-L1) in human glioblastoma specimens and their relationship to other tissue-based and clinical parameters. In specimens from adults with newly diagnosed or recurrent glioblastoma, the investigators reported that diffuse/fibrillary PD-L1 expression of variable extent was found in more than 70% of glioblastoma specimens, with a higher proportion in newly diagnosed cases. Furthermore, more than 70% of cases showed evidence of high tumor-infiltrating lymphocyte (TIL) infiltration, although most were of sparse-to-moderate density. In glioblastoma samples from the Cancer Genome Atlas, the relationships between PD-L1 expression, molecular subtypes, and clinical outcomes were explored. Samples with known molecular subtypes were then classified according to their level of PD-L1 expression (high or low), and a significant difference in the distribution of PD-L1 high and low groups was found across molecular subtypes, with the mesenchymal subtype in particular having a high level of PD-L1 expression and the proneural and G-CIMP subtypes primarily having low expression of PD-L1. The anti-PD-L1 antibody, 5H1, used in this study is not commercially available, although its use has been confirmed in other experiments, whereas some commercially available antibodies have failed to show reliable PD-L1 labeling.1 The authors conclude that PD-L1 expression and TILs were found in most of the glioblastoma samples they evaluated, but a relationship between these parameters and outcome was not observed. Still, the high expression of PD-L1 in these glioblastoma samples suggests that it may be a valid target for further clinical investigation.

Recurrent PDL1 expression and PDL1 (CD274) copy number alterations in breast implant–associated anaplastic large cell lymphomas