Prognostic value of human leukocyte antigen G expression in solid tumors: a systematic review and meta-analysis.

Introduction: Identification of modulators of the immune response with inhibitory properties that could be susceptible for therapeutic intervention is a key goal in cancer research. An example is the human leukocyte antigen G (HLA-G), a nonclassical major histocompatibility complex (MHC) class I molecule, involved in cancer progression. Methods: In this article we performed a systematic review and meta-analysis on the association between HLA-G expression and outcome in solid tumors. This study was performed in accordance with PRISMA guidelines and registered in PROSPERO. Results: A total of 25 studies met the inclusion criteria. These studies comprised data from 4871 patients reporting overall survival (OS), and 961 patients, reporting disease free survival (DFS). HLA-G expression was associated with worse OS (HR 2.09, 95% CI = 1.67 to 2.63; P < .001), that was higher in gastric (HR = 3.40; 95% CI = 1.64 to 7.03), pancreatic (HR = 1.72; 95% CI = 0.79 to 3.74) and colorectal (HR = 1.55; 95% CI = 1.16 to 2.07) cancer. No significant differences were observed between the most commonly utilized antibody (4H84) and other methods of detection. HLA-G expression was associated with DFS which approached but did not meet statistical significance. Discussion: In summary, we describe the first meta-analysis associating HLA-G expression and worse survival in a variety of solid tumors. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022311973.

Plasma fractalkine contributes to systemic myeloid diversity and PD-L1/PD-1 blockade in lung cancer.

Recent studies highlight the importance of baseline functional immunity for immune checkpoint blockade therapies. High-dimensional systemic immune profiling is performed in a cohort of non-small-cell lung cancer patients undergoing PD-L1/PD-1 blockade immunotherapy. Responders show high baseline myeloid phenotypic diversity in peripheral blood. To quantify it, we define a diversity index as a potential biomarker of response. This parameter correlates with elevated activated monocytic cells and decreased granulocytic phenotypes. High-throughput profiling of soluble factors in plasma identifies fractalkine (FKN), a chemokine involved in immune chemotaxis and adhesion, as a biomarker of response to immunotherapy that also correlates with myeloid cell diversity in human patients and murine models. Secreted FKN inhibits lung adenocarcinoma growth in vivo through a prominent contribution of systemic effector NK cells and increased tumor immune infiltration. FKN sensitizes murine lung cancer models refractory to anti-PD-1 treatment to immune checkpoint blockade immunotherapy. Importantly, recombinant FKN and tumor-expressed FKN are efficacious in delaying tumor growth in vivo locally and systemically, indicating a potential therapeutic use of FKN in combination with immunotherapy.

Circulating Low Density Neutrophils Are Associated with Resistance to First Line Anti-PD1/PDL1 Immunotherapy in Non-Small Cell Lung Cancer.

Single-agent immunotherapy has been widely accepted as frontline treatment for advanced non-small cell lung cancer (NSCLC) with high tumor PD-L1 expression, but most patients do not respond and the mechanisms of resistance are not well known. Several works have highlighted the immunosuppressive activities of myeloid subpopulations, including low-density neutrophils (LDNs), although the context in which these cells play their role is not well defined. We prospectively monitored LDNs in peripheral blood from patients with NSCLC treated with anti-PD-1 immune checkpoint inhibitors (ICIs) as frontline therapy, in a cohort of patients treated with anti-PD1 immunotherapy combined with chemotherapy (CT+IT), and correlated values with outcomes. We explored the underlying mechanisms through ex vivo experiments. Elevated baseline LDNs predict primary resistance to ICI monotherapy in patients with NSCLC, and are not associated with response to CT+IT. Circulating LDNs mediate resistance in NSCLC receiving ICI as frontline therapy through humoral immunosuppression. A depletion of this population with CT+IT might overcome resistance, suggesting that patients with high PD-L1 tumor expression and high baseline LDNs might benefit from this combination. The activation of the HGF/c-MET pathway in patients with elevated LDNs revealed by quantitative proteomics supports potential drug combinations targeting this pathway.

Genomic Mapping of Splicing-Related Genes Identify Amplifications in LSM1, CLNS1A, and ILF2 in Luminal Breast Cancer.

Alternative splicing is an essential biological process, which increases the diversity and complexity of the human transcriptome. In our study, 304 splicing pathway-related genes were evaluated in tumors from breast cancer patients (TCGA dataset). A high number of alterations were detected, including mutations and copy number alterations (CNAs), although mutations were less frequently present compared with CNAs. In the four molecular subtypes, 14 common splice genes showed high level amplification in >5% of patients. Certain genes were only amplified in specific breast cancer subtypes. Most altered genes in each molecular subtype clustered to a few chromosomal regions. In the Luminal subtype, amplifications of LSM1, CLNS1A, and ILF2 showed a strong significant association with prognosis. An even more robust association with OS and RFS was observed when expression of these three genes was combined. Inhibition of LSM1, CLNS1A, and ILF2, using siRNA in MCF7 and T47D cells, showed a decrease in cell proliferation. The mRNA expression of these genes was reduced by treatment with BET inhibitors, a family of epigenetic modulators. We map the presence of splicing-related genes in breast cancer, describing three novel genes, LSM1, CLNS1A, and ILF2, that have an oncogenic role and can be modulated with BET inhibitors.

A Proteomic Atlas of Lineage and Cancer-Polarized Expression Modules in Myeloid Cells Modeling Immunosuppressive Tumor-Infiltrating Subsets.

Monocytic and granulocytic myeloid-derived suppressor cells together with tumor-infiltrating macrophages constitute the main tumor-infiltrating immunosuppressive myeloid populations. Due to the phenotypic resemblance to conventional myeloid cells, their identification and purification from within the tumors is technically difficult and makes their study a challenge. We differentiated myeloid cells modeling the three main tumor-infiltrating types together with uncommitted macrophages, using ex vivo differentiation methods resembling the tumor microenvironment. The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules. The relationships between monocytic MDSCs and TAMs, monocytic MDSCs and granulocytic MDSCs, and hierarchical relationships of expression networks and transcription factors due to lineage and cancer polarization were mapped. Highly purified immunosuppressive myeloid cell populations that model tumor-infiltrating counterparts were systematically analyzed by quantitative proteomics. Full functional interactome maps have been generated to characterize at high resolution the relationships between the three main myeloid tumor-infiltrating cell types. Our data highlights the biological processes related to each cell type, and uncover novel shared and differential molecular targets. Moreover, the high numbers and fidelity of ex vivo-generated subsets to their natural tumor-shaped counterparts enable their use for validation of new treatments in high-throughput experiments.

Understanding LAG-3 Signaling.

Lymphocyte activation gene 3 (LAG-3) is a cell surface inhibitory receptor with multiple biological activities over T cell activation and effector functions. LAG-3 plays a regulatory role in immunity and emerged some time ago as an inhibitory immune checkpoint molecule comparable to PD-1 and CTLA-4 and a potential target for enhancing anti-cancer immune responses. LAG-3 is the third inhibitory receptor to be exploited in human anti-cancer immunotherapies, and it is considered a potential next-generation cancer immunotherapy target in human therapy, right next to PD-1 and CTLA-4. Unlike PD-1 and CTLA-4, the exact mechanisms of action of LAG-3 and its relationship with other immune checkpoint molecules remain poorly understood. This is partly caused by the presence of non-conventional signaling motifs in its intracellular domain that are different from other conventional immunoregulatory signaling motifs but with similar inhibitory activities. Here we summarize the current understanding of LAG-3 signaling and its role in LAG-3 functions, from its mechanisms of action to clinical applications.

Transcriptomic Mapping of Non-Small Cell Lung Cancer K-RAS p.G12C Mutated Tumors: Identification of Surfaceome Targets and Immunologic Correlates.

Targeting K-RAS-mutant non-small cell lung cancer (NSCLC) with novel inhibitors has shown promising results with the recent approval of sotorasib in this indication. However, progression to this agent is expected, as it has previously been observed with other inhibitors. Recently, new immune therapeutics, including vectorized compounds with antibodies or modulators of the host immune response, have demonstrated clinical activity. By interrogating massive datasets, including TCGA, we identified genes that code for surface membrane proteins that are selectively expressed in K-RAS mutated NSCLC and that could be used to vectorize novel therapies. Two genes, CLDN10 and TMPRSS6, were selected for their clear differentiation. In addition, we discovered immunologic correlates of outcome that were clearly de-regulated in this particular tumor type and we matched them with immune cell populations. In conclusion, our article describes membrane proteins and immunologic correlates that could be used to better select and optimize current therapies.

PD-L1 in Systemic Immunity: Unraveling Its Contribution to PD-1/PD-L1 Blockade Immunotherapy.

The use of monoclonal antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. However, despite the significant improvement in overall survival and progression-free survival of patients undergoing these immunotherapy treatments, the only clinically accepted biomarker with some prediction capabilities for the outcome of the treatment is PD-L1 expression in tumor biopsies. Nevertheless, even when having PD-L1-positive tumors, numerous patients do not respond to these treatments. Considering the high cost of these therapies and the risk of immune-related adverse events during therapy, it is necessary to identify additional biomarkers that would facilitate stratifying patients in potential responders and non-responders before the start of immunotherapies. Here, we review the utility of PD-L1 expression not only in tumor cells but in immune system cells and their influence on the antitumor activity of immune cell subsets.

Systemic Blood Immune Cell Populations as Biomarkers for the Outcome of Immune Checkpoint Inhibitor Therapies.

The development of cancer immunotherapy in the last decade has followed a vertiginous rhythm. Nowadays, immune checkpoint inhibitors (ICI) which include anti-CTLA4, anti-PD-1 and anti-PD-L1 antibodies are in clinical use for the treatment of numerous cancers. However, approximately only a third of the patients benefit from ICI therapies. Many efforts have been made for the identification of biomarkers allowing patient stratification into potential responders and progressors before the start of ICI therapies or for monitoring responses during treatment. While much attention is centered on biomarkers from the tumor microenvironment, in many cases biopsies are not available. The identification of systemic immune cell subsets that correlate with responses could provide promising biomarkers. Some of them have been reported to influence the response to ICI therapies, such as proliferation and activation status of CD8 and CD4 T cells, the expression of immune checkpoints in peripheral blood cells and the relative numbers of immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells. In addition, the profile of soluble factors in plasma samples could be associated to response or tumor progression. Here we will review the cellular subsets associated to response or progression in different studies and discuss their accuracy in diagnosis.

Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy.

The majority of lung cancer patients progressing from conventional therapies are refractory to PD-L1/PD-1 blockade monotherapy. Here, we show that baseline systemic CD4 immunity is a differential factor for clinical responses. Patients with functional systemic CD4 T cells included all objective responders and could be identified before the start of therapy by having a high proportion of memory CD4 T cells. In these patients, CD4 T cells possessed significant proliferative capacities, low co-expression of PD-1/LAG-3 and were responsive to PD-1 blockade ex vivo and in vivo. In contrast, patients with dysfunctional systemic CD4 immunity did not respond even though they had lung cancer-specific T cells. Although proficient in cytokine production, CD4 T cells in these patients proliferated very poorly, strongly co-upregulated PD-1/LAG-3, and were largely refractory to PD-1 monoblockade. CD8 immunity only recovered in patients with functional CD4 immunity. T-cell proliferative dysfunctionality could be reverted by PD-1/LAG-3 co-blockade. Patients with functional CD4 immunity and PD-L1 tumor positivity exhibited response rates of 70%, highlighting the contribution of CD4 immunity for efficacious PD-L1/PD-1 blockade therapy.

PD-L1 Expression in Systemic Immune Cell Populations as a Potential Predictive Biomarker of Responses to PD-L1/PD-1 Blockade Therapy in Lung Cancer.

PD-L1 tumor expression is a widely used biomarker for patient stratification in PD-L1/PD-1 blockade anticancer therapies, particularly for lung cancer. However, the reliability of this marker is still under debate. Moreover, PD-L1 is widely expressed by many immune cell types, and little is known on the relevance of systemic PD-L1⁺ cells for responses to immune checkpoint blockade. We present two clinical cases of patients with non-small cell lung cancer (NSCLC) and PD-L1-negative tumors treated with atezolizumab that showed either objective responses or progression. These patients showed major differences in the distribution of PD-L1 expression within systemic immune cells. Based on these results, an exploratory study was carried out with 32 cases of NSCLC patients undergoing PD-L1/PD-1 blockade therapies, to compare PD-L1 expression profiles and their relationships with clinical outcomes. Significant differences in the percentage of PD-L1⁺ CD11b⁺ myeloid cell populations were found between objective responders and non-responders. Patients with percentages of PD-L1⁺ CD11b⁺ cells above 30% before the start of immunotherapy showed response rates of 50%, and 70% when combined with memory CD4 T cell profiling. These findings indicate that quantification of systemic PD-L1⁺ myeloid cell subsets could provide a simple biomarker for patient stratification, even if biopsies are scored as PD-L1 null.

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography.

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

Myeloid-Derived Suppressor Cells in the Tumor Microenvironment: Current Knowledge and Future Perspectives.

The current knowledge on tumor-infiltrating myeloid-derived suppressor cells (MDSCs) is based mainly on the extensive work performed in murine models. Data obtained for human counterparts are generated on the basis of tumor analysis from patient samples. Both sources of information led to determination of the main suppressive mechanisms used by these cell subsets in tumor-bearing hosts. As a result of the identification of protein targets responsible for MDSCs suppressive activity, different therapeutics agents have been used to eliminate/reduce their adverse effect. In the present work, we review the current knowledge on suppressive mechanisms of MDSCs and therapeutic treatments that interfere with their differentiation, expansion or activity. Based on the accumulation of new evidences supporting their importance for tumor progression and metastasis, the interest in these cell types is increasing. We revise the methods of MDSC generation/differentiation ex vivo that may help in overcoming problems associated with limited numbers of cells available from animals and patients for their study.

PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.

PDL1 blockade produces remarkable clinical responses, thought to occur by T cell reactivation through prevention of PDL1-PD1 T cell inhibitory interactions. Here, we find that PDL1 cell-intrinsic signaling protects cancer cells from interferon (IFN) cytotoxicity and accelerates tumor progression. PDL1 inhibited IFN signal transduction through a conserved class of sequence motifs that mediate crosstalk with IFN signaling. Abrogation of PDL1 expression or antibody-mediated PDL1 blockade strongly sensitized cancer cells to IFN cytotoxicity through a STAT3/caspase-7-dependent pathway. Moreover, somatic mutations found in human carcinomas within these PDL1 sequence motifs disrupted motif regulation, resulting in PDL1 molecules with enhanced protective activities from type I and type II IFN cytotoxicity. Overall, our results reveal a mode of action of PDL1 in cancer cells as a first line of defense against IFN cytotoxicity.