PD-L1 Gene Research Articles

Prognostic importance of PD-L1 expression in bladder cancer patients.

PD-L1/PD-1 molecules are known as important mediators in immune-escape mechanisms of tumors. PD-L1 is highly expressed in various malignancies, including bladder cancer. However, the prognostic value of PD-L1 in bladder cancer patients remains controversial. To investigate the prognostic significance of PD-L1 expression in tumor tissues of bladder cancer patients. RNA was isolated from FFPE tumor tissues of 48 bladder cancer patients using the monophasic phenol and guanidine isothiocyanate method. Total RNA was converted to cDNA and gene expression levels were analyzed by qRT-PCR. The differential expression levels of the PD-L1 gene between tumor grade and cancer stage groups were analyzed by independent student's t-test and one-way ANOVA. Statistically significantly increased PD-L1 expression was observed in the high-grade tumor group (p < 0.05). No significant difference in PD-L1 expression was found among pTa, pT1, and pT2 groups. In addition, the difference in overall survival was not significantly different between groups. The results showed that high PD-L1 expression in bladder cancer was associated with tumor aggressiveness and grade. Despite the inability of the qRT-PCR to show the PD-L1 expression at different locations of tumor tissue, evaluation of PD-L1 mRNA expression by qRT-PCR, which is a highly sensitive and specific assay, appears to be a robust approach. Furthermore, these findings may contribute to a rationale for recommending anti-PD-L1 immunotherapy as an alternative to standard therapy for bladder cancer patients who are most likely to benefit from it.

PD-L1 expression is regulated by ATP-binding of the ERBB3 pseudokinase domain

How PD-L1 expression is regulated in cancer is poorly understood. Here, we report that the ATP-binding activity of ERBB3 pseudokinase regulates PD-L1 gene expression in colorectal cancers (CRCs). ERBB3 is one of the four members of the EGF receptor family, all with protein tyrosine kinase domains. ERBB3 is a pseudokinase with a high binding affinity to ATP. We showed that ERBB3 ATP-binding inactivation mutant reduces tumorigenicity in genetically engineered mouse models and impairs xenograft tumor growth of CRC cell lines. The ERBB3 ATP-binding mutant cells dramatically reduce IFN-γ-induced PD-L1 expression. Mechanistically, ERBB3 regulates IFN-γ-induced PD-L1 expression through the IRS1-PI3K-PDK1-RSK-CREB signaling axis. CREB is the transcription factor that regulates PD-L1 gene expression in CRC cells. Knockin of a tumor-derived ERBB3 mutation located in the kinase domain sensitizes mouse colon cancers to anti-PD1 antibody therapy, suggesting that ERBB3 mutations could be predictive biomarkers for tumors amenable to immune checkpoint therapy.

CD97 serves as a novel biomarker of immune cell infiltration in hepatocellular carcinoma

BackgroundCD97 is the most widely expressed G protein-coupled receptor in the epidermal growth factor seven-span transmembrane family. It plays a vital role in cell adhesion, migration, and cell connection regulation. We explored the role of CD97 in hepatocellular carcinoma (HCC).MethodsWe evaluated CD97 mRNA expression in HCC using TNMplot and the Gene Expression Omnibus database. The clinical prognostic significance of CD97 in HCC patients was evaluated by gene expression profiling interactive analysis, the Kaplan–Meier plotter, and the UALCAN database. The Tumor Immune Estimation Resource (TIMER) and CIBERSORT databases were used to analyze the relationships among CD97, genes positively related with CD97, and tumor-infiltrating immune cells.ResultsCD97 was highly expressed in HCC tissues and was associated with an adverse prognosis. CD97 and genes positively related with CD97 were positively correlated with the abundance of tumor-infiltrating immune cells and strongly correlated with tumor-infiltrating macrophages (all r ≥ 0.513, P < 0.001). CD97 was positively correlated with M2 macrophage and tumor-associated macrophage markers (both r ≥ 0.464, P < 0.001). CD97 was found to be an immune-related gene in HCC and positively correlated with the TOX, PD-L1, PD-L2, CTLA4, and PD-1 immune checkpoint genes. CD97 copy number alterations affect the level of immune cell infiltration and mRNA expression.ConclusionsCD97 can be used as a potential molecular marker of prognosis in HCC, which is associated with immune cell infiltration.

Molecular, Epigenetic, and Immune Landscape of TP53-mutated (TP53-M) Acute Myeloid Leukemia (AML) and Higher Risk Myelodysplastic Syndromes (HR-MDS)

Introduction: Mutations in TP53 confer an adverse prognosis in patients (pts) with AML and HR-MDS. Comprehensive analyses of the genetic, epigenetic, and immunologic landscape of TP53-M AML and HR-MDS are lacking, especially associations of allelic status of TP53 with immune and epigenetic disease phenotype. Methods: Using a large, randomized study with extensive and prospective clinical, histopathological, cytogenetic, molecular, epigenetic, and immunologic annotations, we compared data from 61 TP53-M (37 AML, 24 HR-MDS) and 144 TP53 wild-type (TP53-WT; 89 AML, 55 HR-MDS) pts. Pts were randomized to azacitidine (AZA) monotherapy or AZA + anti-PDL1 antibody durvalumab, which did not improve clinical outcomes (Zeidan et al. Blood Advances 2022). A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed at Munich Leukemia Laboratory. Only pathogenic TP53 variants with variant allele frequency (VAF) ≥2% were included. Pts with a common SNP or VUS in the TP53 gene were classified as TP53-WT. Multi-hit TP53 mutations were defined as previously published by Grob et al. (Blood 2022) which confirmed that TP53-M AML and HR-MDS can be grouped as one entity. BM aspirates from screening and cycle 3 day 22 (C3D22) were used to study gene expression profiles by RNA-sequencing and immune phenotyping by flow cytometry. Gene set enrichment analysis (GSEA) using the MSigDB and H (Hallmark) gene sets as provided by the msigdbr R package were performed. Flow cytometry data were analyzed into a collection of 170 reportable subpopulations in the immune cell and immune checkpoint panel and 68 reportables in the tumor/blast panel. DNA methylation of peripheral blood samples was assessed using Illumina's Infinium Human Methylation EPIC methylation array pre-treatment and at C2D1. Global DNA methylation scores (GDMS) and focal DNA demethylation in PD-1, PD-L1 and PD-L2 regulatory regions were analyzed. Results: Of the 61 TP53-M and 144 TP53-WT pts, 85.4% and 10.4% had complex karyotype, respectively. TP53-M pts had fewer co-occurring mutations compared with TP53-WT (mean number of mutations: 1.8 [SD: 0.77] vs 2.9 [SD: 1.4]; P <0.001). Distinct mutational patterns by TP53 mutational status were noted (Figure 1). The median VAF for TP53 mutations was 40%, and 90% had a VAF ≥10%. 22 (11.0%) and 41 (20.5%) pts were mono-hit and multi-hit, respectively. Global DNA methylation was independent of TP53 mutation status at baseline when comparing mono-hit, multi-hit and wild-type. AZA therapy led to statistically significant decrease in GDMS independent of TP53 mutation status (Figure 2). Various loci of PD-1, PD-L1 and PD-L2 genes showed differences in methylation status by TP53 mutation status, which were reversed on treatment. However, methylation of 2 PD-L2 gene loci (cg07211259 and cg11299543) remained significantly lower for multi-hit TP53-M compared to TP53-W at C2D1. Patterns of differentially expressed genes were similar for multi-hit TP53-M vs wild-type and for all TP53-M vs wild-type. The number of differentially expressed genes was higher in the AML cohort compared to MDS pts both at baseline (1887 vs 78) and after C3 (116 vs 0) with TP53-M samples having more differentially expressed genes. ZNF560 was the gene with the strongest differential expression by TP53 mutation status. TP53-M pts had higher expression of T-cell genes (e.g., IL7R), markers of proliferation (MKI67), and PD-L1 (CD274) expression compared to TP53-W at baseline. Genes in hallmark clusters of heme metabolism, inflammatory response, cell cycle regulation, and TGF-β signaling pathways were enriched in TP53-M pts in GSEA. TP53-M pts had higher median levels of PD-L1-positive progenitors (5.6% vs 2.4%; p=0.006) and less myeloid progenitor cells (58% vs 75%; p=0.005) than TP53-WT at baseline. However, there was no shift towards a more exhausted T-cell phenotype among TP53-M pts (CD3+CD4+ PD1+ TIM3+ T-cells and CD3+CD8+PD1+TIM3+ T-cells as % of CD3+CD4+ T-cells and CD3+CD8+ T-cells, respectively). Conclusion: Although an increased population of cytotoxic T-cells suggest an activated immune system, we also found the upregulation of inhibitory immune checkpoints such as PD-L1 in TP53-mut pts suggesting an immunosuppressive microenvironment as a potential contributor to the poor prognosis of TP53-M AML and MDS with similar findings across disease type (HR-MDS vs AML) and mono- vs multi-hit TP53 mutation status. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immuno-Genetic Changes Underlie Response to Immune Checkpoint Blockade Therapy in Richter's Syndrome Mouse Models

Richter's syndrome (RS) represents one of the foremost clinical challenges in CLL, due to the disappointing responses to both standard chemotherapy and novel agents. Checkpoint blockade (CPB) with anti-PD-1 antibody treatment has shown promise in RS (but minimal efficacy in CLL), suggesting that distinct immuno-genetic features underlie differential response to immunotherapy of these two entities. We previously generated CRISPR-based multiplexed gene-edited mouse models of RS in immune competent backgrounds with genetic, epigenetic, transcriptional and functional similarity with human disease, thus enabling interrogation of changes in tumor microenvironmental (TME) features underlying CLL transformation. To this end, we analyzed the T-cell and monocyte/macrophage composition of 5 CLL, 3 CLL/RS (i.e. cases with equal co-existence of CLL and RS), and 9 murine RS cases by multi-parametric flow cytometry of splenocyte preparations, and compared them with 5 age-matched wild-type controls (15-18 month old). Whereas the overall proportional abundance of CD3+ T cells was not significantly changed across groups, we detected decreased CD4+ and increased CD8+ T cell populations in RS cases (P≤0.04, ANOVA). Both CD4+ and CD8+ T cells showed a prominent skewing toward the effector memory subset (CD44+ CD62L-), when compared to naïve (CD44- CD62L+) and central memory (CD44+ CD62L+). CD4+ and CD8+ T cell populations from RS cases also showed significant upregulation of exhaustion markers including PD-1, TIGIT, TIM3 and LAG3 (P≤0.05). The monocytic compartment was similarly skewed, with RS-associated enrichment in non-classical monocytes (CD43+ LyC6med/low, known to have lower antigen presentation capacity), when compared to classical inflammatory CD43+ LyC6high cells (P=0.04). Importantly, we measured increased abundance of CD11b+ Ly6C- tumor associated macrophages in RS cases (P=0.02), which expressed significantly reduced MHC class II (P=0.003), and increased PD-1 surface levels (P=0.02). Further, in 11 cases (3 CLL/RS, 8 RS) where at least 5-10% residual CLL cells co-existed with RS within the same animal, and the two populations were discernable by size, we measured increased expression levels of both PD-1 and PD-L1 ligands on RS compared to CLL by flow cytometry (P≤0.001, paired t test), consistent with surface PD-1 observed in human clonally related RS. Interestingly, surface PD-L1 levels were particularly enriched in RS cases carrying clonal deletion in Mga, a negative regulator of MYC (P=0.01, Mann Whitney), in line with the known role of MYC in upregulating PD-L1 gene expression. In light of these changes, we asked whether our animal models could be utilized as a reliable platform to study differential response to CPB. To this end, we leveraged transplantable models in immune competent mice. In our initial study, 40 million RS cells from a Trp53-deleted primary tumor were transplanted into 20 CD45.1 recipients (2 million/recipient), which were randomized either 1 [median disease burden: 2%(1-13.5)] or 2 weeks [37%(7-54)] post-transplant through peripheral blood (PB) flow cytometric assessment of circulating disease. Treatment with 200 micrograms rat IgG2a anti-PD-1 or isotype control (n=5 mice/group) was performed for 3 weeks, every 3 days, and animals euthanized 3 days after the last dose. Whereas animals with highest disease burden at treatment start (2 weeks post-transplant) showed minimal, if any, efficacy of CPB treatment, animals with lower disease burden at time of randomization (1 week post-transplant) showed significant reduction in RS infiltration of PB, spleen and bone marrow at euthanasia (P≤0.04, Mann Whitney). This suggests that disease burden may impact differential response to CPB. In conclusion, our analyses demonstrate marked exhaustion of the T cell and monocytic compartment and increased expression of PD-1 and PD-L1 on tumor cells and TME components upon RS transformation. Initial analyses showed variable benefit from single agent anti-PD-1 therapy in Trp53-mutant disease. We are currently exploring combinatorial strategies with targeted therapy (including PI3K and BTK inhibitors) to improve in vivo response rates in the context of tumor lines with distinct genetics and characterizing the immune microenvironmental changes induced by these treatments.

Genomic landscape, immune characteristics and prognostic mutation signature of cervical cancer in China

PurposeThis study aimed to analyse the genomic alteration profiles and immune characteristics of a cohort of Chinese cervical cancer patients to understand why certain patients benefited from molecular targeted therapies and immunotherapy as well as their prognostic significance.MethodsPD-L1 expression and clinicopathological information were obtained from 98 cervical cancer patients. Differences in PD-L1 expression and gene mutations between squamous cell carcinoma (SCC) and adenocarcinoma (AC) were analysed by the chi-square test or Fisher's exact test. Differences in gene mutations between our cohort and The Cancer Genome Atlas (TCGA) cohort were tested by Fisher's exact test. Logistic regression was used to analyse factors influencing TMB-high.ResultsPositive PD-L1 expression was significantly higher in cervical SCC than in cervical AC (87% vs. 39%, p < 0.001). Frequently mutated genes in cervical cancer included the PIK3CA, KMT2D, and KMT2C genes, among others. PIK3CA gene mutation rates were significantly higher in SCC than in AC (p = 0.004). The TERT gene mutation rate was significantly higher in our cohort than in the TCGA cohort (12% vs. 1%, p < 0.001). The independent predictors of high TMB were KMT2C and LRP1B gene mutations (p < 0.05). We also found that PTEN mutations were associated with worse survival (median PFS, 12.16 vs. 21.75 months, p = 0.0024).ConclusionCervical SCC and AC have different molecular profiles and immune characteristics, suggesting that targeted treatments for SCC and AC patients may improve clinical outcomes. KMT2C and LRP1B gene mutations are independent predictors of TMB-high status in cervical cancer. We also proposed the prognostic value of PTEN mutations.

EP400NL is involved in PD-L1 gene activation by forming a transcriptional coactivator complex

EP400 is an ATP-dependent chromatin remodelling enzyme that regulates DNA double-strand break repair and transcription, including cMyc-dependent gene expression. We previously showed that the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. As the EP400 N-terminal-Like (EP400NL) gene resides next to the EP400 gene locus, this prompted us to investigate whether EP400NL plays a similar role in transcriptional regulation to the full-length EP400 protein. We found that EP400NL forms a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, this EP400NL complex displays H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. We demonstrated that the transcriptional coactivator function of EP400NL is required for serum and IFNγ-induced PD-L1 gene activation. Furthermore, transcriptome analysis indicates that EP400NL contributes to cMyc-responsive mitochondrial biogenesis. Taken together, our studies show that EP400NL plays a role as a transcription coactivator of PD-L1 gene regulation and provides a potential target to modulate cMyc functions in cancer therapy.

Essential thrombocythaemia progression to the fibrotic phase is associated with a decrease in JAK2 and PDL1 levels

It has been postulated that the changes in the molecular characteristics of the malignant clone(s) and the abnormal activation of JAK-STAT signaling are responsible for myeloproliferative neoplasm progression to more advanced disease phases and the immune escape of the malignant clone. The continuous JAK-STAT pathway activation leads to enhanced activity of the promoter of CD274 coding programmed death-1 receptor ligand (PD-L1), increased PD-L1 level, and the immune escape of MPN cells. The aim of study was to evaluate the PDL1 mRNA and JAK2 mRNA level in molecularly defined essential thrombocythaemia (ET) patients (pts) during disease progression to post-ET- myelofibrosis (post-ET-MF). The study group consisted of 162 ET pts, including 30 pts diagnosed with post-ET-MF. The JAK2V617F, CALR, and MPL mutations were found in 59.3%, 19.1%, and 1.2% of pts, respectively. No copy-number alternations of the JAK2, PDL1, and PDCDL1G2 (PDL2) genes were found. The level of PD-L1 was significantly higher in the JAK2V617F than in the JAK2WT, CALR mutation-positive, and triple-negative pts. The PD-L1 mRNA level was weakly correlated with both the JAK2V617F variant allele frequency (VAF), and with the JAK2V617F allele mRNA level. The total JAK2 level in post-ET-MF pts was lower than in ET pts, despite the lack of differences in the JAK2V617F VAF. In addition, the PD-L1 level was lower in post-ET-MF. A detailed analysis has shown that the decrease in JAK2 and PDL1 mRNA levels depended on the bone marrow fibrosis grade. The PDL1 expression showed no differences in relation to the genotype of the JAK2 haplotypeGGCC_46/1, hemoglobin concentration, hematocrit value, leukocyte, and platelet counts. The observed drop of the total JAK2 and PDL1 levels during the ET progression to the post-ET-MF may reflect the changes in the JAK2V617F positive clone proliferative potential and the PD-L1 level–related immunosuppressive effect. The above-mentioned hypothesis is supported by The Cancer Genome Atlas (TCGA) data, confirming a strong positive association between CD274 (encoding PD-L1), CXCR3 (encoding CXCR3), and CSF1 (encoding M-CSF) expression levels, and recently published results documenting a drop in the CXCR3 level and circulating M-CSF in patients with post-ET-MF.

Chimeric immune checkpoint protein vaccines inhibit the tumorigenesis and growth of rat cholangiocarcinoma

Cholangiocarcinoma (CCA) isthe second most common primary liver malignancy and carries a dismal prognosis due to difficulties in achieving an optimal resection, and poor response to current standard-of-care systemic therapies. We previously devised a CTLA4-PD-L1 DNA cancer vaccine (DNA vaccine) and demonstrated its therapeutic effects on reducing tumor growth in a thioacetamide (TAA)-induced rat intrahepatic CCA (iCCA) model. Here, we developed a CTLA4-PD-L1 chimeric protein vaccine (Protein vaccine), and examined its effects in the rat iCCA model. In a therapeutic setting, iCCA-bearing rats received either DNA plus Protein vaccines or Protein vaccine alone, resulting in increased PD-L1 and CTLA-4 antibody titers, and reduced iCCA tumor burden as verified by animal positron emission tomography (PET) scans. Treating iCCA-bearing rats with Protein vaccine alone led to the increase of CTAL4 antibody titers that correlated with the decrease of tumor SUV ratio, indicating regressed tumor burden, along with increased &lt;i&gt;CD8&lt;/i&gt; and granzyme A (&lt;i&gt;GZMA&lt;/i&gt;) expression, anddecreased PD-L1 expression on tumor cells. In a preventive setting,DNA or Protein vaccines were injected in rats before the induction of iCCA by TAA. Protein vaccines induced a more sustained PD-L1 and CTLA-4 antibody titers compared with DNA vaccines, and was more potent in preventing iCCA tumorigenesis. Correspondingly, Protein vaccines, but not DNA vaccines, downregulated PD-L1 gene expression and hindered the carcinogenesis of iCCA. Taken together, the CTLA4-PD-L1 chimeric proteinvaccine may function both as a therapeuticcancer vaccine andas a preventive cancer vaccine in theTAA-induced iCCA rat model.

INCREASED PROGRAMMED DEATH LIGAND-1 EXPRESSION LEADS TO WORSE CLINICAL FEATURES IN PATIENTS WITH MYELODYSPLASTIC SYNDROME

Immune checkpoints are regulators of the immune system response that allow self-tolerance. Molecules such as Programmed Cell Death Protein 1 (PD-1) and its Ligand (PD-L1) participate in the immune checkpoint by signaling co-inhibition of lymphocyte responses. In cancers, PD-L1 expression is associated with the immune evasion mechanism, which favors tumor growth. The role of PD-L1 is already known for several solid tumors and anti-PD-L1 drugs are used for treatment. Myelodysplastic Syndrome (MDS) is a heterogeneous bone marrow disease with an increased risk of progression to Acute Myeloid Leukemia (AML). The pathogenesis of MDS involves several factors, including alterations in immune pathways. However, the role of PD-L1 still lacks understanding in this disease. The aim of this study was to evaluate if PD-L1 gene expression would be linked to clinical and prognostic features in patients with myelodysplastic syndrome. In this study, we evaluated PD-L1 mRNA expression by real-time PCR using TaqMan ® probes. Fifty-three patients classified according to WHO 2016 were analyzed. We observed that patients with dyserythropoiesis have higher PD-L1 expression than patients without dyserythropoiesis (p = 0.050). Furthermore, patients classified as excess of blasts 2 (MDS-EB2) presented an upregulation in mRNA expression of PD-L1 compared to excess of blasts 1 (MDS-EB1) (p = 0.050). Throughout the statistical analysis, we detected three patients with very high levels of PD-L1 expression, being classified as outliers. Analyzing these three patients specifically, all of them had hemoglobin lower than 8g/dL, blast count higher than 8%, dysplasia in at least two sectors and had died by the end of the study. The increase of PD-L1 in MDS may be considered a marker of poor prognosis.

Lymphopenic condition enhanced the antitumor immunity of PD-1-knockout T cells mediated by CRISPR/Cas9 system in malignant melanoma

BackgroundAdoptive transfer of PD-1 knockout T cells mediated by CRISPR/Cas9 technology has been used in various cancers and got satisfactory treatment effectiveness. However, the effectiveness was limited due to the low proliferation ability, antigen recognition ability and short lifetime of T cells in vivo. MethodIn this study, PD-1 knockout T cells mediated by CRISPR/Cas9 system were transferred into lymphopenic mice after sub-lethal dose of total body irradiation. The antitumor effects of PD-1 knockout T cells were comprehensively analyzed by flow cytometry. Moreover, PD-L1 knockout B16 cells were inoculated subcutaneously in lymphopenic mice receiving infusion of naïve T cells to value the role of PD-1/PD-L1 axis on lymphopenia-induced antitumor immunity ResultIn this study, we found that the PD-1-knockout T cells underwent several rounds of homeostatic proliferation in vivo when they were transferred into lymphopenic mice. The number of IFN-γ-releasing CTL was significantly increased and the tumor growth was remarkably inhibited in lymphopenic mice receiving infusion of PD-1 knockout T cells. The expression of PD-L1 on tumor cells rose smartly in lymphopenic mice undergoing homeostatic proliferation. PD-L1 gene knockout on B16 melanoma cells could effectively enhance the antitumor immunity mediated by the homeostatic proliferation of T cells and significantly inhibited the growth of tumor ConclusionThese findings suggested that lymphopenic condition after total body irradiation might be able to create an environment to promote the PD-1 knockout T cells to recognize tumor antigen and undergo homeostatic proliferation, thus induced a more powerful antitumor immunity than adoptively transferring into immunocompetent hosts.

HSP70-Promoter-Driven CRISPR/Cas9 System Activated by Reactive Oxygen Species for Multifaceted Anticancer Immune Response and Potentiated Immunotherapy.

To address the low response rate to immune checkpoint blockade (ICB) therapy, we propose a specific promoter-driven CRISPR/Cas9 system, F-PC/pHCP, that achieves permanent genomic disruption of PD-L1 and elicits a multifaceted anticancer immune response to potentiate immunotherapy. This system consists of a chlorin e6-encapsulated fluorinated dendrimer and HSP70-promoter-driven CRISPR/Cas9. F-PC/pHCP under 660 nm laser activated the HSP70 promoter and enabled the specific expression of the Cas9 protein to disrupt the PD-L1 gene, preventing immune escape. Moreover, F-PC/pHCP also induced immunogenic cell death (ICD) of tumor cells and reprogrammed the immunosuppressive tumor microenvironment. Overall, this specific promoter-driven CRISPR/Cas9 system showed great anticancer efficacy and, more importantly, stimulated an immune memory response to inhibit distant tumor growth and lung metastasis. This CRISPR/Cas9 system represents an alternative strategy for ICB therapy as well as enhanced cancer immunotherapy.

A novel pyroptosis-related LncRNA signature predicts prognosis and indicates tumor immune microenvironment in skin cutaneous melanoma

AimsTo explore the correlation between the pyroptosis-related lncRNAs (PRlncRNAs) and the prognosis of skin cutaneous melanoma (SKCM), and clarify the effects of the PRlncRNAs on the tumor immune infiltration. Main methodsIn this study, we utilized RNA-seq and clinical characteristics data obtained from TCGA and GEO database to perform co-expression analysis and LASSO Cox regression analysis to construct a 12-PRlncRNA prognostic prediction model. We also performed functional analysis, immune infiltration analysis and drug sensitivity analysis, as well as correlation analysis with autophagy/ferroptosis genes, tumor mutational burden, and PD-1/PD-L1 genes. Key findingThe model based on the 12-PRlncRNA signature could effectively predict the prognosis of SKCM. Low risk group had a higher anti-tumor immune level generally compared with high-risk group. The signature was correlated with the expression of autophagy/ferroptosis-related genes and PD1/PD-L1 genes and tumor mutational burden. Additionally, drug sensitivity analysis indicated potential therapeutic targets. SignificanceOur study demonstrated the impact of PRlncRNAs on SKCM. The model established based on the 12-PRlncRNA showed significant prognostic value for SKCM and may be instructive in pyroptosis-related targeted therapy in the clinic.

Genomic landscape, immune characteristics, and a prognostic mutation signature of advanced cervical cancer in China (162)

Objectives: This study aimed to analyze genomic alteration profiles and immune characteristics of a cohort of Chinese advanced cervical cancer patients to understand why certain patients benefited from molecular targeted therapies, immunotherapy, and their prognostic significance. Methods: PD-L1 expression and clinical-pathologic information from 98 cervical cancer samples were obtained. Cervical cancer data were obtained from The Cancer Genome Atlas (TCGA). Differences in PD-L1 expression and gene mutations between squamous cell carcinoma (SCC) and adenocarcinoma (AC) were analyzed using the Chi-square test or Fisher’s exact test. Differences in gene mutations between our cohort and TCGA cohort were tested by Fisher’s exact test. The relationship between gene mutation and TMB was analyzed by the Mann-Whitney U test. Logistic regression was used to analyze factors influencing TMB-high. Kaplan-Meier survival analyses were applied. Results: Positive PD-L1 expression was significantly higher in cervical SCC than in cervical AC (87% vs 39%, p < 0.001). Frequently mutated genes in cervical cancer included PIK3CA, KMT2D, and KMT2C genes, among others. PIK3CA gene mutation rates were significantly higher in SCC than in AC (p = 0.004). TERT gene mutation rate was significantly higher in our cohort than TCGA cohort (12% vs 1%, p < 0.001). SCC had a significantly higher TMB than AC (p < 0.001). The independent predictors of TMB-high were KMT2C and LRP1B gene mutations (p <0.05). In this study, the median progression-free survival (PFS) was 12.16 months in the PTEN mutant group versus 21.75 months in the wild-type group (p=0.0024). Conclusions: Cervical SCC and AC have different molecular profiling and immune characteristics, suggesting that targeted treatments for SCC and AC patients may improve clinical outcomes. KMT2C and LRP1B gene mutations are independent predictors of TMB-high status in cervical cancer. We have also proposed the prognostic value of PTEN mutation. Objectives: This study aimed to analyze genomic alteration profiles and immune characteristics of a cohort of Chinese advanced cervical cancer patients to understand why certain patients benefited from molecular targeted therapies, immunotherapy, and their prognostic significance. Methods: PD-L1 expression and clinical-pathologic information from 98 cervical cancer samples were obtained. Cervical cancer data were obtained from The Cancer Genome Atlas (TCGA). Differences in PD-L1 expression and gene mutations between squamous cell carcinoma (SCC) and adenocarcinoma (AC) were analyzed using the Chi-square test or Fisher’s exact test. Differences in gene mutations between our cohort and TCGA cohort were tested by Fisher’s exact test. The relationship between gene mutation and TMB was analyzed by the Mann-Whitney U test. Logistic regression was used to analyze factors influencing TMB-high. Kaplan-Meier survival analyses were applied. Results: Positive PD-L1 expression was significantly higher in cervical SCC than in cervical AC (87% vs 39%, p < 0.001). Frequently mutated genes in cervical cancer included PIK3CA, KMT2D, and KMT2C genes, among others. PIK3CA gene mutation rates were significantly higher in SCC than in AC (p = 0.004). TERT gene mutation rate was significantly higher in our cohort than TCGA cohort (12% vs 1%, p < 0.001). SCC had a significantly higher TMB than AC (p < 0.001). The independent predictors of TMB-high were KMT2C and LRP1B gene mutations (p <0.05). In this study, the median progression-free survival (PFS) was 12.16 months in the PTEN mutant group versus 21.75 months in the wild-type group (p=0.0024). Conclusions: Cervical SCC and AC have different molecular profiling and immune characteristics, suggesting that targeted treatments for SCC and AC patients may improve clinical outcomes. KMT2C and LRP1B gene mutations are independent predictors of TMB-high status in cervical cancer. We have also proposed the prognostic value of PTEN mutation.

Pilot Study: Immune Checkpoints Polymorphisms in Greek Primary Breast Cancer Patients.

Background: Breast cancer is the most prevalent and second leading cause of cancer-related death in women worldwide. Despite early detection and better treatment therapies, 30% of early-stage breast cancer patients still develop recurrent disease. Breast cancer is a heterogeneous disease comprising several molecular subtypes, commonly classified into clinical subtypes based on the hormone receptor status. These subtypes included luminal A and luminal B, which have different prognoses. Breast cancer development and progression involve many factors. Polymorphisms of PD-1, PD-L1, and PD-L2 genes have been previously associated with high risk and prognosis of cancer. However, no studies have associated PD-1, PD-L1, and PD-L2 polymorphisms with primary breast cancer subtypes. Hence, this study evaluated functional single nucleotide polymorphisms of PD-1, PD-L1, and PD-L2 with primary breast cancer subtypes, luminal A, and luminal B. In addition, we evaluated the PD-L1 protein expression in relation to primary breast cancer subtypes and stages. Results: There were no significant differences in the allele frequencies of PD-1 polymorphisms (rs2227981 G>A, rs7421861 A>G, and rs11568821 C>T) and PD-L1 polymorphisms (rs10815225 C>T and rs2282055 T>G) when compared with the general European population. However, a significant difference was detected in one of the PD-L2 polymorphisms (rs1009759 A>G), with the G allele higher in breast cancer patients than in the general European population. A higher prevalence of the T allele of PD-L1 polymorphism rs2282055 T>G was observed in luminal B breast cancer patients compared with luminal A. No significant difference was detected in other polymorphisms. We also observed that the PD-L1 rs2282055 TT genotype was more prevalent in luminal B breast cancer patients compared with luminal A. Our results found no association of the selected SNPs in the PDCD1 gene with breast cancer risk. Similarly, the protein expression data showed that PD-L1 and PD-L2 are associated with an aggressive phenotype, Luminal B, and advanced breast cancer stage. Conclusion: These findings suggest that immune checkpoint polymorphisms are associated with the risk and subtypes of breast cancer.

Abstract 5884: Comprehensive analyzing the expression of IDO1 and TDO2 in bladder cancer

Abstract Background: Several lines of evidence have been focused on the roles of tryptophan metabolism genes like indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in bladder cancer progression. However, the alternative way of targeting in cancer immunotherapy has been not yet investigated in bladder cancer. Method: We explored and validated the clinicopathological significance and the correlation with immune infiltrates of IDO1 and TDO2 expression in bladder cancer using Bladder urothelial carcinoma (BLCA) and other Gene Expression Omnibus (GEO) datasets (GSE13570, GSE31684, GSE48277) downloaded from https://xenabrowser.net/ and https://www.ncbi.nlm.nih.gov/geo/. The correlation between IDO1, TDO2 expression and the immune infiltrates as well as PD-L1 gene expression in BLCA dataset was explored with TIMER2.0, http://timer.comp-genomics.org/. Immunohistochemistry was performed to validate the relationship between TDO2 and PD-L1 expression in bladder cancer. Results: We figured out the IDO1 and TDO2 expressions were significantly upregulated in basal type compared with other molecular subtypes in BLCA. P53 mutations were associated with upregulation of IDO1 and TDO2 expression (p&amp;lt;0.01) whereas FGFR3 mutation was associated with downregulation of IDO1 and TDO2 expression (p&amp;lt;0.001). The overexpression of IDO1, TDO2 overexpression was significantly associated with advanced disease in BC. IDO1 and TDO2 expression were significantly correlated with purity and immune cell infiltrates, including T cells CD8+, T cells CD4+, B cells, neutrophils, and macrophages as wells as myeloid dendritic cells. Interestingly, TDO2 were positively correlated with cancer-associated fibroblasts (CAFs) (rho=0.5, p=1.14e-24). PD-L1 expression was also correlated with IDO1 (rho=0.64, p=4.42e-49) and TDO2 expression (rho=0.42, p=5.01e-19). Immunohistochemistry revealed the correlation between TDO2 and PD-L1 expression (p=0.011). Conclusion: Our results pointed out that IDO1 and TDO2 could play an essential role in regulating the tumor microenvironment as well as immune tolerance in bladder cancer. Suggesting that IDO1, TDO2 might be a promising novel immunotherapy target for bladder cancer patients. Citation Format: Quoc Thang Pham, Thanh Tu Nguyen, Thao Quyen Nguyen, Duc Tung Luu, Thi Nhu Diem Pham, Thi Thanh Tam Bui, Thanh Tu Duong, Dang Anh Thu Phan, Quoc Dat Ngo. Comprehensive analyzing the expression of IDO1 and TDO2 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5884.

Abstract 4252: PD-L1-targeted ISAC combines myeloid cell activation, immune-checkpoint inhibition and ADCP to improve anti-tumor efficacy over anti-PD-L1 antibodies in preclinical models

Abstract INTRODUCTION: Immune-stimulating antibody conjugates (ISACs) consist of tumor-targeting antibodies conjugated to immune stimulants and are designed to activate the innate and adaptive immune systems against tumor cells following systemic administration. PD-L1 is an immune checkpoint that regulates anti-tumor T cell responses and is expressed on tumor cells as well as tumor-infiltrating immune cells across many tumor types. Here we show that PD-L1-targeted TLR7/8 ISACs elicit robust myeloid cell activation and can act through PD-L1 on either tumor or immune cells to improve anti-tumor responses compared to anti-PD-L1 treatment in preclinical tumor models. METHODS: A panel of proprietary anti-PD-L1 ISACs was evaluated for target-dependent in vitro myeloid cell activation by co-culturing PD-L1 expressing tumor cells with cDC-enriched primary myeloid cells. Anti-tumor efficacy of anti-PD-L1 ISACs was evaluated in vivo in both syngeneic and xenograft tumor models. MB49-PD-L1 KO cells were generated by knocking-out PD-L1 gene using CRISPR/Cas9 system. RESULTS: Anti-PD-L1 antibodies induced robust ADCP by myeloid effector cells and PD-L1/PD-1 blockade in vitro and in vivo. The conjugated PD-L1 ISACs induced robust, target-dependent activation of myeloid cells when co-cultured with tumor cells expressing PD-L1 at physiological levels, as measured by increased secretion of such cytokines as IL-12p70, TNFα, and IFNγ. Systemically administered surrogate PD-L1 ISACs were well tolerated in mice and showed improved anti-tumor efficacy over anti-PD-L1 antibodies, with significant tumor growth delay or complete responses frequently observed in syngeneic (e.g., MB49, MC38-hPD-L1) as well as xenograft (e.g., HCC1954-hPD-L1) tumor models. The improved in vivo efficacy of PD-L1 ISACs was sustained even in the absence of PD-L1 expression on tumor cells in syngeneic MB49-PD-L1 KO model suggesting that PD-L1 ISAC can induce its mechanism of action also through PD-L1 on myeloid cells. No tumor growth was observed after rechallenging mice previously cured with PD-L1 ISACs indicating development of immunological memory following the ISAC treatment. CONCLUSIONS: These preclinical data demonstrate the potential of a PD-L1-targeted ISAC as a novel multifunctional therapeutic that may improve efficacy of PD-L1/PD-1 inhibition by combining three mechanisms of action into a single molecule: TLR-mediated myeloid cell activation, T cell activation through immune-checkpoint inhibition as well as ADCP. Citation Format: Justin A. Kenkel, Rishali Gadkari, Po Y. Ho, Lisa K. Blum, Romas Kudirka, Karla A. Henning, William G. Mallet, Jennifer E. Melrose, Ganapathy Sarma, Steven J. Chapin, Matthew Zhou, Suprit Deol, Cindy Kreder, Yuyi Shen, Bruce Hug, Puneet Anand, Arthur Lee, Hai Li, Shelley E. Ackerman, Brian S. Safina, David Dornan, Michael N. Alonso, Marcin Kowanetz. PD-L1-targeted ISAC combines myeloid cell activation, immune-checkpoint inhibition and ADCP to improve anti-tumor efficacy over anti-PD-L1 antibodies in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4252.

Abstract 2046: Potential suppression of B effector cells by exosomal PD-L1 on anti-cancer stem cell immunity

Abstract We have reported that cancer stem cell (CSC) vaccination induces CSC-specific T and B cell responses associated with therapeutic efficacy which can be enhanced by anti-PD-L1 administration. Tumor cells have been recognized to suppress T cell function via secretion of exosomes that express PD-L1. Whether a similar suppression by CSCs on B cells exists is unknown. In this study, 4T1 (breast cancer) and CT26 (colon cancer) tumor-bearing mice were administered with anti-CD20 mAb to deplete B cells prior to anti-PD-L1 mAb administration. Depletion of B cells as compared to the controls resulted in more aggressive tumor growth, and the anti-tumor efficacy of anti-PD-L1 was significantly reduced for both 4T1 and CT26, indicating the involvement of host B cells in the anti-tumor effect of anti-PD-L1. Administration of anti-PD-L1 partially recovered the humoral immune response, confirming the PD-L1/PD-1 pathway involvement in B cell suppression. We detected elevated expression of PD-1 on activated B cells, and higher PD-L1 expression on ALDHhigh CSCs than on ALDHlow non-CSCs. We co-cultured purified B cells with ALDHhigh CSCs with or without the addition of anti-PD-L1 mAb and found that the reduction of IgG secretion of B cells suppressed by CSCs could be rescued by anti-PD-L1 in a dose-dependent manner. These experiments indicate that CSCs suppress the IgG production by B cells via the PD-L1/PD-1 axis. In a different model, we isolated exosomes from D5 (melanoma) tumor cell culture media by differential ultracentrifugation and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers: CD63, HRS, ALIX, and TSG101. We generated Rab27ako D5 and F10 (melanoma) cells to delete exosomes by the CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells. We also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-targeted CRISPR/Cas9, confirming the knockout of the PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Based on these findings, the use of Rab27ako and PD-L1ko D5 and F10 CSCs will allow us to further characterize the potential suppression of exosomal PD-L1 on host B cells as well as T cells. Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Potential suppression of B effector cells by exosomal PD-L1 on anti-cancer stem cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2046.

Small extracellular vesicles derived from PD-L1-modified mesenchymal stem cell promote Tregs differentiation and prolong allograft survival.

We aimed to explore whether programmed cell death protein-1 ligand (PD-L1) modification on small extracellular vesicles (sEVs) could promote T regulatory cells (Tregs) differentiation. In this study, it was confirmed that under physiological conditions, PD-L1 expression was minimal in the MSCs and absent in the MSC-sEVs. A vector harboring the PD-L1 gene was constructed and transfected into bone marrow mesenchymal stem cells (BM-MSCs). By extracting the sEVs of these modified BM-MSCs and monitoring the expression of the PD-L1 protein, however, PD-L1 expression was substantially increased in the MSCs and concentrated in the sEVs. Then, the rat naïve CD4 + T cells were cocultured with the sEVs derived from the PD-L1-modified MSCs (sEVsPD-L1). By flow cytometry, a higher percentage of Tregs and anti-inflammatory downstream cytokines (including IL-2, IFN-γ, TGF-β, IL-10) was detected in the sEVsPD-L1 group than that in the control group treated by either sEVs in wild type, modified by empty vector, or blank control. Suppressive effect on CD4 + T cell proliferation serves as additional evidence to support the immunoregulation capacity of sEVsPD-L1. The animal model of vascularized composite allograft further confirmed that PD-L1-modified sEVs induce an immune tolerance, by clinically observation, histopathology, T cell fate and cell product. In conclusion, sEVsPD-L1 efficiently promotes Treg cell differentiation in vitro and in vivo, which suggests their therapeutic potential in the treatment of allograft rejection.

SCALE-1: Safety and efficacy of short course neoadjuvant chemo-radiotherapy plus toripalimab for locally advanced resectable squamous cell carcinoma of esophagus.

4063 Background: Adding PD-1 inhibitors to neoadjuvant chemoradiotherapy have shown promising results in treatment of locally advanced esophageal squamous cell carcinoma (ESCC). However, there is a need to explore more effective and safe treatment doses and schedules. The aim of this single arm phase Ib trial is to determine the safety and efficacy of short course neoadjuvant chemo-radiotherapy plus toripalimab following by esophagectomy in patients with locally advanced resectable ESCC. Methods: Patients (pts) with histopathologically confirmed thoracic ESCC, diagnosed as clinical stage of cT3-4aN0M0/cT1-4aN+M0 per AJCC 8th and ECOG PS of 0-1 were eligible. Toripalimab (240mg) was given with paclitaxel (135 mg/m2) and carboplatin (AUC=5) on D1 every 3 weeks for two cycles. Short course neoadjuvant radiotherapy (3000 cGy in 12 fractions on 5 days per week) was administered from D3 to D18. Esophagectomy was scheduled 4 to 6 weeks after the completion of neoadjuvant therapy. Target total enrollment was 20 pts. The primary endpoint was treatment safety. Pre- and post-treatment specimens were collected for PD-L1 expression assay and gene expression profiles (GEPs) analysis using the nCounter platform. Results: From Jan 29, 2021 to Nov 3, 2021, 26 patients were screened and 23 met the inclusion criteria. The majority of them were diagnosed with clinical stage of cT3-T4 (95.6%), cN1-2 (62.5%), and cTNM III-IVA (78.2%). All 23 pts finished neoadjuvant radiotherapy, while two of them didn’t receive or received reduced dose of the second cycle chemotherapy or toripalimab due to toxicity. Notable grade 3 or higher AEs included leukopenia (n=21), neutropenia (n=21), anorexia (n=19) and nausea (n=18). No treatment related oesophagitis, pneumonia and gastrointestinal hemorrhage above Grade 3 were observed. 20 pts underwent surgery after a median of 7 weeks post neoadjuvant therapy. Reasons for not undergoing surgery were patients’ choice (n=2) and tuberculosis reactivation (n=1). 3 pts with Grade III surgical complications were observed during the perioperative period. 11/20 (55%) complete pathological responses (ypT0N0) were observed, 16/20 (80%) pts were major pathologic response (MPR) and the remaining 4 pts achieved partial response. Pre-treatment GEP analysis revealed significantly higher expression levels of CXCL10, CXCL11, OAS2, and lower level of CD209 and KLRB1 in pCR (n=6) than in Non-pCR group (n=4). Moreover, pCR group showed a trend of upregulated signatures of tumor infiltrating leukocytes, cytolytic activity, cytotoxic T cell, T cell markers and Teff score (P= 0.062) after treatment. Conclusions: Combining short course nCRT with toripalimab is feasible and effective in patients with locally advanced resectable ESCC, and might be a promising approach for neoadjuvant treatment. Clinical trial information: ChCTR2100045104.