PD-L1 lncRNA splice isoform promotes lung adenocarcinoma progression via enhancing c-Myc activity
Qu, S;Jiao, Z;Lu, G;Yao, B;Wang, T;Rong, W;Xu, J;Fan, T;Sun, X;Yang, R;Wang, J;Yao, Y;Xu, G;Yan, X;Wang, T;Liang, H;Zen, K;
PMID: 33849634 | DOI: 10.1186/s13059-021-02331-0
Although using a blockade of programmed death-ligand 1 (PD-L1) to enhance T cell immune responses shows great promise in tumor immunotherapy, the immune-checkpoint inhibition strategy is limited for patients with solid tumors. The mechanism and efficacy of such immune-checkpoint inhibition strategies in solid tumors remains unclear. Employing qRT-PCR, Sanger sequencing, and RNA BaseScope analysis, we show that human lung adenocarcinoma (LUAD) all produce a long non-coding RNA isoform of PD-L1 (PD-L1-lnc) by alternative splicing, regardless if the tumor is positive or negative for the protein PD-L1. Similar to PD-L1 mRNA, PD-L1-lnc in various lung adenocarcinoma cells is significantly upregulated by IFNγ. Both in vitro and in vivo studies demonstrate that PD-L1-lnc increases proliferation and invasion but decreases apoptosis of lung adenocarcinoma cells. Mechanistically, PD-L1-lnc promotes lung adenocarcinoma progression through directly binding to c-Myc and enhancing c-Myc transcriptional activity. In summary, the PD-L1 gene can generate a long non-coding RNA through alternative splicing to promote lung adenocarcinoma progression by enhancing c-Myc activity. Our results argue in favor of investigating PD-L1-lnc depletion in combination with PD-L1 blockade in lung cancer therapy.
Pharmaceuticals (Basel, Switzerland)
Konkoly, J;Kormos, V;Gaszner, B;Sándor, Z;Kecskés, A;Alomari, A;Szilágyi, A;Szilágyi, B;Zelena, D;Pintér, E;
PMID: 34959735 | DOI: 10.3390/ph14121336
Transient receptor potential ankyrin 1 (TRPA1), a nonselective cation channel, contributes to several (patho)physiological processes. Smell loss is an early sign in several neurodegenerative disorders, such as multiple sclerosis, Parkinson's and Alzheimer's diseases; therefore, we focused on its role in olfaction and social behaviour with the aim to reveal its potential therapeutic use. The presence of Trpa1 mRNA was studied along the olfactory tract of mice by combined RNAscope in situ hybridisation and immunohistochemistry. The aversive effects of fox and cat odour were examined in parallel with stress hormone levels. In vitro calcium imaging was applied to test if these substances can directly activate TRPA1 receptors. The role of TRPA1 in social behaviour was investigated by comparing Trpa1 wild-type and knockout mice (KO). Trpa1 mRNA was detected in the olfactory bulb and piriform cortex, while its expression was weak in the olfactory epithelium. Fox, but not cat odour directly activated TRPA1 channels in TRPA1-overexpressing Chinese Hamster Ovary cell lines. Accordingly, KO animals showed less aversion against fox, but not cat odour. The social interest of KO mice was reduced during social habituation-dishabituation and social interaction, but not during resident-intruder tests. TRPA1 may contribute to odour processing at several points of the olfactory tract and may play an important role in shaping the social behaviour of mice. Thus, TRPA1 may influence the development of certain social disorders, serving as a potential drug target in the future.
Xue T, Wang WG, Zhou XY, Li XQ.
PMID: - | DOI: 10.1016/j.pathol.2018.08.011
Summary Programmed cell death ligand 1 (PD-L1) is upregulated in various types of haematological malignancies and is associated with immunosuppression. This study aimed to investigate the expression pattern of PD-L1 in Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL). We retrospectively analysed clinicopathological characteristics in 30 cases of EBV-positive DLBCL and immunohistochemically evaluated the level of membrane bound PD-L1 protein. Twenty-eight cases expressed PD-L1 protein 15 of which showed an intense positive staining. In addition, we investigated the relationships between PD-L1 protein and PD-L1 mRNA and MYC, respectively. The expression level of PD-L1 protein was not fully parallel with PD-L1 mRNA, and no significant correlation was observed between PD-L1 protein and MYC. Notably, PD-L1 mRNA was at a low dosage, which indicated that there might be other mechanisms inducing the overexpression of membrane bound PD-L1 protein apart from genetic alterations. Furthermore, the low expression level of MYC may not interfere with the PD-L1 protein expression in EBV-positive DLBCL. In conclusion, overexpression of PD-L1 protein can be observed in EBV-positive DLBCL, and the level was non-parallel with both PD-L1 mRNA and MYC. Moreover, we emphasise that immunohistochemistry is a clinically reasonable method for screening formalin fixed, paraffin embedded (FFPE) tumour samples in this entity.
Guo L, Li W, Zhu X, Ling Y, Qiu T, Dong L, Fang Y, Yang H, Ying J.
PMID: 27390646 | DOI: 10.1186/s40064-016-2513-x
Abstract
PURPOSE:
To estimate the therapeutic potential of PD-L1 inhibition in breast cancer, we evaluated the prevalence and significance of PD-L1 protein expression with a validated antibody and CD274 gene alternation in a large cohort of triple negative breast cancer (TNBC) and correlated with clinicopathological data and patients overall survival.
METHODS:
Immunohistochemistry and in situ mRNA hybridization was used to detect PD-L1 protein and mRNA expression in tumor tissues from 183 TNBC patients respectively. Fluorescence in situ hybridization analysis was performed on PD-L1 strong expression samples to assess copy number on chromosome 9p24.1 of CD274 gene.
RESULTS:
Expression of PD-L1 by immune cells was observed in 4.9 % of TNBC, while expression by tumor cells accounted for 8.7 %. There was a high concordance in PD-L1 protein expression and PDL1 mRNA expression. Samples with PD-L1 strong expression were found to have a CD274 gene copy number gain. PD-L1 expression was correlated with higher tumor grade, but was independent of menopausal status, lymph nodes metastasis, histological subtype and tumor size. In addition, we used precise stratification of PD-L1 expression on tumor or immune cells of certain breast cancer subtype and suggested that patients with PD-L1 expression in basal-like tumors by immune cells or with CD274 gene copy number gain had a longer disease-specific overall survival.
CONCLUSIONS:
Our findings may promote the more precise analysis of PD-L1 expression in breast cancer and aid the selection of patients who may benefit from immune therapy.
Licenziato, L;Minoli, L;Ala, U;Marconato, L;Fanelli, A;Giannuzzi, D;De Maria, R;Iussich, S;Orlando, G;Bertoni, F;Aresu, L;
PMID: 36951124 | DOI: 10.1177/03009858231162209
Canine diffuse large B-cell lymphoma (cDLBCL) is characterized by high mortality and clinical heterogeneity. Although chemo-immunotherapy improves outcome, treatment response remains mainly unpredictable. To identify a set of immune-related genes aberrantly regulated and impacting the prognosis, we explored the immune landscape of cDLBCL by NanoString. The immune gene expression profile of 48 fully clinically characterized cDLBCLs treated with chemo-immunotherapy was analyzed with the NanoString nCounter Canine IO Panel using RNA extracted from tumor tissue paraffin blocks. A Cox proportional-hazards model was used to design a prognostic gene signature. The Cox model identified a 6-gene signature (IL2RB, BCL6, TXK, C2, CDKN2B, ITK) strongly associated with lymphoma-specific survival, from which a risk score was calculated. Dogs were assigned to high-risk or low-risk groups according to the median score. Thirty-nine genes were differentially expressed between the 2 groups. Gene set analysis highlighted an upregulation of genes involved in complement activation, cytotoxicity, and antigen processing in low-risk dogs compared with high-risk dogs, whereas genes associated with cell cycle were downregulated in dogs with a lower risk. In line with these results, cell type profiling suggested the abundance of natural killer and CD8+ cells in low-risk dogs compared with high-risk dogs. Furthermore, the prognostic power of the risk score was validated in an independent cohort of cDLBCL. In conclusion, the 6-gene-derived risk score represents a robust biomarker in predicting the prognosis in cDLBCL. Moreover, our results suggest that enhanced tumor antigen recognition and cytotoxic activity are crucial in achieving a more effective response to chemo-immunotherapy.
Filley A, Henriquez M, Bhowmik T, Tewari BN, Rao X, Wan J, Miller MA, Liu Y, Bentley RT, Dey M.
PMID: 29330750 | DOI: 10.1007/s11060-018-2753-4
Malignant glioma (MG), the most common primary brain tumor in adults, is extremely aggressive and uniformly fatal. Several treatment strategies have shown significant preclinical promise in murine models of glioma; however, none have produced meaningful clinicalresponses in human patients. We hypothesize that introduction of an additional preclinical animal model better approximating the complexity of human MG, particularly in interactions with host immune responses, will bridge the existing gap between these two stages of testing. Here, we characterize the immunologic landscape and gene expression profiles of spontaneous canine glioma and evaluate its potential for serving as such a translational model. RNA in situ hybridization, flowcytometry, and RNA sequencing were used to evaluate immune cell presence and gene expression in healthy and glioma-bearing canines. Similar to human MGs, canine gliomas demonstrated increased intratumoral immune cell infiltration (CD4+, CD8+ and CD4+Foxp3+ T cells). The peripheral blood of glioma-bearing dogs also contained a relatively greater proportion of CD4+Foxp3+ regulatory T cells and plasmacytoid dendritic cells. Tumors were strongly positive for PD-L1 expression and glioma-bearing animals also possessed a greater proportion of immune cells expressing the immune checkpoint receptors CTLA-4 and PD-1. Analysis of differentially expressed genes in our canine populations revealed several genetic changes paralleling those known to occur in human disease. Naturally occurring canine glioma has many characteristics closely resembling human disease, particularly with respect to genetic dysregulation and host immune responses to tumors, supporting its use as a translational model in the preclinical testing of prospective anti-glioma therapies proven successful in murine studies.
Capsazepine decreases corneal pain syndrome in severe dry eye disease
Journal of neuroinflammation
Fakih, D;Guerrero-Moreno, A;Baudouin, C;Goazigo, AR;Parsadaniantz, SM;
PMID: 33975636 | DOI: 10.1186/s12974-021-02162-7
Dry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED. Chronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests. First, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED. These data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.
International journal of molecular sciences
Szallasi, A;
PMID: 37240443 | DOI: 10.3390/ijms24109098
Temperature-sensitive transient receptor potential (TRP) channels (so-called "thermoTRPs") are multifunctional signaling molecules with important roles in cell growth and differentiation. Several "thermoTRP" channels show altered expression in cancers, though it is unclear if this is a cause or consequence of the disease. Regardless of the underlying pathology, this altered expression may potentially be used for cancer diagnosis and prognostication. "ThermoTRP" expression may distinguish between benign and malignant lesions. For example, TRPV1 is expressed in benign gastric mucosa, but is absent in gastric adenocarcinoma. TRPV1 is also expressed both in normal urothelia and non-invasive papillary urothelial carcinoma, but no TRPV1 expression has been seen in invasive urothelial carcinoma. "ThermoTRP" expression can also be used to predict clinical outcomes. For instance, in prostate cancer, TRPM8 expression predicts aggressive behavior with early metastatic disease. Furthermore, TRPV1 expression can dissect a subset of pulmonary adenocarcinoma patients with bad prognosis and resistance to a number of commonly used chemotherapeutic agents. This review will explore the current state of this rapidly evolving field with special emphasis on immunostains that can already be added to the armoire of diagnostic pathologists.
Coppock JD, Volaric AK, Mills AM, Gru AA.
PMID: 30075155 | DOI: 10.1016/j.humpath.2018.07.025
Targeted inhibition of programmed cell death-1 (PD-1) and its ligand (PD-L1) has emerged as first-line therapy for advanced non-small cell lung cancer. While patients with high PD-L1 expression have improved outcomes with anti-PD-1/PD-L1 directed therapies, use as a predictive biomarker is complicated by robust responses in some patients with low-level expression. Furthermore, reported PD-L1 levels in lung cancers vary widely and discrepancies exist with different antibodies. PD-L1 expression was thus compared by immunohistochemistry (IHC) versus RNA in situ hybridization (ISH) in 112 lung cancers by tissue microarray: 51 adenocarcinoma, 42 squamous cell carcinoma, 9 adenosquamous carcinoma, 5 carcinoid, 3 undifferentiated large-cell carcinoma, 1 large-cell neuroendocrine carcinoma, and 1 small cell carcinoma. At least 1% tumor cell staining was considered positive in each modality. A positive concordance of only 60% (67/112) was found between IHC and ISH. 50% (56/112) were positive by IHC and 50% (56/112) by ISH, however 20% (22/112) were ISH positive but IHC negative. Conversely, 21% (23/112) were IHC positive but ISH negative. There was no significant stratification of PD-L1 positivity by histologic subtype. A trend of more PD-L1 positive stage I cancers identified by ISH versus IHC was observed, however was not statistically significant [50% (27/54) by IHC and 64% (35/55) by ISH, P=.18]. No significant difference in survival was identified, with an average of 5.3months in IHC versus 5.2months in ISH positive cases. The results demonstrate discordance between PD-L1 RNA levels and protein expression in non-small cell lung cancers, warranting comparison as predictive biomarkers.
Wu S, Shi X, Sun J, Liu Y, Luo Y, Liang Z, Wang J, Zeng X.
PMID: 28145884 | DOI: 10.18632/oncotarget.14851
Abstract
BACKGROUND:
Lung adenocarcinoma (AD) is a common variant of non-small cell lung cancer (NSCLC). Programmed cell death protein 1/programmed cell death ligand 1 (PD1/PD-L1) are promising immunotherapy targets and its expression may be an important biomarker of predicting clinical response. In this study, we evaluated PD-L1 expression in conjunction with clinicopathological characteristics and outcomes in resected lung adenocarcinoma.
RESULTS:
This study included 133 cases of lung adenocarcinoma. PD-L1 expression rate in lung adenocarcinoma was 16.5% at the mRNA level and 13.5% at the protein level, and the kappa coefficient of the two examination methods was 0.824 (P = 0.219, highly correlated). PD-L1 was highly expressed in male patients and smokers with lung adenocarcinoma (P = 0.019 and 0.002, respectively), while no associations were identified between PD-L1 expression and age, tumor size, clinical stage, positive pleural invasion, lymph node metastasis, or therapy methods. Overexpression of PD-L1 was a significant indicator of shorter recurrence free survival time and overall survival (P = 0.000 and 0.000, respectively). Multivariate analysis revealed that PD-L1 expression was an independent risk factor for poor recurrence free survival and overall survival (P = 0.009 and 0.016, respectively).
MATERIALS AND METHODS:
Expression of PD-L1 was examined with immunohistochemistry, using the VENTANA PD-L1 (SP263) rabbit monoclonal antibody. mRNA levels of PD-L1 were evaluated using in situ hybridization.
CONCLUSIONS:
PD-L1 overexpression is more frequently observed in male patients and smokers in lung adenocarcinoma. PD-L1 expression is an indicator of worse prognosis in surgically resected lung adenocarcinoma patients.
Veterinary immunology and immunopathology
Murphy, JD;Shiomitsu, K;Milner, RJ;Lejeune, A;Ossiboff, RJ;Gell, JC;Axiak-Bechtel, S;
PMID: 36804838 | DOI: 10.1016/j.vetimm.2023.110560
Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-β, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope ). After validation was performed, RNAscope on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-β and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope method was validated, TGF-β H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-β dots/cell. In summary, RNAscope is a valid technique to detect TGF-β and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-β and PD-L1. Increased TGF-β and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-β, PD-L1, and Tregs effect on prognosis.
Tretiakova M, Fulton R, Kocherginsky M, Long T, Ussakli C, Antic T, Gown A.
PMID: 29271413 | DOI: 10.1038/modpathol.2017.188
Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of correlation between expression and clinical outcomes and immunotherapyresponse may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72-0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86-0.94). Manual scores were highly concordant with automated Aperio scores (0.94-0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.
