Lung Cancer Tissue Microarray Research Articles

Neurotrophin-3 Facilitates Stemness Properties and Associates with Poor Survival in Lung Cancer.

Cancer stem cells (CSCs) shape the tumor microenvironment via neuroendocrine signaling and orchestrate drug resistance and metastasis. Cytokine antibody array demonstrated the upregulation of neurotrophin-3 (NT-3) in lung CSCs. This study aims to dissect the role of NT-3 in lung CSCs during tumor innervation. Western blotting, quantitative reverse transcription-PCR, and flow cytometry were used to determine the expression of the NT-3 axis in lung CSCs. NT-3-knockdown and NT-3-overexpressed cells were derived lung CSCs, followed by examining the stemness gene expression, tumorsphere formation, transwell migration and invasion, drug resistance, soft agar colony formation, and in vivo tumorigenicity. Human lung cancer tissue microarray and bioinformatic databases were used to investigate the clinical relevance of NT-3 in lung cancer. NT-3 and its receptor tropomyosin receptor kinase C (TrkC) were augmented in lung tumorspheres. NT-3 silencing (shNT-3) suppressed the migration and anchorage-independent growth of lung cancer cells. Further, shNT-3 abolished the sphere-forming capability, chemo-drug resistance, invasion, and in vivo tumorigenicity of lung tumorspheres with a decreased expression of CSC markers. Conversely, NT-3 overexpression promoted migration and anchorage-independent growth and fueled tumorsphere formation by upregulating the expression of CSC markers. Lung cancer tissue microarray analysis revealed that NT-3 increased in patients with advanced-stage, lymphatic metastasis and positively correlated with Sox2 expression. Bioinformatic databases confirmed a co-expression of NT-3/TrkC-axis and demonstrated that NT-3, NT-3/TrkC, NT-3/Sox2, and NT-3/CD133 worsen the survival of lung cancer patients. NT-3 conferred the stemness features in lung cancer during tumor innervation, which suggests that NT-3-targeting is feasible in eradicating lung CSCs.

RGS20 promotes non-small cell lung carcinoma proliferation via autophagy activation and inhibition of the PKA-Hippo signaling pathway

BackgroundNovel therapeutic targets are urgently needed for treating drug-resistant non-small cell lung cancer (NSCLC) and overcoming drug resistance to molecular-targeted therapies. Regulator of G protein signaling 20 (RGS20) is identified as an upregulated factor in many cancers, yet its specific role and the mechanism through which RGS20 functions in NSCLC remain unclear. Our study aimed to identify the role of RGS20 in NSCLC prognosis and delineate associated cellular and molecular pathways.MethodsImmunohistochemistry and lung cancer tissue microarray were used to verify the expression of RGS20 between NSCLC patients. CCK8 and cell cloning were conducted to determine the proliferation ability of H1299 and Anip973 cells in vitro. Furthermore, Transcriptome sequencing was performed to show enrichment genes and pathways. Immunofluorescence was used to detect the translocation changes of YAP to nucleus. Western blotting demonstrated different expressions of autophagy and the Hippo-PKA signal pathway. In vitro and in vivo experiments verified whether overexpression of RGS20 affect the proliferation and autophagy of NSCLC through regulating the Hippo pathway.ResultsThe higher RGS20 expression was found to be significantly correlated with a poorer five-year survival rate. Further, RGS20 accelerated cell proliferation by increasing autophagy. Transcriptomic sequencing suggested the involvement of the Hippo signaling pathway in the action of RGS20 in NSCLC. RGS20 activation reduced YAP phosphorylation and facilitated its nuclear translocation. Remarkably, inhibiting Hippo signaling with GA-017 promoted cell proliferation and activated autophagy in RGS20 knock-down cells. However, forskolin, a GPCR activator, increased YAP phosphorylation and reversed the promoting effect of RGS20 in RGS20-overexpressing cells. Lastly, in vivo experiments further confirmed role of RGS20 in aggravating tumorigenicity, as its overexpression increased NSCLC cell proliferation.ConclusionOur findings indicate that RGS20 drives NSCLC cell proliferation by triggering autophagy via the inhibition of PKA-Hippo signaling. These insights support the role of RGS20 as a promising novel molecular marker and a target for future targeted therapies in lung cancer treatment.

SCARA3 inhibits cell proliferation and EMT through AKT signaling pathway in lung cancer

BackgroundScavenger receptor class A member 3 (SCARA3) is decreased in prostate cancer and myeloma. However, functions of SCARA3 in various cancers remain unclear. In this study, we tried to evaluate the functional study of SCARA3 in lung cancer.MethodsThe expression level of SCARA3 in the TCGA-database, lung cancer tissue microarray and lung cancer cells and the prognosis of lung cancer patients were measured. Lung cancer tissue microarray was analyzed pathologically using immunohistochemistry, and quantitative analysis of SCARA3 in normal lung cells and lung cancer cells was analyzed using western blot analysis. Survival curves for lung cancer patients were prepared with the Kaplan-Meier method. Migration and invasion of SCARA3 overexpressed lung cancer cells were determined using a Transwell chamber system. Proliferation of lung cancer cells was determined based on cell viability assay using cell culture in vitro and a tumorigenicity model of BALB/C nude mouse in vivo.ResultsThe expression of SCARA3 was abnormally reduced in TCGA-database, lung tissue microarray, and various lung cancer cells. However, overexpression of SCARA3 reduced the proliferation of lung cancer. The ability of SCARA3 to inhibit cancer cell proliferation was maintained even in vivo using a mouse xenograft model. In addition, overexpression of SCARA3 reduced migration and invasion ability of lung cancer cells and induced decreases of EMT markers such as β-catenin, vimentin, and MMP9. We aimed to prove the role of SCARA3 in the treatment of Lung cancer, and shown that the expression level of SCARA3 is important in cancer treatment using cisplatin. The enhancement of the effect of cisplatin according to SCARA3 overexpression is via the AKT and JNK pathways.ConclusionsThis study confirmed an abnormal decrease in SCARA3 in lung cancer. Overexpression of SCARA3 potently inhibited tumors in lung cancer and induced apoptosis by increasing sensitivity of lung cancer to cisplatin. These results suggest that SCARA3 is a major biomarker of lung cancer and that the induction of SCARA3 overexpression can indicate an effective treatment.

Unsupervised Segmentation in NSCLC: How to Map the Output of Unsupervised Segmentation to Meaningful Histological Labels by Linear Combination?

Background: Segmentation is, in many Pathomics projects, an initial step. Usually, in supervised settings, well-annotated and large datasets are required. Regarding the rarity of such datasets, unsupervised learning concepts appear to be a potential solution. Against this background, we tested for a small dataset on lung cancer tissue microarrays (TMA) if a model (i) first can be in a previously published unsupervised setting and (ii) secondly can be modified and retrained to produce meaningful labels, and (iii) we finally compared this approach to standard segmentation models. Methods: (ad i) First, a convolutional neuronal network (CNN) segmentation model is trained in an unsupervised fashion, as recently described by Kanezaki et al. (ad ii) Second, the model is modified by adding a remapping block and is retrained on an annotated dataset in a supervised setting. (ad iii) Third, the segmentation results are compared to standard segmentation models trained on the same dataset. Results: (ad i–ii) By adding an additional mapping-block layer and by retraining, models previously trained in an unsupervised manner can produce meaningful labels. (ad iii) The segmentation quality is inferior to standard segmentation models trained on the same dataset. Conclusions: Unsupervised training in combination with subsequent supervised training offers for histological images here no benefit.

ACC2 is under-expressed in lung adenocarcinoma and predicts poor clinical outcomes.

Acetyl-CoA Carboxylases (ACCs) are key fatty acid metabolic enzymes responsible for catalyzing the carboxylation of acetyl-CoA to malonyl-CoA. The role of ACC1 has been associated with tumor biology, but the role of ACC2 in cancer remains largely uncharacterized. We conducted a transcriptomic analysis using GEPIA and Oncomine to study the expression of ACC2 in different cancers. Immunohistochemistry was used to examine the expression of ACC2 in lung cancer tissue microarray, and the correlation between ACC2 expression and clinical parameters was analyzed. Following ACC2 knockdown by RNA interference in A549 and HCC827 cells, Cell Counting Kit‑8 and transwell assays were used to detect cell proliferation and migration. Real-time PCR was used to detect cell cycle-related genes in A549 cells. GEO dataset and KM-plotter database were used to analyze the relationship between ACC2 expression and the prognosis in lung cancer patients. We found that ACC2 is under-expressed in cancerous tissue and the expression of ACC2 is negatively correlated with tumor size, regional lymph-node metastases, and clinical stage of lung adenocarcinoma patients. In addition, knocking down ACC2 in A549 cells and HCC827 cells can promote cell proliferation and migration, and cell cycle-related genes MAD2L1 and CCNB2 were up-regulated after ACC2 was knockdown in A549 cells. Finally, we found that lung adenocarcinoma patients with under-expressed ACC2 have a worse prognosis. Our results suggest that ACC2 is a potential diagnostic and prognostic marker that negatively correlated with clinical outcomes in lung adenocarcinoma.

Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study

BackgroundEmerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is...

Knockdown of SALL4 inhibits the proliferation, migration, and invasion of human lung cancer cells in vivo and in vitro.

BackgroundThis study aimed to investigate the SALL4 expression in lung cancer, determine if SALL4 regulates the biological functions of lung cancer cells at the cellular level, and clarify the possible mechanisms involved.MethodsImmunohistochemistry was used to detect the SALL4 expression messenger RNA (mRNA) in 62 cases of lung cancer tissue microarray. The correlation of SALL4 with the clinical pathological parameters and overall life cycle of patients and the impact of disease-free life cycle was analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the SALL4 expression in lung cancer cell lines and nude mouse models. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, colony-forming assay, and flow cytometry were used to detect the effects of interference with SALL4 expression on lung cancer cell proliferation and transplant tumor models; the effect of interference with SALL4 expression on the growth of transplanted tumors in vivo was also examined.ResultsSALL4 was highly expressed in lung cancer tissues and cell lines and was closely related to the patient’s TNM stage and lymph node metastasis. Compared to patients with a high SALL4 expression, those with a lower SALL4 expression had a longer overall and disease-free survival. The expression of SALL4 is an independent risk factor for the prognosis of lung cancer patients. Interference with SALL4 expression can significantly inhibit cell proliferation and clonal formation. Interfering with the expression of SALL4 can arrest the cells in the G0/G1 phase by inhibiting the expression of the cell cycle–related proteins, cyclin B, cyclin E, and cyclin D1. Furthermore, wound-healing and Transwell assays showed that interference with SALL4 expression could significantly inhibit the migration and invasion of lung cancer cells, while experiments in nude mice showed that interference with SALL4 expression could significantly inhibit the size and weight of transplanted tumors.ConclusionsSALL4 was highly expressed in lung cancer cell lines. Interference with the expression of SALL4 can effectively inhibit the proliferation, migration and invasion of lung cancer cells, promote cell cycle arrest, and play the function of tumor suppressor genes. SALL4 may be a new target for the diagnosis and treatment of lung cancer.

High-Plex and High-Throughput Digital Spatial Profiling of Non-Small-Cell Lung Cancer (NSCLC).

Simple SummaryCharacterizing the tumour microenvironment (TME) has become increasingly important to understand the cellular interactions that may be at play for effective therapies. In this study, we used a novel spatial profiling tool, the Nanostring GeoMX Digital Spatial Profiler (DSP) technology, to profile non-small-cell lung cancer (NSCLC) for protein markers across immune cell typing, immune activation, drug targets, and tumour modules. Comparative analysis was performed between the tumour, adjacent tissue, and microenvironment to identify markers enriched in these areas with spatial resolution. Our study reveals that this methodology can be a powerful tool for determining the expression of a large number of protein markers from a single tissue slide.Profiling the tumour microenvironment (TME) has been informative in understanding the underlying tumour–immune interactions. Multiplex immunohistochemistry (mIHC) coupled with molecular barcoding technologies have revealed greater insights into the TME. In this study, we utilised the Nanostring GeoMX Digital Spatial Profiler (DSP) platform to profile a non-small-cell lung cancer (NSCLC) tissue microarray for protein markers across immune cell profiling, immuno-oncology (IO) drug targets, immune activation status, immune cell typing, and pan-tumour protein modules. Regions of interest (ROIs) were selected that described tumour, TME, and normal adjacent tissue (NAT) compartments. Our data revealed that paired analysis (n = 18) of matched patient compartments indicate that the TME was significantly enriched in CD27, CD3, CD4, CD44, CD45, CD45RO, CD68, CD163, and VISTA relative to the tumour. Unmatched analysis indicated that the NAT (n = 19) was significantly enriched in CD34, fibronectin, IDO1, LAG3, ARG1, and PTEN when compared to the TME (n = 32). Univariate Cox proportional hazards indicated that the presence of cells expressing CD3 (hazard ratio (HR): 0.5, p = 0.018), CD34 (HR: 0.53, p = 0.004), and ICOS (HR: 0.6, p = 0.047) in tumour compartments were significantly associated with improved overall survival (OS). We implemented both high-plex and high-throughput methodologies to the discovery of protein biomarkers and molecular phenotypes within biopsy samples, and demonstrate the power of such tools for a new generation of pathology research.

Effects of stomatin protein expression on proliferation and apoptosis of lung cancer cells

Objective: To investigate the effect of stomatin protein expression on the proliferation and apoptosis of lung cancer cells. Methods: The expressions of stomatin mRNA in human bronchial epithelial cells (HBE) and lung cancer cells (H520, A549, 95D, H460, Glc-82, 973 and H1299) were detected by Real-time PCR. Immunohistochemistry (IHC) was used to detect stomatin protein expression in 4 lung cancer tissue microarrays with 259 cases of lung cancer and adjacent normal tissues. After knocking down the expression of stomatin in A549 cells, the proliferation was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, the apoptosis was detected by flow cytometry, the expression levels of total protein kinase B (AKT) and phosphorylated AKT at Ser473 were detected by Western blot. BALB/c nude mice were used to detect the tumorigenic ability of stomatin downregulated A549 cells (3 mice) and control cells (3 mice), and the protein expressions of stomatin, Ki67 and CD31 in tumor tissues were detected by IHC. Results: The M (range) of stomatin mRNA expression level in H520, A549, 95D, H460, Glc-82, 973, H1299 and HBE cells were 2.71 (2.66), 3.55 (3.16), 0.26 (0.22), 2.08 (1.98), 0.87 (0.35), 1.72 (2.53), 1.10 (1.82) and 0.01 (0.02), respectively. The mRNA expression levels of stomatin in H520, A549 and H460 cells were higher than that of HBE cells (all P<0.05), whereas there was no statistical difference among 95D, Glc-82, 973, H1299 and HBE cells (all P>0.05). IHC of lung tissue microarrays showed that the positive rate of stomatin expression in human lung cancer tissues was 34.7% (90/259), which was significantly higher than that in adjacent normal tissues [1.9% (5/259)] (P<0.05). In stomatin positive lung cancer tissues, the M (IQR) of tumor size for lower stomatin expression tissues (67 cases) was [41.22 (2 761.50) cm], which was smaller than that of higher stomatin expression tissues [(23 cases, 57.98(1 333.50) cm) (P<0.05). After knocking down stomatin expression, the fourth day absorbance value of stomatin-downregulated A549 cells was 0.55±0.07, which was lower than that of control cells (0.79±0.16) (P=0.012). The proportion of early apoptotic cells of stomatin-downregulated A549 cells [8.83 (53.00)] was higher than that of control cells [4.17 (25.00)] (P=0.026). The Ser473 phosphorylated AKT protein expression level in stomatin-downregulated A549 cells was 0.68±0.16, which was decreased compared with control cells (1.16±0.39) (P<0.05). The M (IQR) of total AKT expression level in stomatin-downregulated A549 cells was 4.25 (17.00), without statistically significant difference from control cells [4.75 (19.00)] (P>0.05). After inoculation of stomatin-downregulated A549 cells in nude mice for 43 days, the tumor volume was (37.93±3.12) mm(3), which was significantly smaller than that of the control group [(454.04±32.39) mm(3)] (P<0.001). And the expression levels of stomatin, nuclear proliferation antigen Ki67, and platelet-endothelial cell adhesion molecule CD31 were 1.78±0.69, 5.19±3.84, and 10.77±1.67, respectively, which were all decreased compared with control group (17.52±8.76, 54.14±41.02, and 19.72±6.97, respectively) (all P<0.05). Conclusion: Stomatin promotes lung cancer cell proliferation and inhibits cell early apoptosis by regulating AKT signaling pathway.

P1.14-22 SASH1, a Novel Prognostic and Predictive Factor for PARP Inhibitors in Lung Cancer

Genomic instability is a universal hallmark of all cancers. Many of the most commonly used chemotherapeutic agents target this genomic instability by directly damaging the DNA, which results in tumour cell death. Our previous work has revealed that loss of SASH1 is associated with impaired apoptosis and increased cellular proliferation. A new generation of drugs have been developed that target the DNA repair enzyme PARP to induce DNA damage and cell death. SASH1 (SAM and SH3 domain containing protein 1) has been described as a tumour suppressor and in support of this SASH1 mRNA levels are decreased in lung, breast, thyroid and colorectal cancers. Our data demonstrates that SASH1 functions in the repair of DNA damage and loss of SASH1 protein expression could be used as a companion diagnostic for PARP inhibitors. SASH1 IHC staining of lung cancer was correlated with patient survival. DNA damage repair was assessed following the depleted of SASH1 (siRNA). SASH1 protein levels in cell lines were correlated to PARP inhibitor sensitivity. A lung cancer tissue microarray (TMA) of 225 patients was assessed for SASH1 protein level. Low SASH1 levels were associated with an improved patient prognosis in adenocarcinoma on univariate analysis (p = 0.03). Analysis of DNA repair pathways demonstrated that SASH1 plays a role in homologous recombination (HR). Based on this observation, the impact of SASH1 expression on sensitivity to PARP inhibitors was explored. An inverse correlation between SASH1 levels and sensitivity to Olaparib was identified in lung cancer cell lines Figure 1 (R2 = 0.882). We subsequently analysed Olaparib sensitivity in a panel of SASH1 depleted lung cancer cells that demonstrated increased Olaparib sensitivity. Our results indicate that SASH1 protein expression is a prognostic factor in lung cancer, high levels being associated with a worse prognosis in adenocarcinoma. Low SASH1 expression is associated with loss of HR and has the potential to be a predictive biomarker for sensitivity to PARP inhibitors in this disease.

Oncogenic enhancer of zeste homolog 2 is an actionable target in patients with non-small cell lung cancer.

BackgroundThe aims of this study were to investigate the link between enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDAC) in preclinical studies and in human lung cancer tissue microarrays.MethodsEnhancer of zeste homolog 2 and HDAC1 mRNA expression in two lung adenocarcinoma (LUAD) datasets (MDACC and TCGA) were correlated with patient outcomes. We evaluated the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor, suberoylanilide hydroxamic acid (SAHA). The response to SAHA was assessed at baseline and after alteration of EZH2 or HDAC mRNA expression in LUAD cell lines.ResultsDirect correlation was found between EZH2 and HDAC1 expression (P < 0.0001). When EZH2 expression was knocked down‐ or upregulated, there was a corresponding decrease or increase in expression of HDAC expression, respectively. Cell lines with high EZH2 expression responded to SAHA treatment with a mean inhibition rate of 73.1% compared to 43.2% in cell lines with low EZH2 expression (P < 0.0001). This correlation was confirmed in non‐small cell lung cancer (NSCLC) specimens from MDACC (Spearman's correlation r = 0.416; P < 0.0001) and TCGA datasets (r = 0.221; P < 0.0001). Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of both datasets had the lowest survival compared to the patients with low expression of either or both markers.ConclusionOur findings show that overexpression of EZH2 is a negative prognostic indicator. Increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting NSCLC patients for treatment with HDAC inhibitors.

Abstract LB-318: Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow

Abstract Background: Emerging data suggests that predictive biomarkers based on the spatial arrangement of multiple cell types in FFPE tissue sections will be an important component of precision medicine in immune-oncology. Multiplexed immunofluorescence (mIF) facilitates such assessments. If mIF is to play a translational role in research and ultimately clinical practice, it is vital to refine, standardize, and validate an end-to-end workflow that supports large scale multi-site trials and clinical laboratory processes. To this end, six institutions collaborated to develop an automated 6-plex assay focused on the PD-1/PD-L1 axis and assessed its inter- and intra-site reproducibility. Specific attention was paid to assessment of %PD-L1 expression by immune cells (ICs), as pathologists have poor concordance for this parameter as noted in the Blueprint 2 and multi-institutional NCCN studies on PD-L1 IHC. Methods: A 7-color mIF panel (PD-L1, PD-1, CD8, CD68, FoxP3, Cytokeratin, and DAPI) was optimized on a Leica Bond Rx autostainer. Serial sections of tonsil and a lung cancer tissue-microarray (TMA), antibodies and TSA-Opal detection reagents (Akoya Biosciences) were distributed. Cell pellet arrays were also distributed and used to normalize batch variation in intensity measurements. Tonsil and TMA sections were stained at each site and imaged at 20x using a Vectra Polaris. Cells were segmented and phenotyped using image analysis algorithms. In tonsil sections, the average intensity of the top quartile of cells positive for each marker was assessed to identify potential variation in staining intensity. In lung TMAs, cell densities and %PD-L1 expression in immune cells (CD68+ and CD8+ cells) was determined. Results: The average staining intensity coefficients of variation (CV) for all markers within sites was 10% in tonsil samples. Inter-site concordance for tumor cell and immune cell subset densities in TMAs, had an average R2 value of 0.86 and slope of 0.96. Inter-site concordance for %PD-L1+ ICs had an average R2 value of 0.81, and slope of 0.82, in contrast to ICC values of &amp;lt;0.3 in the NCCN and Blueprint 2 studies. Conclusions: We demonstrate a reproducible end-to-end process for mIF characterization of the PD-1/PD-L1 axis including automated staining, multispectral imaging, and machine-learning-trained image analysis algorithms. This approach improved reproducibility of %PD-L1 IC assessment and brought it in line with %PD-L1 tumor cell assessment by pathologists. Sources of variation were identified and will be discussed. The described approach may serve as a template for assessing reproducibility of emerging mIF panels for other investigative teams, with an eye toward translating such approaches into clinical trials and ultimately into the clinic. Citation Format: Clifford Hoyt, Kristin Roman, Liz Engle, Chichung Wang, Carmen Ballesteros-Merino, Shawn M. Jensen, John McGuire, Yi Zheng, Carla Coltharp, Mei Jiang, Justin Lucas, Edwin Parra, Ignacio Wistuba, Darren Locke, Bernard A. Fox, David L. Rimm, Janis Marie Taube. Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-318.

Abstract 513: Investigating the expression of indolamine deoxygenase I (IDO1) and other immune markers in tissue microarrays

Abstract Purpose of the study: To gain insight to the proportion of solid tumors that express IDO1, CD73 and PD-L1 in lung cancer, colon adenocarcinoma, renal cell carcinoma (RCC) and melanoma, and the correlation between expression of these immune checkpoints and immune cell infiltration within the tumor. Experimental procedures: Using a battery of properly validated antibodies, we have performed an in situ analysis of IDO1, CD73 and PD-L1 expression by immunohistochemistry in a panel of colon cancer (n=68), lung cancer (n=36), RCC (n=67) and melanoma (n=48) tissue microarray samples. Tumor infiltrating T cells, CD3+ and CD8+, were also detected by IHC and counted in serial sections for each sample. We search for a possible a correlation between the expression of these immunosuppressor proteins and the number of infiltrating CD3+ and CD8+ T cells as well as their expression in the different tumor components: tumor cells, stroma and endothelium. Results: Our IHC analysis allowed us to classify the analyzed samples into two main groups: those with expression of IDO1, CD73 and PD-L1, which were also the ones with a high presence of immune cells within the tumors, “hot tumors”, and those that showed to be negative for all three proteins, and that correlated with lower infiltration of T cells, “cold tumors”. We also analyzed the location of the proteins within the tumors, and found an effect between endothelial expression of Ido1 and reduced infiltration in lung and RCC tumors. Conclusions: The response to combined immune checkpoint and IDO1 therapy could depend on the proper stratification of patients; therefore, it is of significant interest to determine the proportion of solid tumors that express IDO1, CD73 and PD-L1, and the correlation of the expression of these three immune check points to immune cell infiltration within the tumor. Thus, proper patient stratification based on the expression of these immune checkpoints may improve the efficacy of IDO1 inhibitor based immune therapies. Citation Format: Ana Cerezo, Gloria Martinez-del Hoyo, Sandra Peregrina, Tamara Mondejar, Eduardo Caleiras, Patricia Gonzalez, Eva Lospitao, Sonia Hernandez-Tiedra, Laura Diezma, Estela Casas, Frank Dorsey, Karim Benhadji, Raymond Gilmour, Sandaruwan Geeganage, Susana Velasco. Investigating the expression of indolamine deoxygenase I (IDO1) and other immune markers in tissue microarrays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 513.

Lung Adenocarcinoma Syndecan-2 Potentiates Cell Invasiveness.

Altered expression of syndecan-2 (SDC2), a heparan sulfate proteoglycan, has been associated with diverse types of human cancers. However, the mechanisms by which SDC2 may contribute to the pathobiology of lung adenocarcinoma have not been previously explored. SDC2 levels were measured in human lung adenocarcinoma samples and lung cancer tissue microarrays using immunohistochemistry and real-time PCR. To understand the role of SDC2 in vitro, SDC2 was silenced or overexpressed in A549 lung adenocarcinoma cells. The invasive capacity of cells was assessed using Matrigel invasion assays and measuring matrix metalloproteinase (MMP) 9 expression. Finally, we assessed tumor growth and metastasis of SDC2-deficient A549 cells in a xenograft tumor model. SDC2 expression was upregulated in malignant epithelial cells and macrophages obtained from human lung adenocarcinomas. Silencing of SDC2 decreased MMP9 expression and attenuated the invasive capacity of A549 lung adenocarcinoma cells. The inhibitory effect of SDC2 silencing on MMP9 expression and cell invasion was reversed by overexpression of MMP9 and syntenin-1. SDC2 silencing attenuated NF-κB p65 subunit nuclear translocation and its binding to the MMP9 promoter, which were restored by overexpression of syntenin-1. SDC2 silencing in vivo reduced tumor mass volume and metastasis. These findings suggest that SDC2 plays an important role in the invasive properties of lung adenocarcinoma cells and that its effects are mediated by syntenin-1. Thus, inhibiting SDC2 expression or activity could serve as a potential therapeutic target to treat lung adenocarcinoma.

Comparison of programmed death-ligand 1 (PD-L1) immunostain for nonsmall cell lung carcinoma between paired cytological and surgical specimens.

Background:programmed death-ligand 1 (PD-L1) is a ligand for the inhibitory programmed cell death protein 1 (PD-L1), which are targeted by several anti-PD-1 and PD-L1 drugs for lung cancer treatment. In clinical practice, many lung cancer cases only have cytology samples available to test PD-L1. Our current study compared the PD-L1 immunohistochemistry (IHC) between paired cytological and surgical samples.Materials and Methods:Formalin-fixed lung cancer tissue microarray and paired cell blocks and surgical specimens from the same patients with a confirmed diagnosis of lung squamous cell carcinoma (SCC, n = 29) and adenocarcinoma (AC, n = 23) were sectioned for PD-L1 IHC.Results:PD-L1 was expressed on tumor cells in 16 of 29 (55%) SCC surgical specimens and 18 of 29 (62%) paired cytologic specimens with 83% matched immunostains. PD-L1 was expressed on tumor cells in 13 of 23 (57%) AC surgical specimens and in 17 of 23 (74%) paired cytologic specimens with 79% matched immunostains. The PD-L1 was expressed on inflammatory cells in 20 of 23 (87%) AC surgical specimens and in 15 of 23 (65%) paired cytologic specimens with 70% matched immunostains. The PD-L1 was expressed on inflammatory cells in 18 of 29 (62%) SCC surgical specimens and in 12 of 29 (41%) paired cytologic specimens with 79% matched immunostains.Conclusions:PD-L1 immunostain in cytology samples matched very well with paired surgical samples in both SCC and AC cases. The cytologic samples present slightly higher sensitivity for PD-L1 immunostain on tumor cells as compared to surgical biopsies.

Abstract 1720: Analysis of multiplexed immunotherapy targets and secreted ligands using computational tumor microenvironment profiling

Abstract As the number and diversity of cellular and signaling pathway therapeutic targets in immuno-oncology increases, more therapy combinations are aimed at multiple cell types in the tumor microenvironment (TME). To develop rational combinations of therapies that will be effective in any individual patient, multivariate biomarker assays will be required to understand each patient's specific tumor and immune profile. Understanding the mechanistic effects of immunotherapy includes the tissue context of direct immunotherapy targets and downstream effector molecules such as secreted cytokines. Although the content of tumor-infiltrating immune cells and gene expression of the total tumor can be ascertained through several methods, spatial arrangements and proximity of cells and co-localization of biomarkers can only be achieved with tissue-based assays. Duplex chromogenic in situ hybridization assays using the RNAscope platform allows for the visualization of gene expression of mRNA targets directly in tissue for multiple cell types or biomarkers. Computational Tissue Analysis (cTA™) was used to quantify cellular spatial distributions including regional density maps, proximity measurements, and colocalization of biomarkers in both the tumor nests and the surrounding stroma that would be challenging by visual inspection of duplex RNAscope assays in non-small cell lung cancer (NSCLC) tissue microarrays (TMAs). Often, neither immune effector cells nor the therapeutic target is sufficient in isolation to understand blocked anti-tumor immune responses and therefore the potential efficacy of a single or combination therapeutic strategy. Multiplex RNAscope assays and cTA™ in combination allow the assessment of end points that measure spatial relationships between cell types expressing certain biomarkers. As these are performed in the context of the tissue microarchitecture within and across multiple tissue sections, they are a significant advancement over traditional tissue-based manual end points. Citation Format: Jenifer Caldara, Brooke Hirsch, Joseph Krueger, Chris Bunker, Jeff Kim, Annelies Laeremans, Mohammed Qutaish, Allison Harney. Analysis of multiplexed immunotherapy targets and secreted ligands using computational tumor microenvironment profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1720.

A regulatory circuit composed of DNA methyltransferases and receptor tyrosine kinases controls lung cancer cell aggressiveness.

Overexpression of DNMT1 and KIT is prevalent in lung cancer, yet the underlying molecular mechanisms are poorly understood. While the deregulated activation of DNMT1 or KIT has been implicated in lung cancer pathogenesis, whether and how DNMT1 and KIT orchestrate lung tumorigenesis are unclear. Here, using human lung cancer tissue microarrays and fresh frozen tissues, we found that the overexpression of DNMT1 is positively correlated with the upregulation of KIT in tumor tissues. We demonstrated that DNMT1 and KIT form a positive regulatory loop, in which ectopic DNMT1 expression increases, whereas targeted DNMT1 depletion abrogates, KIT signaling cascade through Sp1/miR-29b network. Conversely, an increase of KIT levels augments, but a reduction of KIT expression ablates, DNMT1 transcription by STAT3 pathway leading to in-parallel modification of the DNA methylation profiles. We provided evidence that KIT inactivation induces global DNA hypomethylation, restores the expression of tumor suppressor p15INK4B through promoter demethylation; in turn, DNMT1 dysfunction impairs KIT kinase signaling. Functionally, KIT and DNMT1 co-expression promotes, whereas dual inactivation of them suppresses, lung cancer cell proliferation and metastatic growth in vitro and in vivo, in a synergistic manner. These findings demonstrate the regulatory and functional interplay between DNA methylation and tyrosine kinase signaling in propelling tumorigenesis, providing a widely applicable approach for targeting lung cancer.

Lung carcinoma progression and survival versus amino- and carboxyl-parathyroid hormone-related protein expression.

Expression of the carboxyl PTHrP region of parathyroid hormone-related protein (PTHrP) is a positive prognostic indicator in women with lung cancer, but amino PTHrP is a negative indicator in other lung cancer patients. This project investigated whether PTHrP could be expressed as predominantly amino PTHrP or carboxyl PTHrP in individual lung carcinomas. It also assessed domain-specific effects on cancer progression and patient survival. PTHrP immunoreactivities were analyzed versus survival in a human lung cancer tissue microarray (TMA). Growth was compared in athymic mice for isogenic lung carcinoma xenografts differing in expression of amino and carboxyl PTHrP domains. In the TMA, 33 of 99 patient tumors expressed only one PTHrP domain, while 54 expressed both. By Cox regression, the hazard ratio for cancer-specific mortality (95% confidence interval) was 2.6 (1.28-5.44) for amino PTHrP (P = 0.008) and 0.6 (0-2.58) for carboxyl PTHrP (P = 0.092). Xenografts of H358 lung adenocarcinoma cells that overexpressed amino PTHrP grew twice as fast as isogenic low PTHrP tumors in athymic mice, but growth of tumors expressing amino plus carboxyl PTHrP was not significantly different than growth of the control tumors. In summary, the presence of amino PTHrP signifies worse prognosis in lung cancer patients. In mouse xenografts, this effect was abrogated if carboxyl PTHrP was also present. Amino PTHrP and carboxyl PTHrP can vary independently in different lung carcinomas. Carboxyl PTHrP may temper the stimulatory effect of amino PTHrP on cancer progression.

LHX6, An Independent Prognostic Factor, Inhibits Lung Adenocarcinoma Progression through Transcriptional Silencing of β-catenin

Introduction: Our previous study identified LIM homeobox domain 6 (LHX6) as a frequently epigenetically silenced tumor-suppressor gene in lung cancer. However, its clinical value has never been evaluated, and the in-depth anti-tumor mechanism remains unclear.Methods: Public database was used for lung cancer, lung adenocarcinoma and lung squamous carcinoma patients and tissue microarray data was used for lung adenocarcinoma patients to study prognostic outcome of LHX6 expression by Kaplan-Meier and Cox-regression analysis. In vitro proliferation, metastasis and in vivo nude mice model were used to evaluate the anti-tumor effect of LHX6 on lung adenocarcinoma cell lines. The mechanisms were explored using western blot, TOP/FOP flash assays and luciferase reporter assays. LHX6 expression and clinical stages data were collected from The Cancer Genome Atlas database (TCGA).Results: Expression of LHX6 was found to be a favorable independent prognostic factor for overall survival (OS) of total lung adenocarcinoma patients (P=0.014) and patients with negative lymph nodes status (P=0.014) but not related the prognostic outcome of lung squamous cell carcinoma patients. The expression status of LHX6 significantly correlated to histological grade (P<0.01), tumor size (P=0.026), lymph node status (P=0.039) and clinical stages (P<0.01) of lung adenocarcinoma patients. Functionally, LHX6 inhibited the proliferation and metastasis of lung adenocarcinoma cells in vitro and in vivo. Furthermore, LHX6 suppressed the Wnt/β-catenin pathway through transcriptionally silencing the expression of β-catenin, and the promoter region (-1161 bp to +27 bp) was crucial for its inhibitory activity.Conclusions: Our data indicate that the expression of LHX6 may serve as a favorable prognostic biomarker for lung adenocarcinoma patients and provide a novel mechanism of LHX6 involving in the tumorigenesis of lung adenocarcinoma.

Cytokeratin 18 knockdown decreases cell migration and increases chemosensitivity in non-small cell lung cancer.

Cytokeratin 18 (CK18) is a structural protein that is normally expressed in many single-layer epithelia. Previous studies have indicated that aberrant CK18 expression is associated with cancer progression. However, the functions of CK18 in lung cancer have not been fully elucidated. Here, we investigate the roles of CK18 in non-small cell lung cancer (NSCLC). CK18 protein expression was evaluated by immunohistochemistry in a lung cancer tissue microarray containing 129 cancer samples, and correlations between CK18 expression and clinicopathological characteristics and prognosis were analyzed. We then studied the effects of CK18 knockdown on cell motility and chemosensitivity in lung cancer cells. High CK18 expression was detected in 101/129 (78.3%) lung cancers. CK18 expression was significantly correlated to clinical stage, lymph node metastasis, the number of pathologically positive lymph nodes and recurrence and metastasis. Kaplan-Meier survival analysis showed that CK18 was a prognostic factor for overall survival (P=0.016) and disease-free survival (P=0.014). In addition, CK18 knockdown decreased cell migration and enhanced the sensitivity of lung cancer cells to paclitaxel. These findings indicate that CK18 plays an important role in lung cancer progression and may be a therapeutic target for NSCLC.