Lung Squamous Cell Carcinoma Samples Research Articles

Abstract 5293: A method to identify somatic mutations from tumor samples in the absence of matched normal tissue

Abstract Tumor-specific molecular profiling by next-generation sequencing (NGS) from tissue or blood (liquid biopsy) can have a tremendous potential to guide personalized healthcare for cancer treatments. However, based on the current clinical practices1, often tumor or blood may be sequenced without the matched germline normal tissue or buffy coat. Since the tumor cell content of the specimens can vary widely, as well as the fact that sequencing will identify both germline and somatic variations, confident identification of somatic mutations becomes very challenging in the absence of a matched normal. Identification of somatic variations in cell-free DNA becomes even more difficult due especially to the low yields of tumor-derived DNA in plasma samples. An ensemble tree-based machine learning method was developed to classify tumor-specific somatic mutations in the absence of matched normal (CSMutan). This algorithm leverages multiple public databases and variant allele frequencies as features to classify somatic mutations and germline variants. Using The Cancer Genome Atlas (TCGA) and in-house datasets, models were built for each of the three different types of tumor specimens: i.e., fresh frozen, formalin-fixed, paraffin-embedded (FFPE), and plasma samples. The performance of the models was evaluated using cross-validation and/or testing on independent sets of samples. Lung adenocarcinoma samples from TCGA were used to build the fresh frozen model, which achieved an area under the ROC curve (AUC) of 0.9968 on an independent data set of lung squamous cell carcinoma samples, also from TCGA. In-house sample sets were used to build the FFPE model and the plasma model. The FFPE model achieved an ROC AUC of 0.993, while the model trained on plasma samples was able to classify with an AUC of 0.997 based on ten-fold cross-validation. Although sequencing matched normal tissue is the most preferred and accurate method for identifying high-confidence somatic mutations, this work demonstrates that it is possible to identify somatic mutations in tissue or blood with high accuracy when the matched normal samples may not be available. The future directions for this method are to add additional, and more diverse, cancer samples for both the training and independent validation so that potentially higher accuracy and generalizability can be obtained. 1. Meyerson, M., Gabriel, S. & Getz, G. Advances in understanding cancer genomes through second-generation sequencing. Nature Reviews Genetics 11, 685-696 (2010) Citation Format: Lijing Yao, Preeti Lal, Li-Tai Fang, John Lee, John Palma, Andre Rosenthal, Bernd Hinzmann, Alex Lovejoy, Hugo Y. K. Lam. A method to identify somatic mutations from tumor samples in the absence of matched normal tissue [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 5293.

Long non-coding RNAs RP5-821D11.7, APCDD1L-AS1 and RP11-277P12.9 were associated with the prognosis of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC), a type of non-small cell lung carcinoma, has a poor therapeutic response, high relapse rate and poor prognosis. The present study was designed to reveal the key long non-coding RNAs (lncRNAs) associated with the prognosis of LUSC. The lncRNA expression profiles of LUSC and adjacent samples were downloaded from The Cancer Genome Atlas database. Based on the edgeR and DEseq packages, the differentially expressed lncRNAs (DELs) between LUSC and adjacent samples were obtained and the intersecting DELs were regarded as significant DELs. Subsequently, a prognostic risk model was established using Cox regression analysis and its classification effect was detected by survival analysis. Using survival analysis, the effect of the prognostic risk model was assessed in the validation set and other types of cancer. Finally, the co-expression genes of key lncRNAs were screened using the Multi-Experiment Matrix tool and the STRING database, and their functions were predicted via enrichment analysis using the Database for Annotation, Visualization and Integrated Discovery tool. A total of 2,041 significant DELs between LUSC and adjacent samples were screened. The prognostic risk model consisting of RP5-821D11.7, APCDD1L-AS1 and RP11-277P12.9 was established, which had a good classification effect. Cox multivariate regression analysis demonstrated that risk score may serve as an independent prognostic factor. Furthermore, certain co-expression genes of RP5-821D11.7 (including proliferating cell nuclear antigen), APCDD1L-AS1 (including semaphorin 5A, semaphorin 6D, ADAMTS like 1, ADAM metallopeptidase with thrombospondin type 1 motif 6, slit guidance ligand 3, and tenascin C) and RP11-277P12.9 (including Wnt family member 2B) were identified. Additionally, ‘positive regulation of cell migration’ and ‘proteinaceous extracellular matrix’ were enriched. In conclusion, the expression levels of the lncRNAs RP5-821D11.7, APCDD1L-AS1 and RP11-277P12.9 may affect the prognosis of LUSC.

Mutational Landscape of DDR2 Gene in Lung Squamous Cell Carcinoma Using Next-generation Sequencing

BackgroundLung cancer represents the leading cause of cancer-related death worldwide. Despite great advances in lung cancer management with the recent emergence of molecular targeted therapies for non-squamous non–small-cell lung cancer, no dramatic improvements have been achieved in lung squamous cell carcinoma (SCC). Mutations in discoidin domain receptor 2 (DDR2) gene were recently identified as promising molecular targets in this histology. The aim of this study is to describe the DDR2 mutational landscape of lung SCC and investigate the associated clinical factors. MethodsNext-generation sequencing of the DDR2 gene was performed on 271 samples of lung SCC. Patients followed in our institution from January 2011 to August 2014 were retrospectively selected for data collection. Other driver gene alterations (EGFR, KRAS, BRAF, HER2, and PI3KCA) were analyzed using pyrosequencing. ResultsA total of 11 patients harboring a DDR2 mutation was detected among the 271 sequenced lung SCC samples (4%). We describe 10 unreported mutations, comprising a novel DDR2 exon 7 splice mutant. DDR2 mutations were not mutually exclusive with other driver gene alterations. One hundred thirty-six patients were included for clinical comparison and logistic regression analysis. No difference was detected between DDR2-mutant and DDR2 wild-type lung SCC regarding clinical characteristics or survival. ConclusionDDR2 mutations were observed in 4% of cases of lung SCC of European descent. DDR2-mutated tumors can exhibit other driver gene alterations. No clinical characteristics were significantly associated with DDR2 mutation.

1715P - PIK3CA mutation and PDL1 expression in lung squamous cell carcinoma surgically resected

Background: Squamous-cell carcinoma (SCC) of the lung is the second most frequent histology in non-small cell lung cancer (NSCLC). Over the last decade new approaches targeting specific pathways in NSCLC have emerged but very few advances were made in its treatment. The PI3K-AKT-mTOR pathway is implicated in multiple cancer processes and PIK3CA mutations are being investigated in SCC as potential therapeutic target. The aim of the study was to evaluate the histological characteristics, mutations in PIK3CA and PD-L1 expression in these tumors. Methods: Surgically resected lung SCC samples (FFPE) from 100 patients, stage I-III, were included in this study. Clinicopathologic characteristics included tumor size, TNM, smoking status, lymphovascular and pleural invasion, histopathological grade, stromal lymphoplasmacytic reaction and type of tumoral growing. DNA was isolated from 92 samples according to standard procedures and PIK3CA mutation analysis was done using Cobas 4800 platform. PD-L1 expression was analyzed in 74 cases by immunohistochemistry with PDL1 22C3 pharmDX assay. The PD-L1 expression was evaluated by tumor proportion scores (TPS) as IASLC guidelines. Results: The mean age was 68 years (53-86), 14 females and 86 males. About staging: 53 patients had stage I, 27 stage II, and 19 stage III. 53% and 35% showed vascular and pleural invasion respectively. Low to moderate stromal lymphoplasmacytic reaction was found in 34% and severe in 11%. PI3KCA mutation was found in 9/92 (9,8%) patients: E545X (n = 2), E542K (n = 5), H1047X (n = 1), C420R (n = 1). PD-L1-expression was found in 31 out of 74 cases (42%): 14 cases with TPS between 1-49%; 17 cases with TPS >50%. 5/9 (55%) of PIK3CA mutated cases were PD-L1 positive (2 of them >50%). In PIK3CA non-mutated cases 26 out of 67 (39%) showed PD-L1 expression: 11 cases with TPS between 1-49%; 15 cases with TPS >50%. Conclusions:PIK3CA mutation was found in 9,8% of SCC of the lung, most of them in exon 9. PD-L1 expression was found in 42% of the SCC in our series. 55% of PIK3CA mutated patients were positive for PD-L1 expression. No correlation has been found between PD-L1 expression and PIK3CA mutation in SCC. It is important to know more about the relation of these factors to select the patients for immunotherapy and new target agents. Legal entity responsible for the study: IIS Funding: None Disclosure: All authors have declared no conflicts of interest.

Correlation between RNA-Seq and microarrays results using TCGA data

RNA sequencing (RNA-Seq) and microarray are two of the most commonly used high-throughput technologies for transcriptome profiling; however, they both have their own inherent strengths and limitations. This research aims to analyze the correlation between microarrays and RNA-Seq detection of transcripts in the same tissue sample to explore the reproducibility between the techniques. Using data of RNA-Seq v2 and three different microarrays provided by The Cancer Genome Atlas, 11,120 genes of 111 lung squamous cell carcinoma samples were simultaneously detected by the four methods. Then we analyzed the Pearson correlation between microarrays and RNA-Seq. Finally, in the six comparison results, 9984 (89.8%) genes, irrespective of which two methods were used, simultaneously showed the existence of correlation, whereas only 83 (0.1%) genes proved to have no significant correlation in either comparison. In addition, the comparisons between 3266 (29.3%) genes showed high correlation (R≥0.8) in all six comparisons, only for 1643 (14.8%) genes correlation were not as high in either comparison. Meanwhile, transcripts with extreme high or low expression levels were more highly discrepant across the methods. In conclusion, we found that, for most transcripts, the results obtained by RNA-Seq and microarrays were highly reproducible.

MiRNA profiling of primary lung and head and neck squamous cell carcinomas: Addressing a diagnostic dilemma

ObjectiveTo determine whether microRNA (miRNA) profiling of primary lung and head and neck squamous cell carcinomas could be useful to identify a specific miRNA signature that can be used to further discriminate between primary lung squamous carcinomas and metastatic lesions in patients with a history of head and neck squamous cell cancer. MethodsSpecimens of resected primary head and neck and lung squamous cell carcinomas were obtained from formalin-fixed, paraffin-embedded blocks. Paraffin blocks were sectioned and deparaffinized, and total RNA was isolated and profiled. Quantitative polymerase chain reaction was performed to verify array results. ResultsTwelve head and neck and 16 lung squamous cell carcinoma samples met quality control metrics and were included for analysis. Forty-eight miRNAs were differentially expressed (P < .05) between the 2 groups. Of these, 30 were also significantly associated (q < .25) with tumor type in 2 independent sets of primary head and neck and lung squamous carcinomas profiled by The Cancer Genome Atlas consortium, including miR-34a and miR-10a. The ratio of miR-10a and miR-10b was especially predictive of primary cancer site in all 3 data sets, with area under the (receiver operating characteristics) curve values ranging from 0.922 to 0.982. Quantitative polymerase chain reaction confirmed the association of miR-34a expression and the miR-10:miR-10b ratio with tumor type. ConclusionsMicroRNA expression may be useful for discriminating between head and neck and lung squamous cell carcinomas, including miR-34a and the miR-10a:miR-10b ratio. This differentiation has clinical importance because it could help determine the appropriate therapeutic approach.

Implication of downregulation and prospective pathway signaling of microRNA-375 in lung squamous cell carcinoma

BackgroundLung cancer is one of the most typical cancers in the world. Altered expression profiles of microRNA-375(miR-375) are linked to many diseases including lung cancer. However, the relationship between miR-375 and lung squamous cell carcinoma (LUSC) is controversial. MethodsWe first evaluated the 23 LUSCs and the paired normal lung tissues by qRT-PCR. Then we analyzed the LUSC samples with miR-375 expression based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Furthermore, bioinformatics analysis was performed to explore the biological role of miR-375 in LUSC. ResultsThe expression of miR-375 was remarkably reduced in LUSC tissues compared with that in paired lung tissues by qRT-PCR (P=0.003). Additionally, the TCGA dataset suggested that miR-375 was significantly downregulated in 478 LUSC tissues compared with 45 normal lung tissues (P<0.0001), as well as the result derived from GEO datasets (the pooled SMD=−1.01; 95%CIs-1.66 to −0.33, P=0.004). Furthermore, a total of 1348 miR-375-related differently expressed genes were identified by the analytical integration, which were involved in critical pathways of LUSC like neuron differentiation, plasma membrane part and sequence-specific DNA binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway examination also unveiled the involvement of target genes in morphine addiction and drug metabolism- other enzymes and neuroactive ligand-receptor interaction. Finally, the expression of WNT5A was inversely correlated with miR-375 expression according to TCGA dataset (r=−0.2342, P<0.0001). ConclusionsmiR-375 exerts a strong tumor-suppressive effect in LUSC and provided novel insight into the biological function in tumorigenesis and progression of LUSC.

Epigenetically silenced PTPRO functions as a prognostic marker and tumor suppressor in human lung squamous cell carcinoma.

Protein tyrosine phosphatase receptor-type O (PTPRO), a member of the PTP family, has been frequently reported as potential tumor suppressor in many types of cancer. However, the exact function of PTPRO in lung squamous cell carcinoma (LSCC) remains unclear. Bisulfite sequencing and methylation specific polymerase chain reaction (PCR) were used to identify the methylation status of PTPRO in LSCC cells, and quantitative methylation specific PCR was used to evaluate the methylation levels of PTPRO in LSCC patients. Stably expressing PTPRO vectors were constructed and transfected into H520 and SK-MES-1 cells, followed by MTT and colony formation assays, and analysis of tumor weight and volume in in vivo mouse xenograft models. The present study demonstrated that the CpG island of PTPRO exon 1 was obviously hypermethylated in LSCC cells and tissues. The mRNA expression of PTPRO could be restored by treatment with a demethylation agent. Increased methylation and decreased mRNA levels of PTPRO were observed in LSCC samples compared with adjacent healthy tissues, and were associated with poor prognosis of patients. The mRNA expression of PTPRO was negatively correlated with its methylation level in tumors. Functionally, ectopic PTPRO expression in LSCC cells significantly inhibited the proliferation rates, and colony formation, in comparison with control and non-transfected cells. In vivo assays confirmed the inhibitory effect of PTPRO on LSCC cell growth. In conclusion, these data provided evidence that epigenetic regulation of PTPRO impairs its tumor suppressor role in LSCC, and restoration of PTPRO may be a potential therapeutic strategy.

Differentiated regulation of immune-response related genes between LUAD and LUSC subtypes of lung cancers.

Lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) are the two major subtypes of lung cancer, with LUSC exhibits faster progression rate than LUAD. To investigate the roles of immune-response related genes (IRGs) in lung cancer progression, we used LUAD and LUSC samples at different cancer progression stages, and identified that the expression profiles of IRGs could serve as a better classification marker for cancerous tissues of both LUAD and LUSC. We found that the expression changes of IRGs were different between LUAD and LUSC. Cell cycle promoting genes, including KIFs and proteasomes, showed faster up-regulation in LUSC, whereas immune response promoting genes, including MHC molecules and chemokines, were more rapidly repressed in LUSC. Comparative pathway analysis revealed that members of the Toll-like receptor and T cell receptor signaling pathways exhibited diverged expression changes between LUAD and LUSC, especially at the early cancer stages. Our results revealed the difference of LUAD and LUSC from the immune response point of view, and provided new clues for the differential treatment of LUAD and LUSC.

MicroRNAs modulate the expression of the SOX18 transcript in lung squamous cell carcinoma.

Recent statistics show that lung cancer is the second most common malignant tumor in the world (14% of all cancers in the USA), both in terms of morbidity and mortality. The mortality of this type of tumor shows an increasing trend (28% for men and 26% for women). Lung squamous cell carcinoma(LSCC) is the second‑largest histological subtype of non‑small cell lung cancers(NSCLCs) after adenocarcinoma. SRY‑related HMG‑box18(SOX18) protein is an important transcription factor involved in the development of the cardiovascular system and the lymphatic ducts. In addition, it was observed that SOX18 functions in wound healing processes and the development of atherosclerosis. Likewise, an increased level of this protein was found in melanomas and malignant pancreatic, stomach and breast tumors. Furthermore, high expression of SOX18 in gastric cancer stromal cells was found to be associated with a poor patient prognosis. In the present study, we analyzed the expression of the SOX18 protein and the mRNA level in postoperative samples of LSCC and non‑malignant lung tissues(NMLTs), and a disparity in both levels was observed. Because of the fact that microRNAs(miRNAs) play important roles in the initiation and progression of lung cancer, the main aim of this study was to identify the miRNAs that interact with the SOX18 transcript in NSCLC cases. SOX18 mRNA expression level was significantly lower in the LSCC tissues than that noted in the NMLTs (p<0.01). However, protein levels were higher in the LSCC cases compared to these levels in the NMLTs (p<0.0001). We showed that miR‑7a and miR‑24‑3p were expressed more highly in the NMLTs than levels in the LSCC samples, and that they could be switched off in lung cancer tissue. Additionally, correlations between RQ‑values of SOX18 in NMLTs and LSCC samples (r=0.43, p=0.019), and between miR‑7a and miR24‑3p in NMLT cases (r=0.4, p=0.057) aswellas in the LSCC samples (r=0.51, p=0.012) were noted. In conclusion, miRNAs interact with the mRNA of the SOX18 gene, but the mechanism by which they could be inhibited in cancer cells requires further examination.

Abstract 407: Functional analysis of discoidin domain receptor 2 mutation and expression in squamous cell lung cancer

Abstract The treatment strategy for lung cancer has been rapidly progressed, especially in molecular targeted therapy. However, most treatments are established for adenocarcinoma of lung, but not squamous cell carcinoma (SQCC). SQCC occupies approximately 30% among entire lung cancer patients, but the prognosis is worse than that of adenocarcinoma patients. Discoidin domain receptor (DDR) 2 mutations have been recently reported as one of the candidates of molecular targets for lung SQCC, and they are associated with sensitivity to dasatinib, which is already applicable to treatment of chronic myeloid leukemia. However, precise roles of DDR2 on SQCCs of lung have not been clarified. We performed Sanger sequencing of DDR2 gene in a set of 44 primary lung SQCC samples and 7 lung SQCC cell lines, and identified a novel mutation (T681I) in a lung SQCC patient and a lung SQCC cell line, EBC-1. Endogenous protein expression level of DDR2 measured by Western blot technique was high in a lung SQCC cell line, PC-1. Overexpression of wild type and T681I of DDR2 promoted cancer cell invasion of a human lung SQCC cell line, H226B. In animal model, lung metastasis was enhanced and survival was shortened after transplantation of DDR2 wild type overexpressing cells into NOD/SCID/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. Using the cDNA microarray and protein phosphorylation array assay, we found that ectopic expression of DDR2 induced TGF-β1 protein and mRNA expression accompanied with phosphorylation of c-Jun, p38 MAP kinase, and MMP1 elevation. During the process of metastasis, TGF-β pathway plays pivotal roles especially on disruption of basement membrane through induction of MMPs from fibroblasts mediated through both Smad and non-Smad pathways. Since phosphorylations of c-Jun and p38 MAPK were reported to be involved in non-Smad pathway, DDR2 would induce TGF-β mainly through non-Smad pathway, resulting in cancer invasion and metastasis. Taken together, elevation of DDR2 protein might contribute on tumor progression as well as some of DDR2 mutations in lung SQCC. Citation Format: Naomi Kobayashi-Watanabe. Functional analysis of discoidin domain receptor 2 mutation and expression in squamous cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 407.

Mutations and expression of the NFE2L2/KEAP1/CUL3 pathway in Chinese patients with lung squamous cell carcinoma.

Recent studies have reported an abnormally high alteration rate in the nuclear factor erythroid 2-like 2 (NFE2L2)/kelch-like ECH-associated protein 1 (KEAP1)/cullin 3 (CUL3) pathway. But the status of this pathway in Chinese patients with lung squamous cell carcinoma (SqCC) has not been thoroughly studied, and there are many uncertainties regarding the expression of pathway intermediates. cDNA sequencing and TaqMan qRT-PCR were carried out in paired cancer and adjacent normal samples obtained from 100 Chinese patients with lung SqCC. Immunohistochemical staining was performed in 50 other paraffin-embedded specimens. We detected 47 mutations in 36 patients (36%), and 143 single nucleotide polymorphism (SNP) in 59 patients (59%), of which 41 mutations and 31 SNPs resulted in amino acid (AA) and possibly functional changes. By combining qRT-PCR and immunohistochemistry staining, we confirmed that the expression of NFE2L2 and KEAP1 were highly increased, while the expression of CUL3 was not significantly changed in lung SqCC samples from Chinese patients. Considering the frequent mutations and abnormal expression, the NFE2L2/KEAP1/CUL3 pathway may play an important role in the therapy of Chinese patients with lung SqCC.

Identification of differentially expressed genes between lung adenocarcinoma and lung squamous cell carcinoma by gene expression profiling.

The present study aimed to identify the differentially expressed genes (DEGs) between lung adenocarcinoma and normal lung tissues, and between lung squamous cell carcinoma and normal lung tissues, with the purpose of identifying potential biomarkers for the treatment of lung cancer. The gene expression profile (GSE6044) was downloaded from the Gene Expression Omnibus database, which included data from 10 lung adenocarcinoma samples, 10 lung squamous cell carcinoma samples, and five matched normal lung tissue samples. After data processing, DEGs were identified using the Student's t-test adjusted via the Benjamini-Hochberg method. Subsequently, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, and a global network was constructed. A total of 95 upregulated and 241 downregulated DEGs were detected in lung adenocarcinoma samples, and 204 upregulated and 285 downregulated DEGs were detected in lung squamous cell carcinoma samples, as compared with the normal lung tissue samples. The DEGs in the lung squamous cell carcinoma group were enriched in the following three pathways: Hsa04110, Cell cycle; hsa03030, DNA replication; and hsa03430, mismatch repair. However, the DEGs in the lung adenocarcinoma group were not significantly enriched in any specific pathway. Subsequently, a global network of lung cancer was constructed, which consisted of 341 genes and 1,569 edges, of which the top five genes were HSP90AA1, BCL2, CDK2, KIT and HDAC2. The expression trends of the above genes were different in lung adenocarcinoma and lung squamous cell carcinoma when compared with normal tissues. Therefore, these genes were suggested to be crucial genes for differentiating lung adenocarcinoma and lung squamous cell carcinoma.

Identification of FGF19 as a prognostic marker and potential driver gene of lung squamous cell carcinomas in Chinese smoking patients.

Comprehensive genomic characterizations of lung squamous cell carcinoma (LSCC) have been performed, but the differences between smokers (S-LSCC) and never smokers (NS-LSCC) are not clear, as NS-LSCC could be considered as a different disease from S-LSCC. In this study we delineated genomic alterations in a cohort of 21 NS-LSCC and 16 S-LSCC patients, and identified common gene mutations and amplifications as previously reported. Inclusion of more NS-LSCC patients enabled us to identify unreported S-LSCC- or NS-LSCC-specific alterations. Importantly, an amplification region containing FGF19, FGF3, FGF4 and CCND1 was found five-times more frequent in S-LSCC than in NS-LSCC. Amplification of FGF19 was validated in independent LSCC samples. Furthermore, FGF19 stimulated LSCC cell growth in vitro. These data implicate FGF19 as a potential driver gene in LSCC with clinic characteristics as smoking.

Identification of crucial regulatory relationships between long non-coding RNAs and protein-coding genes in lung squamous cell carcinoma

PurposeThis study aimed to analyze the relationships of long non-coding RNAs (lncRNAs) and protein-coding genes in lung squamous cell carcinoma (LUSC). MethodsRNA-seq data of LUSC deposited in the TCGA database were used to identify differentially expressed protein-coding genes (DECGs) and differentially expressed lncRNA genes (DE-lncRNAs) between LUSC samples and normal samples. Functional enrichment analysis of DECGs was then performed. Subsequently, the target genes and regulators of DE-lncRNAs were predicted from the DECGs. Additionally, expression levels of target genes of DE-lncRNAs were validated by RT-qPCR after the silence of DE-lncRNAs. ResultsIn total, 5162 differentially expressed genes (DEGs) were screened from the LUSC samples, and there were seven upregulated lncRNA genes in the DEGs. The upregulated DECGs were enriched in GO terms like RNA binding and metabolic process. Meanwhile, the downregulated DECGs were enriched in GO terms like cell cycle. Furthermore, the lncRNAs PVT1 and TERC targeted multiple DECGs. PVT1 targeted genes related to cell cycle (e.g. POLA2, POLD1, MCM4, MCM5 and MCM6), and reduced expression of PVT1 decreased expression of the genes. TERC regulated several genes (e.g. NDUFAB1, NDUFA11 and NDUFB5), and reduced expression of TERC increased expression of the genes. Additionally, PVT1 was regulated by multiple transcription factors (TFs) identified from DECGs, such as HSF1; and TERC was modulated by TFs, such as PIR. ConclusionA set of regulatory relationships between PVT1 and its targets and regulators, as well as TERC and its targets and regulators, may play crucial roles in the progress of LUSC.

Adoptive immunotherapy using T lymphocytes redirected to glypican-3 for the treatment of lung squamous cell carcinoma.

There are unmet medical needs for patients with lung squamous cell carcinoma (LSCC). Therefore, in this study, we explored the antitumor potential of third-generation glypican 3 (GPC3)-redirected chimeric antigen receptor (CAR)-engineered T lymphocytes (CARgpc3 T cells) in tumor models of LSCC. First, we demonstrated by immunohistochemistry (IHC) that GPC3 was expressed in 66.3% of LSCC samples and in 3.3% of lung adenocarcinoma (LAD) samples but not in normal lung tissues. In the presence of GPC3-positive LSCC cells, CARgpc3 T cells were highly activated and increased in number. CARgpc3 T cells could specifically lyse GPC3-positive LSCC cells in vitro. In two established LSCC xenograft models, CARgpc3 T cells could almost completely eliminate the growth of GPC3-positive cells. Additionally, the CARgpc3 T cells were able to persist in vivo and efficiently infiltrate the cancerous tissues. Taken together, these findings indicate that CARgpc3 T cells might be a novel potential therapeutic agent for the treatment of patients with LSCC.

Abstract 2978: An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data

Abstract The Cancer Genome Atlas (TCGA) contains various types of genomic data from a wide variety of cancers, several of which affect the same tissue site. Here we analyze copy number and RNA-Seq data from TCGA for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Level 1 (raw) data, rather than Level 3 (segmented) data, from TCGA was used for an integrated analysis of copy number and gene expression profiles of the two cancers. Using the copy number and SNP- probes, re-processed tumor profiles were more consistent with a control set in terms of median number of copy number events, sample ploidy, and breakpoint genes than with the published “level 3” TCGA data. Probe-level data were analyzed using Nexus Copy Number SNP-FASST2 (a multi-state HMM algorithm that uses both SNP and copy number probes in making state assignments), with systematic correction applied to correct for GC biases. Additionally we performed manual baseline adjustment to correct for sample ploidy based on whole-genome B-alelle frequency data for each sample. Overall, the median number of copy number events in the LUAD TCGA data set was reduced from 371 (in the level 3 set) to 299, and from 681 (in the level 3 set) to 177 in the LUSC TCGA data set. After manual inspection, more than 38% of the TCGA LUAD samples and 50% of the TCGA LUSC samples available at level 3 were found to have incorrect baseline ploidy assignments. The resultant re-analyzed copy number data sets were used for an integrated analysis between the two tumor types. Comprehensive comparative analysis using Fisher's Exact Test revealed statistically significant differences (percent differential = 25%, p&amp;lt;0.001) in copy number profiles between the two lung tumor types; copy number changes include differential loss of chromosome 1p, loss of chromosome 3p, gain of chromosome 3q, loss of chromosome 4 and loss of chromosome 5. These correlated to changes in overall survival: individuals with loss of chromosome 1p and/or loss of 5q resulted in a significantly poorer prognosis (p&amp;lt;0.05). While more frequent in the LUSC sample population, this change in overall survival outcome was extended to samples with chromosome 1p loss in LUAD samples as well. Integration with RNA-Seq expression data from each tumor type revealed statistically significant correlations (p&amp;lt;0.05) with these copy number alterations, identifying potential driver genes of interest among each subtype and lung tumors in general. Citation Format: Andrea J. OHara, Raja Keshavan, Zhiwei Che, Soheil Shams. An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2978. doi:10.1158/1538-7445.AM2015-2978

RBMS3 is a tumor suppressor gene that acts as a favorable prognostic marker in lung squamous cell carcinoma.

Recent research indicates that RBMS3 may act as a tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) and esophageal squamous cell carcinoma (ESCC). It has been reported that RBMS3 directly binds to the promoter region of c-Myc in ESCC and that β-catenin from both whole cell extracts and nuclear fractionation was significantly downregulated in RBMS3-transfected NPC cells compared to control cells. The aim of this study was to evaluate the clinical significance of the RBMS3 gene expression in relation to the expression of Wnt pathway components in patients with lung squamous cell carcinoma (LSCC). RBMS3, c-Myc and cytoplasmic β-catenin were detected in 39.76, 56.63 and 89.16% of 83 LSCC samples by immunohistochemistry, respectively, in 83 primary LSCC samples. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased RBMS3 mRNA and expression in 33.33% (10/30) and 36.67% (11/30) tumor tissues, respectively. Statistical correlation analysis showed RBMS3 to be negatively correlated with c-Myc (r=-0.384, p<0.001) and not correlated with cytoplasmic β-catenin in the LSCC samples. Multivariate Cox proportional hazards model analysis showed that the combined marker RBMS3/c-Myc was an independent prognostic indicator of overall survival (p=0.001; HR 3.470; IC 95%, 1.652-7.290), and c-Myc was a prognostic indicator of disease-free survival (p<0.001; HR 3.182; IC 95%, 1.961-8.920). RBMS3 is a novel TSG in LSCC, and its downregulation facilitates development and progression of LSCC. Therefore, it is suggested that Rbms3 as a tumor marker may play an important role in diagnosis of LSCC.

Abstract 526: Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung

Abstract The amplification of the distal portion of chromosome 3q in lung cancer is a major signature of neoplastic transformation, particularly in the squamous cell carcinoma (SCC) of the lung. A number of potential drivers in the 3q amplicon have been identified and are proposed to contribute to the development of lung SCC. miRNAs have emerged as a new class of small, non-coding RNAs that regulate gene expression and might act as integral parts of the molecular architecture of oncogene and tumor suppressor networks in human cancer, including non-small cell lung cancer (NSCLC). miR 1224-5p, 1248 and 944 are located at 3q amplicon and overexpressed in 50%, 26% and 53% of 30 primary SCCs when compared to normal tissues. The overexpression of these miRNAs was further validated in 237 TCGA lung SCC samples compared with 35 normal tissues (p=0.007, 1.18E-16 and 7.71E-20, respectively). The expression of the three miRNAs was higher in lung SCC compared to lung adenocarcinomas (p=8.19E-05, 1.04E-33 and 3.37E-95, respectively), suggesting unique roles of these miRNAs in the lung SCC development. Using miRNA mRNA target prediction tools (miRANDA and TargetScan) and in the combination with a literature search, we found RUNX1T1 and SMAD4 were candidate tumor suppressor gene target for miRNA-1224-5p and miRNA-1248. Downregulation of RUNX1T1 and SMAD4 were confirmed in TCGA lung SCC dataset. Using real-time RT-PCR, we found that the same three miRNAs 1224-5p, 1248 and 944 are overexpressed in 3q amplified lung SCC cell lines H520 and HCC95. When miRNA inhibitors were used to repress the expression of miR-1224-5p or miR-1248 in H520, we found that RUNX1T1 and SMAD4 protein expression levels were increased, suggesting that these tumor suppressor genes are regulated by miR-1224-5p or miR-1248. Lastly, knockdown of miR-1224-5p or miR-1248 using miRNA inhibitors led to 35-60% growth inhibition in H520 and HCC95 cells. Together, these results indicate potential roles for miR-1245-5p and 1248 in regulating cell proliferation by directly targeting tumor suppressor genes RUNX1T1 or SMAD4 in lung SCC. This work provides, for the first time, evidence for overexpression of miRNAs at the 3q26-29 amplicon that might be implicated in the oncogenesis of lung SCC. The work is supported by the NIH Grant R01 CA102353 to PPM and Lung Cancer Research Foundation grant (2011) to JQ. Note: This abstract was not presented at the meeting. Citation Format: Jun Qian, Xiangming Ji, Yong Zou, Megan Hoeksema, Heidi Chen, Pierre P. Massion. Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2014-526

Abstract 1526: Integrative and comparative genomic analysis of East-Asian lung squamous cell carcinomas

Abstract Purpose Lung squamous cell carcinoma (SqCC) is the second most prevalent type of lung cancer. Currently, no targeted-therapeutics are approved for treatment of this cancer, largely due to a lack of systematic understanding of the molecular pathogenesis of the disease. To identify therapeutic targets and perform comparative analyses of lung SqCC, we probed somatic genome alterations of lung SqCC cases from Korean patients. Patients and Methods We performed whole-exome sequencing of DNA from 104 lung SqCC samples from Korean patients and matched normal DNA. In addition, copy number analysis and transcriptome analysis were conducted for a subset of these samples. Clinical association with cancer-specific somatic alterations was investigated. Results This cancer cohort is characterized by a very high mutational burden with an average of 261 somatic exonic mutations per tumor and a mutational spectrum showing a signature of cigarette-smoke exposure. Seven genes demonstrated statistical enrichment for mutation (TP53, RB1, PTEN, NFE2L2, KEAP1, MLL2 and PIK3CA). Comparative analysis between Korean and North American lung SqCC demonstrated similar spectrum of alterations in these two populations, in contrast to the differences seen in lung adenocarcinoma. We also uncovered recurrent occurrence of therapeutically actionable FGFR3-TACC3 fusion in lung SqCC. Conclusion These findings provide new steps towards the identification of genomic target candidates for precision medicine in lung SqCC, a disease with a significant unmet medical need. Note: This abstract was not presented at the meeting. Citation Format: Youngwook Kim, Peter S. Hammerman, Jaegil Kim, Gad Getz, Matthew Meyerson, Keunchil Park. Integrative and comparative genomic analysis of East-Asian lung squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2014-1526