Lung Squamous Cell Research Articles

Altered Treg and IL-1A Expression in the Immune Microenvironment of Lung Squamous-cell Cancer after EGFR Blockade

背景与目的精确靶向表皮生长因子受体（epidermal growth factor receptor, EGFR）的治疗在肺鳞癌、口腔和肠胃癌中取得了一定疗效，但会引发系统性炎症。本实验旨在探究EGFR抑制剂治疗癌引发的肿瘤内部免疫变化。方法我们通过含H-ras基因逆转录病毒转染EGFR基因缺失或野生型小鼠角质细胞，将改造的细胞同源移植至小鼠以成瘤，吉非替尼治疗荷瘤小鼠，流式细胞仪检测T细胞比例与程序性死亡受体1（programmed death 1, PD-1）表达，RT-PCR检测细胞因子与趋化因子的表达。结果敲除EGFR基因形成的肿瘤较野生型小，并且肿瘤微环境中浸润FoxP3+调节性T细胞（regulatory cells, Treg）细胞较少，FoxP3 RNA较少，程序性死亡受体1（programmed death 1, PD-1）阳性CD4+细胞比例降低。表明肿瘤细胞可以自主调节肿瘤微环境。野生型成瘤模型使用吉非替尼治疗1周显示，相对于对照组肿瘤较小；在这短期的药理模型中，同样观察到FoxP3+细胞、FoxP3 RNA减少的趋势，同时IL-1A/IL-1RA比例明显升高，表明相对短暂的系统性抑制EGFR信号通路可改变靶向肿瘤的免疫微环境。结论肿瘤细胞自发（基因）或系统性（药理作用）的抑制EGFR信号通路可减少肿瘤的生长和肿瘤微环境中Treg的渗透。EGFR依赖性Treg细胞增强肺鳞癌的生长，是EGFR抑制剂治疗的靶标。

Association of lymph node involvement with the prognosis of pathological T1 invasive non-small cell lung cancer

BackgroundLymph node involvement could help to predict the prognosis of pathological T1 (pT1, diameters of ≤3 cm) non-small cell lung cancer (NSCLC). This study assessed the clinicopathological factors and associated lymph node involvement in invasive lung adenocarcinoma (IAC) and squamous cell lung cancer (SCC) and the overall and disease-free survival associated with these factors.MethodsThree hundred and twenty-five patients with pathological T1 NSCLC (253 IAC and 72 SCC) were retrospectively analyzed from a pool of 1094 primary lung cancer patients. The data were assessed using multiple logistic regression, Kaplan-Meier curves and multivariable analyses.ResultsAmong patients with a ≤30-mm tumor lesion (N = 325), N1 and N2 lymph node involvement was found in 28 (8.6%) and 34 (10.4%) patients, respectively. Lymph node metastasis occurred in 13.0% (33/253) of pT1 IAC patients and 40.3% (29/72) of SCC patients. Carcinoembryonic antigen (CEA) levels, SCC by histology, and tumor lesions larger than 1.0 cm were associated with lymph node involvement (P < 0.0001, <0.0001, and 0.048, respectively). In IAC patients, negative lymph nodes were associated with better overall survival compared with lymph node-positive ones (P = 0.021). No significant difference was observed in SCC patients regardless of lymph node status (P = 0.40). Multivariable Cox analysis revealed that lymph node involvement was an independent prognostic predictor of overall IAC patient survival (P = 0.041), but not of SCC patient survival (P = 0.470). Chemotherapy was administered to 72.2% (52/72) of SCC patients, a significantly higher rate when compared with that of IAC patients (42.3%, 107/253).ConclusionsLymph node metastasis was inversely associated with the overall survival of IAP patients, but not with the survival of SCC patients. Patients with pT1 SCC exhibited a significantly higher rate of lymph node involvement when compared with IAC patients. Thus, a systematic lymph node dissection should be performed in pT1 IAC patients, especially in patients with IAC larger than 1.0 cm, for additional treatment selections to improve survival.

The landscape of the tumor-infiltrating B cell and its association with T-cell and tumor microenvironment in human cancers.

76 Background: Compared to recent advances in our knowledge of T cell biology with success of immunotherapy, little progress has been made in understanding of the effects of B cells in tumor microenvironment and their interactions with T cells. Preclinical studies reported that B cells may have immune suppressive roles in tumor microenvironment via induction of T cell exhaustion. However, this association has not been shown in human tissues. We explored the landscape of tumor infiltrating B and T cells and their association with tumor microenvironment in various human cancers for which the FDA approved the use of immune checkpoint inhibitors. Methods: Expression patterns for 812 immune related genes from the TCGA database were utilized to define tumor infiltrating cells in 2951 patients with bladder urothelial carcinoma, renal clear cell carcinoma, skin cutaneous melanoma, lung squamous cell carcinoma, lung adenocarcinoma, and head and neck squamous cell carcinoma. Odds ratios (ORs) of the numbers of tumors with versus without activated B cell infiltration by the presence of activated CD8T cell infiltration were calculated. Results: Immune landscape of the six human cancers showed a consistent inverse association between tumor infiltrating activated B and CD8 T cells (OR = 0.18, p &lt; 0.001). B cell infiltration was associated with increased expressions of immune checkpoints PD-L1, PD-1 and CTLA-4 and regulatory cytokines TGF-β, IL-10 and IL-35, which are known to be secreted by regulatory B cells. Angiogenic markers, such as angiopoietins, VEGF, MMP-9, CXCL10, CXCL11 and Tie2, showed differential expression patterns between B cell high and low groups. Conclusions: This is the first study that reports the inverse association between tumor infiltrating B and CD8 T cells in human tissues. The strong associations between B cell infiltration and increased expressions of suppressive cytokines and immune checkpoints suggest regulatory B cells may play a role in the T cell suppression in tumor microenvironment. Our results implicate that depleting B cells, leading to possible disinhibition of T cell activation, may be a future therapeutic option in potentiating T cell mediated immunity.

Phase II study of nivolumab in patients with advanced non-small cell lung cancer (NSCLC) in Korea.

92 Background: Nivolumab (BMS-936558/ONO-4538), a fully human IgG4, PD-1 immune-checkpoint inhibitor antibody, has shown durable clinical activity in several tumor types. Recently, two phase III studies (CheckMate-017 and -057) demonstrated that nivolumab improved overall survival (OS) than docetaxel in second-line of squamous (SQ) and non-squamous (NSQ) Non-Small Cell Lung Cancer (NSCLC), respectively. Here, we report the results of a phase II study to evaluate the efficacy and safety of nivolumab in Korean patients (pts) with previously treated advanced SQ and NSQ NSCLC. Methods: This study requires pts aged ≥ 20 years with ECOG Performance Status (PS) of 0 or 1, stage IIIB/IV or recurrent NSCLC and at least one prior chemotherapy including platinum containing regimen. Pts received nivolumab 3 mg/kg IV Q2W until progression or unacceptable toxicity. The primary endpoint in this study was the objective response rate (ORR) (RECIST v1.1). Results: Nivolumab was administered to 100 NSCLC pts (SQ: 44, NSQ: 56), male/female: 78 (SQ: 44, NSQ: 34)/22 (SQ: 0, NSQ: 22); PS 0/1: 14 (SQ: 6, NSQ: 8)/86 (SQ: 38, NSQ: 48); aged 29 to 80 [median: 66.5] years (SQ: 40 to 80 [median: 69.5], NSQ: 29 to 77 [median: 63.5]); Stage IIIB/IV/recurrence: 6 (SQ: 5, NSQ: 1)/91 (SQ: 37, NSQ: 54)/3 (SQ: 2, NSQ: 1)). In SQ and NSQ NSCLC, ORR was 15.9% (7/44) and 23.2% (13/56), respectively. Median progression-free survival was 2.6 mo and 5.3 mo, respectively. Complete Response was observed in 2.3% (1/44) and 1.8% (1/56), respectively. Median OS was 12.3 mo and 16.3 mo, respectively. Median follow-up was 8.9 mo and 12.3 mo, respectively. Most common adverse drug reaction (ADR) was decreased appetite 15.9% (7/44), followed by pyrexia 9.1% (4/44) in SQ NSCLC, and decreased appetite 12.5% (7/56), followed by pruritus 10.7% (6/56), fatigue 8.9% (5/56), pyrexia 5.4% (3/56) and nausea 5.4% (3/56) in NSQ NSCLC. Grade 3-4 ADR was observed in 6.8% (3/44) and 10.7% (6/56) of SQ and NSQ NSCLC, respectively. No interstitial lung disease and no grade 5 ADRs were observed in this study. Conclusions: Nivolumab was considered to be effective and used safely in Korean pts with SQ and NSQ NSCLC as well as in non-Korean pts with SQ and NSQ NSCLC. Clinical trial information: NCT02175017.

Nivolumab-Induced Rapid Tumor Remission of Pulmonary Adenocarcinoma

We The immune checkpoint modulator, nivolumab (BMS-936558/ONO-4538), is the first PD-1 inhibitor to gain regulatory approval, for the treatment of patients with unresectable melanoma. Nivolumab received FDA approval for the treatment of melanoma in December 2014. On 24 April 2015, the Committee for Medical Products for Human Use of the European Medicine Agency recommended approval of Nivolumab for metastatic melanoma as a monotherapy. In March 2015, the US FDA approved it for the treatment of squamous non-small cell lung cancer. This case reports about a patient with metastatical pulmonary adenocarcinoma who benefited from the therapy with nivolumab. Already after 10 weeks of treatment with nivolumab, we registered rapid tumor remission.

Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix.

Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression.A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma.A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC.Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition.

Epigenome-wide association study of smoking and DNA methylation in non-small cell lung neoplasms.

Tobacco smoke is a well-established lung cancer carcinogen. We hypothesize that epigenetic processes underlie carcinogenesis. The objective of this study is to examine the effects of smoke exposure on DNA methylation to search for novel susceptibility loci. We obtained epigenome-wide DNA methylation data from lung adenocarcinoma (LUAD) and lung squamous cell (LUSC) tissues in The Cancer Genome Atlas (TCGA). We performed a two-stage discovery (n = 326) and validation (n = 185) analysis to investigate the association of epigenetic DNA methylation level with cigarette smoking pack-years. We also externally validated our findings in an independent dataset. Linear model with least square estimator and spline regression were performed to examine the association between DNA methylation and smoking. We identified five CpG sites highly associated with pack-years of cigarette smoking. Smoking was negatively associated with methylation levels in cg25771041 (WWTR1, p = 3.6 × 10−9), cg16200496 (NFIX, p = 3.4 × 10−12), cg22515201 (PLA2G6, p = 1.0 × 10−9) and cg24823993 (NHP2L1, p = 5.1 × 10−8) and positively associated with the methylation level in cg11875268 (SMUG1, p = 4.3 × 10−8). The CpG-smoking association was stronger in LUSC than LUAD. Of the five loci, smoking explained the most variation in cg16200496 (R2 = 0.098 [both types] and 0.144 [LUSC]). We identified 5 novel CpG candidates that demonstrate differential methylation patterns associated with smoke exposure in lung neoplasms.

Abstract LB-072: Impact of baseline serum cytokines on survival in patients (pts) with advanced squamous (SQ) non-small cell lung cancer (NSCLC) treated with nivolumab (nivo) or docetaxel (doc): Exploratory analyses from CheckMate 063 and CheckMate 017

Abstract Background: Nivo, a fully human IgG4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, is approved in the United States and European Union for the treatment of pts with advanced SQ NSCLC refractory to prior chemotherapy, based on results of phase 2 (CheckMate 063) and phase 3 (CheckMate 017) trials. In these studies, PD-1 ligand (PD-L1) expression was neither predictive nor prognostic of survival benefit. Here we present results of exploratory analyses from these studies evaluating the potential correlation of baseline serum cytokines with overall survival (OS) in pts with SQ NSCLC. Methods: Baseline serum cytokine concentrations in evaluable pts from CheckMate 063 and CheckMate 017 treated with nivo (n = 222) or doc (n = 118) were analyzed using a custom HumanMAP quantitative multiplexed immunoassay (Myriad RBM, Austin, TX). Multivariate analyses using a stepwise variable selection were performed in a Cox model using a 6:4 training:test validation in nivo-treated subjects. The performance of the association with OS of the identified cytokine set in nivo-treated subjects was tested using time-varying receiver-operating characteristic (ROC) analysis. SQ-cytoscore, generated to quantify the effect of the identified cytokine set on OS, was computed as follows: 1) calculation of the tertile bin distribution of each cytokine in the set in all pts and assignment of a point score (0, 1, or 2) for each tertile expression of each cytokine; 2) calculation of SQ-cytoscore for each pt as the sum of points; 3) categorization of pts as SQ-cytoscore “high” or “low” based on the median cutoff. OS was analyzed in pts with high vs low SQ-cytoscore treated with nivo and doc using the Kaplan-Meier method and 18-mo data cutoffs for CheckMate 063 (June 2015) and CheckMate 017 (August 2015). Results: Of 26 evaluable baseline cytokines, a set of 14 was identified to be associated with OS. Median OS was longer in pts with high vs low SQ-cytoscore in both nivo- and doc-treated cohorts (nivo [n = 102 vs 120]: 15.6 vs 5.3 months, HR:0.48, 95%CI:0.36-0.64, P&amp;lt;0.0001; doc [n = 70 vs 48]: 9.1 vs 4.9 months (HR:0.39, 95%CI:0.27-0.56, P&amp;lt;0.0001). Among pts with high SQ-cytoscores (n = 172), median OS was 15.6 vs 9.1 months (HR:0.63, 95%CI:0.45-0.88, P = 0.0051) for nivo- vs doc-treated pts, respectively. Among pts with low SQ-cytoscores (n = 168), median OS was 5.3 vs 4.9 months (HR:0.51, 95%CI:0.37-0.71, P = 0.0009) for nivo- vs doc-treated pts, respectively. Conclusion: In pts with advanced SQ NSCLC pooled from 2 clinical trials, a set of serum cytokines (details to be presented) potentially associated with OS benefit was identified. The benefit of a high SQ-cytoscore appeared more profound in pts treated with nivo vs doc. These preliminary findings require prospective validation in future studies. Citation Format: Benedetto Farsaci, William J. Geese, Kaushal D. Desai, Chelsea Jin, Scott J. Antonia, Hervé Lena, Leora Horn, David Planchard, Karen L. Reckamp, Thomas E. Stinchcombe, Scott Gettinger, Hossein Borghaei, Matthew D. Hellmann, Christopher Harbison, Dong Xu, M. Anne Blackwood-Chirchir, Naiyer Rizvi. Impact of baseline serum cytokines on survival in patients (pts) with advanced squamous (SQ) non-small cell lung cancer (NSCLC) treated with nivolumab (nivo) or docetaxel (doc): Exploratory analyses from CheckMate 063 and CheckMate 017. [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 LB-072.

Abstract LB-140: Scaling discovery proteomics to large lung cancer cohorts using data independent acquisition

Abstract The success of label free protein quantification relies on larger cohort sizes to accurately profile protein expression patterns enabling specific targets to be translated to confirmatory studies. Thus, proteome profiling experiments must satisfy two primary requirements: maximizing breadth and depth of protein sampling as well as facilitating automated data processing. The approach presented here integrates discovery experiments used to create reference databases and rapid data independent acquisition (DIA) methods to examine large clinical cohorts using &amp;lt; 3 hours of instrument time per individual patient's resected tumor specimen. The combination facilitates exhaustive, automated data mining for protein identification and relative quantitation. Samples used to evaluate the robustness of the study were collected from lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). A sample total of 159 (50 LUSC, 46 LUAD, and 63 adjacent control lung tissues) were analyzed using UHPLC separations coupled to a hybrid quadrupole-orbital ion trap mass spectrometer (QExactive Plus) for DIA. Automated data processing was performed by matching peptides in spectral libraries developed from expression proteomics experiments using pooled tissue proteomes. Data were evaluated to determine differences between LUSC, LUAD, and adjacent control tissue, which were then compared to previous literature to prioritize candidate biomarkers and evaluate the performance of DIA LC-MS/MS for tumor proteome profiling. Hierarchical clustering visualized potential tumor classification schemes based on proteomic phenotypes. In a further study, analysis of cores from a tissue microarray produced quantitative data for &amp;gt; 3,000 proteins per core, indicating that the technology can be applied to minimal amounts of formalin fixed paraffin embedded tumor tissue. Because TMAs are assembled to address specific clinical questions, further analysis of these cohorts is an extremely valuable use for DIA; tumor proteome profiles can be rapidly accumulated and mapped to the clinical variables used to select samples for the TMA. Data independent acquisition provides a method for discovery proteomics that balances a sufficient depth of coverage with the ability to analyze large cohorts of patients (n = 100-400) within 1-2 months on a single instrument. Initial data have been produced for lung squamous cell carcinoma and lung adenocarcinoma, which indicate its utility for assessment of patient groups. Citation Format: Scott Peterman, Bin Fang, Melissa Hoffmann, Amol Prakash, Paul A. Stewart, Richard Liu, Matthew Smith, Joseph Johnson, Steven Eschrich, Guolin Zhang, Eric Haura, John Koomen. Scaling discovery proteomics to large lung cancer cohorts using data independent acquisition. [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 LB-140.

Abstract 2873: Identification of FXR1-associated protein complexes in lung cancer

Abstract RNA-binding proteins (RBPs) are the master regulators of mRNA processing and translation and are often aberrantly expressed in cancer. We have recently identified Fragile X Mental Retardation-Related 1 (FXR1) as a novel cancer gene in non-small cell lung cancer (NSCLC) and its expression is correlated with poor prognosis in multiple human cancers. FXR1 encodes an RNA binding protein (RBP) that belongs to the family of fragile X-related proteins including the fragile X mental retardation protein (FMR1) and FXR2. Inactivation of FMR1 expression is the cause of the Fragile X syndrome in humans. Little is known for the function of FXR1 in human cancers. In this study, endogenous FXR1 or a Flag-tagged FXR1 was immunoprecipitated from H520, a lung squamous carcinoma cell line, and analyzed by shotgun proteomics. The Flag-tagged FXR1 was also transfected into human HEK293 cells and followed by co-immunoprecipitation and shotgun proteomic analysis. In total we found 206 proteins enriched in H520 with more than two-fold change spectral counts over the IgG control. Of the 206 proteins, 49 were detected in HEK293 cells as well and 157 were only detected in H520. KEGG pathway analysis indicated enrichment of proteins involved in ribosomal function, RNA transport and proteasome. To identify lung cancer related proteins, we interrogated the mRNA expression of 206 proteins in The Cancer Genome Atlas (TCGA) lung cancer dataset (1013 tumors,109 normal), in a combined NSCLC dataset (1392 tumors, 240 normal) from the Gene Expression Omnibus (GEO) and in a previously reported dataset of putative FXR1 target mRNAs and found 75 altered genes in common. Among these, we found that 24 genes were not only unregulated in all NSCLC samples (n = 2405, FDR&amp;lt;0.007) but also positively correlated with FXR1 (p&amp;lt;0.05). Gene ontology analysis revealed 22 genes were enriched in cellular macromolecule metabolic process including six genes in cell cycle regulation and seven genes in mRNA processing (p = 0.0017). Disease association analysis showed six genes were enriched in immunologic deficiency syndromes (p = 0.002), consistent with post-transcriptional suppression of tumor necrosis factor-alpha (TNFα) by FXR1 and further implying a role for FXR1-associated complex in the suppressed immune response pathway we previously identified in squamous cell carcinoma of the lung. Lastly, survival analysis indicated that this 25-gene signature (BUB3,CSNK2A1,DARS,DHX9,FXR1,HDAC2,HNRNPAB,HSP90AA1,KPNB1,LMNB1,MCM4,MSH6,NUP205,PRKDC,PSMC5,PSMD3,SFPQ,SMARCC1,SMC4,SNRPD1,STK38L,TNPO1,UCK2,XPO1 and XRCC5) was significantly associated with worse overall survival in NSCLC (HR = 1.84, p = 0.01, adjusted for age, gender, stage and smoking history). Together, these data suggest the 24 proteins/mRNAs are closely associated with FXR1 in lung cancer and may form critical core complexes that are associated with NSCLC tumorigenesis. This work was supported by RO1 CA102353 to PPM. Citation Format: Jun Qian, Yong Zou, Megan Hoeksema, Bradford Harris, Heidi Chen, Pierre Massion. Identification of FXR1-associated protein complexes in 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 2873.

Abstract 593: Highly cytotoxic EGFR/CD16A TandAbs specifically recruit NK cells to potently kill various types of solid tumors

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

Abstract The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [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 1377.

Abstract 3875: Proteome-wide approach to identify novel biomarkers in oral tumorigenesis

Abstract Oral cancer is the most common type of intraoral head and neck cancer in humans, with a worldwide prevalence of more than 400,000 cases. In India, oral cancer ranks in top three in comparison with other cancer types. The majority of oral cancer patients are diagnosed at an advanced disease stage. Although, there have been many advances in oral cancer treatment, the overall survival rate of oral cancer patients only improved marginally over the past 30 years. The treatment regimen of oral cancer is mainly based on the tumor, node and metastasis (TNM) classification and histopathological diagnosis. These methods are subjective and often have low sensitivity to detect the disease in early stages. Hence, there is an urgent need for early diagnosis to develop biomarkers; to identify high risk individuals, to improve cancer detection at early stages, to predict disease outcome and response to therapy. Proteomics have been successfully employed in studies of lung, breast, prostate, gastrointestinal cancer, and head and neck squamous cell carcinoma. Reports are not much available to describe well characterized biomarkers which will aid for early detection of oral cancer. Racial differences were reported in many cancers including its morbidity, mortality, treatment response and survival rates. In Indian patient population, there is a significant knowledge gap exists in utilizing proteomics for biomarker discovery. The aim of this study is to identify novel candidate genes which may be used as prognostic or diagnostic biomarkers for Indian oral squamous cell carcinoma. The tongue tumor and adjacent normal samples were subjected to 2D - Differential in Gel Electrophoresis (2D-DIGE). Differentially expressed proteins in tumor samples were identified using nano-LC-MS/MS and validated by quantitative real time PCR. We found 800 protein spots were differentially expressed and seventy proteins were identified by mass spectrometry. Among them twelve proteins including Myosin isoforms, hemoglobin beta, Annexin A1, Creatinine kinase, Carbonic anhydrase, HSPA8 showed decreased expression while eighteen proteins including Apolipoprotein A1, HSPA5, Alpha 1 antitrypsin, Annexin isoforms, Peptidyl prolyl cis trans isomerase, Serpin isoforms, Tropomyosin, Thioredoxin, Alpha enolase, Calpain, Glutathione S Transferase, Heat shock protein beta 1, S100 isoforms showed increased expression in tongue tumor tissues. Most of the differentially regulated proteins were known to be involved in calcium homeostasis, cell cycle, cell growth and migration. The major outcome of this study was the identification of novel protein molecules which might be used as predictive, prognostic and diagnostic biomarkers for oral cancer. These proteins can also serve as a therapeutic intervention targets for oral cancer progression. This study would be the first study to map the whole proteome profile of the cancerous as well as normal tongue cheek tissue in Indian scenario. Citation Format: Sivagnanam Ananthi, Naga Padma Lakshmi CH, Anbarasu K, Mahalingam Sundarasamy. Proteome-wide approach to identify novel biomarkers in oral tumorigenesis. [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 3875.

Abstract 849: Improved geometric deconvolution of bulk tumor genomic data

Abstract We develop new methods for improved computational deconvolution of clonal populations from heterogeneous bulk genomic data. Deconvolution methods have become an important tool in resolving intratumor heterogeneity from bulk tumor genomic data, but prevailing methods are still able only to resolve only coarse-grained approximations of true clonal substructure. We seek to improve a genomic deconvolution strategy called “simplicial complex inference” that is designed to take advantage of similarity between the clonal subpopulations of single tumors or tumor regions to better infer portraits of those clonal subpopulations and their distribution across tumor samples. We evaluate the method through application to DNA copy-number (CN) data from a 472-tumor ovarian cancer (OV) panel and a 408-tumor lung squamous small cell carcinoma (LUSC) panel from The Cancer Genome Atlas (TCGA). We deconvolved the OV data into a mixture of six clonal models, two per data sample, each interpreted as a consensus copy number profile of one recurring progression stage found in a subset of OV samples. We analyzed the resulting models for enrichment of labels derived from RNASeq-based subtyping (Verhaak et al., 2013), revealing significant correspondence by chi-squared test between tumor subclusters identified by the deconvolution and OV subtypes (p = 0.0168). We further analyzed the clonal models to identify regions of significant amplification or loss, which we tested for enrichment of gene ontology (GO) terms and pathway terms through the DAVID toolkit. Ontological analysis showed significant enrichment for GO terms connected to genetic regulation and proliferation including “DNA binding,” “DNA packing,” and “protein-DNA complex” (p &amp;lt; 0.001). The LUSC data was partitioned into nine clonal models, three per sample. A log-rank test showed significant correspondence between mixture subclustering and survival for LUSC patients (p &amp;lt; 0.001). Regions of significant CN gain or loss identified in the clonal models were found by DAVID to be significantly enriched for pathway terms including “Cytokine network” (p = 0.0025) and “Cytokines and Inflammatory Response” (p = 0.0043). These findings suggest that more sophisticated models of clonal population structures can enhance our ability to deconvolve heterogeneous tumor genomic data and lead to better inference of clonal substructure. Citation Format: Theodore Roman, Lu Xie, Russell Schwartz. Improved geometric deconvolution of bulk tumor genomic data. [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 849.

Abstract 2818: An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers

Abstract Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models. Citation Format: Xueqian Gong, Wenjuan Wu, Li-Chun Chio, Susan Pratt, Constance King, Yue Webster, Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Philip Iversen, Christoph Reinhard, Shih-Hsun Chen, Richard Bechmann, Sheng-Bin Peng, Sean Buchanan. An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers. [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 2818.

Abstract 971: A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma

Abstract Long non-coding RNAs (lncRNAs) are RNAs that are longer than 200 base pairs and do not translate into proteins but have been shown to play roles in all aspects of biological regulation. LncRNAs have also been shown to regulate gene expression including chromatin organization, transcriptional regulation, and post-transcriptional control. In addition, they are now emerging to be important players in our understanding of cancer and have been shown to promote tumorigenesis. However, the mechanisms driving the advancement of tumor progression to metastasis is poorly understood. To identify lncRNAs critical to tumorigenesis, our lab conducted a pan-cancer analysis of normal and primary tumor tissues from The Cancer Genome Atlas to discover lncRNAs that are commonly altered across cancer types, which we termed ‘onco-lncRNAs’. During our analysis, we discovered a novel unannotated onco-lncRNA, onco-lncRNA-230, that was significantly up regulated in head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC) tumors relative to matched adjacent normal tissues, when available. We chose to focus on onco-lncRNA-230 as it displayed more than seven fold increase in LUAD and more than thirty seven fold change in LUSC tumors relative to adjacent normal. First, we validated this finding by quantitative PCR which revealed greater than a two fold enrichment of onco-lncRNA-230 expression in two out of seven LUAD and all six LUSC cells lines compared to a normal lung cell line. Because we observed the greatest increase in expression in HCC95 LUSC cells, we next sought to determine if modulating the expression of onco-lncRNA-230 promotes oncogenic phenotypes. Silencing of onco-lncRNA-230 in HCC95 cells did not change cellular proliferation but significantly increased apoptosis. Furthermore, silencing onco-lncRNA-230 resulted in a decrease in cellular invasion by transwell assay. Taken together, this represents the first study reporting that onco-lncRNA-230 is dysregulated in multiple cancer types, significantly up-regulated in lung cancer patient samples and lung cancer cell lines, and confers oncogenic phenotypes. Moving forward, we intend to explore how onco-lncRNA-230 mechanistically promotes lung cancer with the objective of utilizing onco-lncRNA-230 as a novel cancer diagnostic and therapy. Citation Format: Cynthia Y. Tang, Jessica M. Silva-Fisher, Ha X. Dang, Nicole M. White, Christopher A. Maher. A novel long noncoding RNA, onco-lncRNA 230, induces apoptosis and invasion in lung squamous cell carcinoma. [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 971.

Research progress of the medicine treatment of advanced lung squamous cell carcinoma

In the last decade, many molecular targeted drugs,such as epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), have been successively applied in clinical use, and they have brought about a substantial prolongation of survival life and improvement in life quality of those patients with lung adenocarcinoma, but that is not real in squamous cell lung cancer.Immunotherapy clinical trials for programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) pathway in non-small cell lung cancer (NSCLC) have been started since 2012. In 2015, an anti-PD-1 antibody,nivolumab,was proved by FDA to treat advanced squamous NSCLC with progression on or after platinum-based chemotherapy. This article reviews the recent advantages in the treatment of advanced lung squamous cell carcinoma. Key words: Lung neoplasms; Carcinoma, squamous cell; Drug therapy; Molecular targeted therapy; Immune checkpoint inhibitors

Economic evaluation of nivolumab for the treatment of second-line advanced squamous NSCLC in Canada: a comparison of modeling approaches to estimate and extrapolate survival outcomes

Background Lung cancer is the most common type of cancer in the world and is associated with significant mortality. Nivolumab demonstrated statistically significant improvements in progression-free survival (PFS) and overall survival (OS) for patients with advanced squamous non-small cell lung cancer (NSCLC) who were previously treated. The cost-effectiveness of nivolumab has not been assessed in Canada. A contentious component of projecting long-term cost and outcomes in cancer relates to the modeling approach adopted, with the two most common approaches being partitioned survival (PS) and Markov models. The objectives of this analysis were to estimate the cost-utility of nivolumab and to compare the results using these alternative modeling approaches.Methods Both PS and Markov models were developed using docetaxel and erlotinib as comparators. A three-health state model was used consisting of progression-free, progressed disease, and death. Disease progression and time to progression were estimated by identifying best-fitting survival curves from the clinical trial data for PFS and OS. Expected costs and health outcomes were calculated by combining health-state occupancy with medical resource use and quality-of-life assigned to each of the three health states. The health outcomes included in the model were survival and quality-adjusted-life-years (QALYs).Results Nivolumab was found to have the highest expected per-patient cost, but also improved per-patient life years (LYs) and QALYs. Nivolumab cost an additional $151,560 and $140,601 per QALY gained compared to docetaxel and erlotinib, respectively, using a PS model approach. The cost-utility estimates using a Markov model were very similar ($152,229 and $141,838, respectively, per QALY gained).Conclusions Nivolumab was found to involve a trade-off between improved patient survival and QALYs, and increased cost. It was found that the use of a PS or Markov model produced very similar estimates of expected cost, outcomes, and incremental cost-utility.

Abstract OT1-01-07: nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC)

Abstract Background: Nivo is an inhibitory antibody against programmed death receptor-1 (PD-1), a regulator of antitumor immunity. Nivo is approved for treatment of unresectable or metastatic melanoma and disease progression (PD) following ipilimumab or, in BRAF V600 mutation–positive melanoma, following a BRAF inhibitor, and for metastatic squamous non-small cell lung cancer (NSCLC) following PD during or after platinum-based chemotherapy. Nivo and other immune checkpoint inhibitors are also being investigated in other tumor-types. nab-P is a novel taxane formulation and does not require prophylaxis with immunosuppressive steroids. It has demonstrated superior efficacy over control regimens in phase III studies of MBC, pancreatic cancer, and NSCLC. This open-label, 6-arm, multicenter phase I trial will evaluate the safety of Nivo with nab-P in 3 cancer types (2 arms/disease): MBC, advanced NSCLC (+ carboplatin), and advanced pancreatic cancer (± gemcitabine). The study design for the MBC portion is described below. Methods: Eligibility criteria include histologically/cytologically confirmed HER2-negative MBC; 1 prior chemotherapy for MBC, including an anthracycline unless clinically contraindicated; no relapse &amp;lt; 12 months after taxane adjuvant therapy; measurable disease by RECIST v1.1; ECOG performance status 0-1; adequate organ function; and preexisting peripheral neuropathy grade &amp;lt; 2. Patients (pts) with MBC will be treated in 2 arms: nab-P 100 mg/m2 on days 1, 8, and 15 of each 28-day cycle plus Nivo 3 mg/kg on days 1 and 15 starting at cycle 3 or nab-P 260 mg/m2 on day 1 of each 21-day cycle plus Nivo 5 mg/kg on day 15 starting at cycle 3. Pts will be treated until PD or allowed to continue treatment beyond RECIST v1.1–defined PD if they continue to meet study eligibility; do not have rapid PD or clinical deterioration or unacceptable toxicities; and can benefit from continuation of study treatment in the treating physician's opinion and will not delay an imminent intervention to prevent serious complications of PD. The primary endpoints of the study are the number of pts with dose-limiting toxicities (DLTs) in each treatment arm (part 1) and the percentage of pts with grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to a TEAE (parts 1 and 2). Part 1 of the study will assess whether the starting dose of Nivo is deemed safe (≤ 1 DLT in 6 pts); otherwise, the Nivo dose will be de-escalated and assessed in a new cohort at the next lower dose level. The Nivo dose in combination with nab-P deemed safe in a treatment arm may be further assessed in part 2 of the study, with enrollment expanded to an additional ∼ 14 pts/arm (total of 20 Nivo-treated pts/arm). Secondary study endpoints include TEAEs leading to dose reduction, delay, interruption, or treatment discontinuation; progression-free survival; overall survival; disease control rate; overall response rate; and duration of response (per RECIST v1.1). Exploratory endpoints include tumor-associated PD-L1 expression, modulation of immune activation in the tumor and peripheral blood in response to Nivo treatment, Nivo serum levels, and development of anti-globulin antibodies. ClinicalTrials.gov identifier NCT02309177. Citation Format: Waterhouse D, Gutierrez M, Bekaii-Saab T, DeRosa W, Wainberg Z, George B, Duval Fraser C, Ko A, Pierce DW, Stergiopoulos S, Soliman H. nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-07.

New oncogenes drivers in lung cancer—new therapeutic targets

Many advances have been achieved during the last decade in the field of lung cancer molecular biology, leading to the identification of potential new oncogene drivers and new therapeutic targets. However, only two targetable biomarkers are currently approved for lung cancer treatment: EGFR activating mutations and EML4-ALK rearrangements. Adenocarcinoma is the most common type of non-small cell lung cancer and was the first to be studied. Indeed, most of lung adenocarcinoma biomarkers were identified several years ago. Nevertheless, new therapies targeting these biomarkers are still investigated. More recently, new oncogene drivers have been identified in the field of lung squamous cell carcinoma and small cell lung cancer, and new agents are being developed to target these biomarkers.