bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A4: An expression map of long noncoding RNAs in human lung and non-small cell lung cancer
Authors: Becker-Santos Daiana D. , Gibb Ewan E. , Vucic Emily A. , Enfield Katey S.S. , Stewart Greg L. , MacAulay Calum E. , Lam Stephen , Lam Wan L.
Published: March 2013

Background: Although still largely unexplored, long non-coding RNAs (lncRNAS) are emerging as an integral functional component of the human transcriptome. LncRNAs are mRNA-like transcripts of at least 200 nucleotides (nts) with no protein-coding capacity. Similar to their protein-coding counterparts, lncRNAs are frequently spliced and polyadenylated, but act at the RNA level. The range of functions described for lncRNAs is extensive, and includes key biological roles in chromatin remodeling, alternative splicing and mRNA degradation. Given their biological functions, dysregulation of lncRNAs is rising as an important feature of many disorders, including malignant transformation. However, the extent of the contribution of differential lncRNA expression to normal lung tissue and lung cancer has not been investigated in a comprehensive manner.

Hypothesis: We hypothesized that lncRNAs are expressed in a lung tissue-specific manner and that non-small-cell lung cancer (NSCLC) exhibits aberrant lncRNA expression patterns.

Methods: Serial Analysis of Gene Expression (SAGE) libraries were used to characterize polyadenylated transcripts in lung tissue compared to a panel of 25 different normal human tissues, and to a cohort of 12 NSCLCs. To generate lncRNA expression profiles, we developed a lncRNA discovery pipeline to map-tag-to-lncRNA matches. To identify differentially expressed lncRNAs we used a permutation test based statistical analysis. Expression pattern in lung tumors were compared to profiles from a variety of cancer types in order to identify lncRNA changes prominent in lung cancer.

Results: Here we show that large-scale expression profiling through SAGE, is an effective resource for investigating the expression pattern of polyadenylated lncRNAs. Applying a novel lncRNA discovery pipeline we reveal extensive, tissue-specific lncRNA expression in normal lung compared to a panel of several different normal human tissues. Importantly, our study reveals that NSCLC demonstrate significantly altered lncRNA expression patterns and identify highly dysregulated transcripts associated with this malignancy as oppose to other types of cancer.

Conclusion: Collectively, our findings support an important role for tissue-specific lncRNAs in lung cancer. Characterization of the functional role of these transcripts will have a considerable impact on our understanding of lung cancer development and progression, and may reveal clinically important biomarkers.
Abstract A41: SOX11 transcriptionally regulates a cytokine-based gene signature in lung cancer-associated fibroblasts (LCAF) to promote tumor growth
Authors: Vicent Silvestre , Gillespie Anna K. , Vakaa Dedeepya , Khatri Purvesh , Chen Ron , Gevaert Olivier , Clarke Nicole , Plevritis Sylvia , Butte Atul , Sweet-Cordero Alejandro
Published: March 2013

Increasing evidence points to an important role for the tumor microenvironment in cancer progression. However, the role of tumor stroma in the pathogenesis of lung cancer is poorly understood. Desmoplasia is a common pathologic feature of many NSCLC. We noted a similar phenotype in a mouse model of lung cancer driven by expression of oncogenic Kras. Lung cancer-associated fibroblasts (LCAFs) and normal lung fibroblasts (NLFs) from the mouse model of Kras-driven lung cancer were isolated and queried at the functional level using a xenograft model. First, we observed that LCAFs promote tumor growth more favorably than NLFs in vivo when co-injected with mouse tumor cells. Moreover, NLFs passaged in vivo acquire features of LCAFs as indicated by an increased tumor-promoting capacity on mouse and human NSCLC xenografts. In addition, conditioned media from LCAFs stimulated cell proliferation in vitro more significantly than that of NLFs. Expression profiling of NLFs and LCAFs using microarrays uncovered a gene set that includes multiple secreted proteins and genes related to inflammatory processes. Moreover, the SRY-related HMG transcription factor Sox11 was highly up-regulated in LCAFs compared to NLFs. Over-expression and knock-down experiments have confirmed that Sox11 regulates the expression of many of the secreted proteins expressed in LCAFs. The relevance of these findings for human NSCLC is suggested by the observation that the mouse LCAF gene expression signature was predictive of survival in a large cohort of human NSCLC. Besides, Sox11 and several of the secreted proteins expressed in LCAFs, along with their putative receptors, were up-regulated in human NSCLC samples. In conclusion, we have identified a potentially novel regulator of the transition between NLFs and LCAFs and, thus, uncovered a new signaling hub that could be used for therapeutic intervention.
Abstract A7: NOTCH 1 and NOTCH 3 expressions for early stage of non-small cell lung cancer
Authors: Cho Eun Kyung , Lee Jae Hoon , Ha Seung Yeon , Park Sanghui , Lee Jae-ik , Hong Junshik , Jung Minkyu , Sym Sun Jin , Park Jinny , Shin Dong Bok
Published: March 2013

Backgrounds: Notch gene Encode 300kDa single-pass transmembrane receptors. It can variously serve as an oncogene or a tumor suppressor and a repressor or inducer of terminal differentiation. It also regulates cell division, differentiation and survival of stem and/or progenital cells in wide range tissues. We examined Notch 1 and 3 to study the correlation of Notch expression and prognosis for non-small cell lung cancer.

Methods: Paraffin-embedded tissue microarrays were constructed. We analyzed Notch 1 and 3 by immunohistochemical stain on surgical tumor specimens.

Results: Results: One hundred fifty-nine of 185 resected tissues for lung cancer (median age 62 years) were available for immunohistochemical stain. The types of tumor histology were adenocarcinoma; 54, squamous cell carcinoma:79, others;26. 121 patients were taken lobectomy and 38 were done pneumonectomy. Notch 1 and 3 receptors were detected in 119 (74.8%) and 130 (81.7%) of total 159 (M:F=114:45) tumor tissues, respectively. Notch 1 receptors showed higher expressions on squamous cell carcinoma than non-squamous cell carcinoma (58.8% vs. 37.8%, p=0.03). However, it was not correlated with sex, smoking status or advanced stages. Five year-relapse free survival (RFS) and overall survival (OS) were 57.4 % and 59.3 % in 159 patients, respectively. Patients with any expression of Notch1 or 3 have correlated with neither RFS nor OS. 5 year OS for patients who showed Notch 3 expression were 62 % and the others were 47.8%, however, it was not statistically significant (p= 0.165). Both of their expressions were detected in 106 of 159 (66.7%) tumor tissues. Sixteen (10 %) had negative expressions in both of them. The patients with both positive expressions had the tendency of longer OS than both negative patients. Their 5 year OS were 60.8 % vs. 38.2% (p=0.068) and 5 year RFS were 56.5% vs. 49.7 % (p=0.35).

Conclusions: Notch 1 showed higher expression on squamous cell carcinoma. Neither Notch 1 nor 3 have correlated with RFS or OS, but patients with both expressions had the tendency of longer overall survival.
Abstract A9: Pharmacological stimulation of DLL1-Notch signaling as an antitumor immunotherapy
Authors: Huang Yuhui , Lin Luping , Biktasova Asel K. , Malhotra Anshu , Shanker Anil , Carbone David P. , Dikov Mikhail M.
Published: March 2013

Deficiencies in immune function are one of mechanisms for tumor escape from host immune surveillance, and therapies aimed at overcoming these defects are of major importance in cancer immunotherapy. However, the underlying causes of these defects are multiple and remain not fully understood. We report a previously undescribed mechanism for tumor-associated defects in T cell function mediated by reducing levels of Delta-like (DLL) 1 and DLL4 in the hematopoietic microenvironment, and show that elevated levels of circulating vascular endothelial growth factor (VEGF) characteristic for cancer patients is one of the mediators of this effect. Remarkably, selective activation of DLL1-Notch signaling alone by over-expression of DLL1 in bone marrow precursors or by soluble multivalent DLL1 treatment significantly enhances tumor-specific immune responses and inhibits tumor growth in multiple tumor models. The data suggest that tumor growth suppresses T cell differentiation and function via inhibition of DLL-mediated Notch signaling in the hematopoietic compartment. Our findings uncover a novel molecular mechanism of immune suppression and suggest a pharmacologic intervention based on activation of DLL1-Notch signaling to reverse this effect, stimulate immune responses and suppress tumor growth. Given that recently DLL1 was also implicated in abnormal tumor vessel formation and growth inhibition [Zhang JP, ea. Cancer Lett. 2011;309:220–227], systemic stimulation of Notch by multivalent DLL1 might represent an efficient therapeutic strategy targeting multiple mechanisms of tumor growth.
Abstract B11: EGFR polymorphism as a predictor of clinical outcome in advanced lung cancer patients treated with EGFR-TKI
Authors: Jung Minkyu , Lee Chul Ho , Park Hyung Soon , Lee Ji Hyun , Kim Joo Hang , Kang Young Ae , Kim Se Kyu , Chang Joon , Kim Dae Jun , Cho Byoung Chul
Published: March 2013

Background: Mutations in the epidermal growth factor receptor (EGFR) have been confirmed as predictors of efficacy for EGFR-tyrosine kinase inhibitors (TKIs). We investigated whether polymorphisms of the EGFR gene were associated with clinical outcome in NSCLC patients treated with EGFR-TKI.

Methods: A polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one and single nucleotide polymorphisms in the promoter region (SNP -216 GG or GT) were evaluated in 71 NSCLC patients by PCR-RFLP and DNA sequencing. Genetic polymorphisms were correlated with clinical outcomes of EGFR-TKIs.

Results: SNP-216G/T polymorphisms were associated with the efficacy of EGFR-TKI. The response rate for the SNP-216G/T was significantly higher than that for the GG (62.5% vs. 27.4%, P=0.044). SNP-216G/T genotype was also associated with longer progression-free survival compared with GG genotype (16.7 months vs. 5.1 months, P=0.005). However, Genotypes for the CA-SSR1 was not associated with the efficacy of EGFR-TKI.

Conclusions: SNP-216G/T polymorphism is potential predictor for clinical outcome in NSCLC patients treated with EGFR-TKI.
Abstract B13: FGFR2 as a therapeutic target in squamous cell lung carcinoma
Authors: Liao Rachel G. , Hammerman Peter , Sivachencko Andrey , Greulich Heidi , Getz Gad , Liu Qingsong , Gray Nathanael , Meyerson Matthew
Published: March 2013

Squamous cell lung carcinoma (lung SCC)) is the second most common subtype of non-small cell lung cancer, with 40,000 new cases diagnosed every year in the United States. Unlike lung adenocarcinoma, few targetable genomic events are known drivers of lung SCC, and therefore, therapeutic options are limited for patients with this disease. As part of The Cancer Genome Atlas (TCGA) project, we have analyzed the entire coding sequence of 196 lung SCCs for mutated genes which may be amenable to targeted therapeutics. In the 196 patient samples analyzed to date, we have observed mutations in the Fibroblast Growth Factor Receptor 2 (FGFR2) kinase gene in ten cases, or 5% of samples. The observed FGFR2 mutations are present in both the extracellular domain and the kinase domain of the protein, an observation that is consistent with activating mutations in FGFR2 that are known to drive endometrial cancer. We therefore hypothesized that these mutations are oncogenic and may be driving transformation in a subset of lung SCC patients. Expression of mutated FGFR2 in NIH-3T3 cells led to anchorage-independent colony formation, which was inhibited by treatment with the pan-FGFR inhibitor FIIN-1. A known mechanism of FGFR2 activation is the constitutive dimerization of receptors with mutated extracellular domains via intermolecular disulfide bond formation, and we have observed this phenomenon in two of our ECD mutants as well. These data suggest that both ECD and kinase mutations in the FGFR2 gene are sufficient to transform cells. This finding, along with recently published data suggesting that FGFR1 amplification is also able to drive lung SCC development, demonstrates that the FGFR family may be a promising therapeutic target in lung SCC.
Abstract B20: Thyroid transcription factor 1 is embedded in a microRNA network
Authors: Mu David
Published: March 2013

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is an essential fetal lung developmental factor which can be recurrently activated by gene amplification in adult lung cancer. These findings imply that TTF-1 is a gain-of-function oncogene selected for gene amplification due to an undefined biological pressure in lung cancer cells. However, recent data in the literature showed that TTF-1 suppressed lung cancer progression in a mouse model system and that the chromosomal area harboring the TTF-1 amplicon also underwent allelic DNA loss in lung cancer. Despite the complexity surrounding TTF-1 in lung cancer, characterization of TTF-1 gene regulatory mechanisms is warranted as lung adenocarcinomas frequently amplified for TTF-1 are the most common histologic type of lung cancer. We have discovered the first microRNA that directly regulates TTF-1 by interacting with its 3′-untranslated region. By gene expression profiling, we identified other putative targets of the microRNA. In line with the microRNA/target relationship, the expression patterns of the microRNA and TTF-1 were in an inverse relationship in human lung cancer. Exploration of human lung cancer genomics data uncovered that TTF-1 gene amplification was significantly associated with DNA copy number loss at one of the two genomic loci encoding the precursor RNA of mature microRNA. This implies the existence of genetic selection pressure to lose the repressive microRNA that would otherwise suppress amplified TTF-1. We detected a signaling loop between transforming growth factor beta (TGFβ and the microRNA and this loop reinforced suppression of TTF-1, consistent with the concept that TTF-1 possesses anti-epithelial mesenchymal transition (EMT) activity. Paradoxically, the microRNA also targets an EMT-promoting gene HMGA2 which is known to be repressed by TTF-1. Our data suggest that there is a group of microRNAs that are directly regulated by TTF-1, reinforcing our hypothesis that TTF-1 in embedded in a microRNA network. In summary, these data connect the lung transcriptional program to the microRNA network.
Abstract B21: E7050, a Met kinase inhibitor, reverses three different mechanisms of hepatocyte growth factor-induced resistance to tyrosine kinase inhibitors in EGFR mutant lung cancer cells
Authors: Nakagawa Takayuki , Takeuchi Shinji , Yamada Tadaaki , Kita Kenji , Yano Seiji
Published: March 2013

Purpose: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant lung cancer cells by activating Met and downstream PI3K/Akt pathway. In addition, HGF accelerates expansion of minor clones with Met amplification and facilitates EGFR-TKI resistance. Purpose of this study is to determine whether E7050, a Met kinase inhibitor, could overcome these three resistant mechanisms to EGFR-TKIs.

Experimental design: Effect of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant selective (WZ4002) EGFR-TKIs was determined using EGFR-mutant human lung cancer cell lines, PC-9 and HCC827 with exon 19 deletion, and H1975 with T790M secondary mutation. As an in vivo models, PC-9 cells mixed with HGF producing fibroblasts, MRC-5, were subcutaneously inoculated into SCID mice and therapeutic effect of E7050 combined with gefitinib was examined.

Results: We found that E7050 circumvented resistance to all of reversible, irreversible, and mutant selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented emergence of gefitinib resistant HCC827 cells induced by continuous exposure to HGF. In vivo model, E7050 combined with gefitinib dramatically regressed the tumor growth, associating with inhibiting Akt phosphorylation in cancer cells.

Conclusions: E7050, a Met kinase inhibitor, reverses three different mechanisms of gefitinib resistance induced by HGF, suggesting usefulness of E7050 for overcoming HGF-induced resistance to gefitinib and new generation EGFR-TKIs.
Abstract B3: Relevance of genotyping non-small-cell lung cancer patients on response to platinum-basedchemotherapy and tyrosine kinase inhibitors
Authors: Campos-Parra Alma D. , Cruz Graciela , Zuloaga Carlos , Aviles Alejandro , Vázquez Manríquez María E. , Borbolla-Escoboza José R. , Cardona Andrés , Abelardo Meneses , Arrieta Oscar
Published: March 2013

Background: Subdividing non-small cell lung cancer (NSCLC) based on molecular alterations such as EGFR, KRAS and ALK genes is important for selecting treatment involving EGFR and EML4-ALK tyrosine kinase inhibitors (TKI). However, little information is available comparing patients' response and progression-free survival in the presence or absence of EGFR, KRAS mutations or the EML4-ALK fusion gene when being treated with chemotherapy.

Methods: NSCLC patients were treated with chemotherapy and/or TKIs. Tests were performed for EGFR and KRAS gene mutation as well as EML4-ALK fusion genes. Progression-free survival and overall survival association with type of treatment and mutational status was analyzed.

Results: The factors associated with a response to chemotherapy were the presence of EGFR and KRAS mutation (p = 0.006 and p = 0.028, respectively). Factors associated with TKI response were adenocarcinoma (HR 2.7: 1.6–4.6 95%CI; p<0.001), EGFR mutation (HR 0.5: 0.3–0.8 95%CI; p = 0.009) and wild-type KRAS (HR 1.7: 1.1–2.8 95%CI; p = 0.013). Mean progression-free survival in the chemotherapy group was 5.3 months (4.8–5.7 95%CI).

Conclusion: EGFR and KRAS mutation status appeared to subdivide NSCLC patients into TKI and chemotherapy response groups.
Abstract B30: Src tyrosine kinase inhibitors activate protective, ULK1-dependent autophagy by downregulating oncomir-106-393-cluster expression in non-small cell lung cancer cells
Authors: Rothschild Sacha I. , Gautschi Oliver , Batliner Jasmin , Jost Melanie , Fey Martin F. , Gugger Mathias , Tschan Mario P.
Published: March 2013

Autophagy has been proposed to play a role as cytoprotective mechanism for tumor cell survival under unfavorable conditions and upon anticancer treatment. In lung cancer autophagy is mostly studied as possible resistance mechanism to therapy. There is increasing evidence that protective autophagy occurs upon various types of anti-cancer treatments, including chemo- and radiation therapy. Src tyrosine kinase inhibitors (TKIs) have been shown to inhibit cell migration and invasion in non-small cell lung cancer (NSCLC) cell lines. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. In the current study we show a marked induction of autophagic activity upon incubation of NSCLC cell lines with Src TKIs as measured by an increased LC3-I to -II conversion, a significant increase in GFP-LC3 dot formation, and a decrease in p62/SQSTM1 protein expression. Increased autophagic activity was found in three NSCLC cell lines (A549, H460, H1299) with two different Src TKIs (saracatinib, dasatinib). Interestingly, the addition of pharmacological autophagy inhibitors such as chloroquine or bafilomycin resulted in cell death in combination with Src TKI treatment. Moreover, we found that Src TKI-induced autophagy is associated with ULK1 expression in all three cell lines investigated. This effect was Src-specific since knocking down endogenous Src using RNAi resulted in a similar induction of ULK1. Using shRNA targeting ULK1 we showed that Src inhibitor induced autophagy is ULK1-dependent. Furthermore, ULK1 is a novel target of microRNA (miR)-106a as shown by ULK1 3′-UTR luciferase experiments and ectopic expression of miR-106a and antimiR-106a in NSCLC cell lines resulting in decreased and increased ULK1 expression upon Src inhibition, respectively. Lastly, in human lung adenocarcinoma compared to matched normal lung tissue (n = 23) miR-106a levels were significantly increased (p<0.0001) whereas ULK1 mRNA expression levels were significantly lower (p<0.0002) in tumor tissue. In conclusion, Src inhibitor-induced protective autophagy might explain their low success in clinical trials. Autophagy induced by Src TKIs depends on ULK1 and combining Src with autophagy inhibitors results in massive cell death as compared to single treatments. Furthermore, downregulation of the ULK1 targeting miR-106a upon Src inhibition allows for the induction of protective autophagy. Combining Src and autophagy inhibitors or Src inhibitors and miR-106a expression may represent attractive treatment options for NSCLC.
Abstract B34: Overcoming resistance to EGFR-Tyrosine Kinase Inhibitor therapy in non-small cell lung cancer
Authors: Sierra Rafael , Chang Anderson , Moffat Jason , Neel Benjamin G. , Tsao Ming-Sound
Published: March 2013

Lung cancer is the leading cause of cancer death in developed countries both for men and women; and the second most common type of cancer diagnosed, with less than 15% of patients surviving beyond 5 years. Since more than 60% of lung adenocarcinomas and more than 90% of lung squamous cell carcinomas express high levels of EGFR mRNA and protein, it has been the focus of efforts to develop new agents that target the EGFR pathway. Clinical trials have demonstrated that patient selection plays a major role for patient response. Patients that present amplification or activating mutations (L878R or exon 19 deletions) of EGFR, have higher response rates. Selection improved response rates from less than 10% to over 60–80%. Despite the promising results, all patients that receive TKI therapy develop resistance to treatment in less than one year. The mechanisms of resistance to EGFR TKi's described so far are those dependant on the overactivation/amplification of other receptor tyrosine kinases (RTK) capable to sustain anti-apoptotic signaling pathways. Our study proposes the use of genome-wide screenings using an 80,000 shRNA library on EGFR resistant cells can identify new genes that mediate the resistance to EGFR targeted therapy and provide the basis for designing new therapeutic approaches to avoid the emergence of resistance. So far, we have generated a shortlist of gene candidates that confer synergistic synthetic lethal interactions with EGFR targeted therapies and that are also highly expressed in resistant cells. We are currently overexpressing candidate genes in sensitive cells searching for those that can confer resistance to EGFR targeted therapies. Our study proposes target genes that can synergize with the current treatments to create new therapeutical strategies that can avoid the acquisition of resistance to targeted therapies. Overall, the information gained from this type of study can be applicable to tumors where ERBB receptor(s) play an important role and targeted therapies are currently used.
Abstract B35: Activation of Putative Compensatory Pathways upon Deletion of Erbb3 in Mutant EGFR-driven Lung Cancer
Authors: Song Xiaoling , Fan Pang-Dian , Guha Udayan , Threadgill David , Varmus Harold , Politi Katerina
Published: March 2013

Lung cancer is the leading cause of cancer-related death in the USA and worldwide. Mutant epidermal growth factor receptor (EGFR)-driven non-small cell lung cancer represents 15% of lung adenocarcinomas. Tumors bearing EGFR mutations are sensitive to treatment with specific tyrosine kinase inhibitors (TKIs) and show radiographic responses in about 70% of cases. However, it is not known why the remaining 30% of the tumors do not respond to these drugs, and patients who initially respond to TKI treatment eventually develop drug resistance on average within a year. These facts underscore the need for a better understanding of the molecular mechanisms that underlie transformation of the lung epithelium by mutant EGFR.

Mutant EGFR-induced signaling is initiated by the formation of EGFR homodimers or heterodimers with other members of the EGFR family (ERBB2, ERBB3 or ERBB4). To investigate the role of EGFR family members in the tumorigenic process driven by mutant EGFR, we used a tetracycline-inducible transgenic model to test the requirement for Erbb3 in mutant EGFR-induced lung tumorigenesis. In this model, deletion of Erbb3 had no effect on tumorigenesis induced by mutant EGFR, suggesting that it is not required to initiate tumorigenesis. Tumors that develop in the absence of Erbb3 remain sensitive to TKIs. Analysis of the biochemical consequences of Erbb3 deletion revealed increased levels of phosphorylation of EGFR and Erbb2 in tumors arising in the absence of Erbb3. Moreover, these tumors show increased activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Together these data suggest that recruitment of compensatory pathways may overcome the need for Erbb3 in tumorigenesis driven by mutant EGFR. Further experiments to test this are ongoing. Knowledge of the role EGFR heterodimerization partners play in mutant EGFR-driven lung cancer can help guide the selection of which EGFR family members to target in the treatment of this disease.
Abstract B7: Use of ponatinib to restrict growth of non-small cell lung cancer (NSCLC) cells overexpressing FGFR1
Authors: Ren Mingqiang , Hong Mei , Wang Suiquan , Cowell John , Hao Zhonglin
Published: March 2013

For advanced stage NSCLC, targeted molecular therapy based on an understanding of the oncogenic driver mutations is emerging as the new front line care replacing chemotherapy where the oncogenic driver mutation is clearly defined e.g EGFR mutation or EML4-ALK translocation. However, the proportion of patients that fall into this category is small and are mostly non-smokers with adeno histology. Identifying new oncogenic drivers in NSCLC is therefore paramount.

In our initial Oncomine database search, we found that FGFRs are overexpressed in various NSCLC tissue specimens. Quantitative real time PCR and Western blot analysis subsequently confirmed overexpression of FGFR1 in about one fourth of NSCLC cell lines analyzed. Among these, H520, H1299 and A549 had significantly higher levels of FGFR1 expression compared to H3122 which carries an EML4-ALK fusion protein. Real time PCR also demonstrated FGFR1 overexpression compared to normal human bronchial epithelial cells transformed by Herpes Papillary Virus E6/E7 (HBE4). Furthermore, as a sign of epithelial to mesenchymal transition, these cells switched from expressing the FGFR1 3b to the 3c form. In a colonogenic assay the growth of cells carrying FGFR1 overexpression/activation is markedly suppressed by ponatinib, a pan FGFR inhibitor as expected. Similarly, ponatinib treatment of NSCLC cell lines using 1 μM concentrations effectively suppressed the phosphorylation level of FGFR1 and its known down-stream targets including pSrc and pPLCγ without significant impact on the total protein expression. Treatment of these cells harboring FGFR1 activation with ponatinib arrests cells at the G1 phase as shown in the flow cytometry analysis. To determine whether the observed effects are specifically due to the suppression of FGFR1 by ponatinib, an shRNA knock down of FGFR1 was performed. Consistently, shFGFR1 significantly inhibited the growth of H520 and H1299 cells, whereas knock-down did not significantly affect the growth of H3122. Similar to ponatinib treatment, shFGFR1 caused more cells to arrest in G1 phase. In a MTT assay, ponatinib synergistically induced growth suppression/killing of H520 cells with cisplatin and paclitaxel. We are currently determining whether ponatinib is effective in inhibiting NSCLC cells derived directly from freshly resected lung cancer specimens.

In conclusion, the FGFR1 pathway is constitutively active in a proportion of NSCLC cell lines and ponatinib is a potent inhibitor of constitutive FGFR1 activation. Ponatinib is potentially useful as a molecularly targeted agent against this portion of NSCLC when used alone or in combination with traditional chemotherapy regimens.
Abstract B8: Targeting p21-activated kinases in human lung cancer
Authors: Ong Christy C. , Jubb Adrian M. , Haverty Peter M. , Zhou Wei , Harris Adrian L. , Belvin Marcia , Friedman Lori S. , Koeppen Hartmut , Hoeflich Klaus P.
Published: March 2013

P21-activated kinases (PAKs) are important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAKs have been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion and actin cytoskeleton organization. To better understand the role of PAK1 in lung tumorigenesis, PAK1 genomic copy number and expression were determined for a panel of adenocarcinoma and squamous non-small cell lung cancers (NSCLC) and small cell lung cancers (SCLC). 64% of squamous NSCLC samples were positive for PAK1 expression and 52% of all cases showed staining of moderate (2+) or strong (3+) immunohistochemical intensity in the malignant cells. Nuclear localization of PAK1 was also evident in a significant proportion of squamous NSCLCs. Adjacent normal lung tissue did not express appreciable levels of PAK1. Selective PAK1 inhibition was associated with delayed cell cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small molecule inhibitors and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis (IAP) proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked IAP (XIAP) efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in NSCLC and a role for PAK1 in cellular survival and proliferation in this indication.
Abstract B9: Cost effectiveness of gene expression profiling for tumor site origin
Authors: Hornberger John , Degtiar Irina , Gutierrez Hialy , Shewade Ashwini , Henner W. David , Becker Shawn , Raab Stephen
Published: March 2013

Background: Gene expression profiling (GEP) reliably supplements traditional clinicopathological information on the tissue of origin (TOO) in metastatic or poorly differentiated cancer. A cost-effectiveness analysis of GEP TOO testing versus usual care was conducted from a third-party payer perspective in the United States.

Methods: A retrospective, observational study examined treatment changes in patients whose physicians had received the GEP TOO test results to help diagnose the tissue-site of their patient's malignancy and to guide appropriate therapy. Changes in planned chemotherapy, surgery, radiation therapy, added blood tests, imaging investigations, and referral to hospice care before and after the GEP TOO test results were recorded. The effect of changes in chemotherapy on survival were based on randomized controlled trials informing appropriate use of chemotherapy cited in National Comprehensive Cancer Network (NCCN) and Up-to-Date guidelines. Drug and administration costs were based on average doses reported in NCCN guidelines. Centers for Medicare and Medicaid Services (CMS) fee schedules were used to obtain other unit costs. Quality-of-life weights were obtained from literature sources. Changes in overall survival, costs, and cost per quality-adjusted life year (QALY) gained were estimated using bootstrap methods.

Results: Use of chemotherapy regimens consistent with guidelines for the final tumor-site diagnosis increased significantly from 42% to 65% (net difference 23%; p< 0.001). Overall survival was projected to increase from 15.9 months to 19.5 months (mean difference 3.6 months, 95%CI [2.0, 5.1]). The average increase in survival adjusted for quality of life was 2.7 months (95%CI [1.4, 3.9]), and average third-party payer costs per patient increased by $10,360 (95% CI [$5,668, $15,053]). The cost per QALY gained was $46,858 (95% CI [$17,995, $75,718]).

Conclusions: GEP TOO testing significantly altered clinical practice patterns for treating metastatic cancer of uncertain primary. It is projected to increase overall survival, QALYs, and costs, resulting in an expected cost per QALY of less than $50,000.
Abstract IA10: Pathology of lung neuroendocrine tumors: In search of evidence for classification
Authors: Linnoila Ilona
Published: March 2013

Neuroendocrine (NE) tumors comprise approximately 25% of all tumors in human lung. As a group they continue to provide challenges at biological, molecular and pathological level for uniform classification. The prototype of NE tumors is the highly aggressive small cell lung cancer (SCLC) that accounts 15% of human lung cancers. For years lung cancers were clinically classified mainly into SCLCs and non-SCLCs (NSCLCs) while it has become evident that lung NE tumors actually form a complex spectrum from SCLC to carcinoid with shifting boundaries. The current WHO classification recognizes SCLC, its variant form combined SCLC, typical and atypical carcinoids as well as large cell NE carcinoma (LCNEC) and its variant form combined large cell carcinoma, the last two cancers under the main NSCLC category of large cell carcinoma. With the exception of the LCNEC, it is expected that the diagnoses will be rendered by routine hematoxylin-eosin stain according the established criteria. Benign entities include diffuse idiopathic pulmonary NE cell hyperplasia which is described in the chapter of preinvasive lesions. Over time there have been several efforts to place lung NE tumors into two to four-tier categories according to their malignant potential (high versus low grade) or degree of differentiation often leaving behind confusing terminology that is later misused. Unlike surgically resected NSCLCs, most NE tumors are frequently diagnosed from biopsies which remains a seldom recognized fact that may create its own biases. The diagnosis that experts most often agree with is SCLC, while distinctions between atypical carcinoid and LCNEC that are in the middle of the spectrum are often disputed. Many molecular studies have also “correctly” classified the endpoints of the NE spectrum while grouping in the middle of it has produced varied results. Moreover, several studies have suggested that NSCLCs with NE features (NSCLC-NEs) is a distinct carcinoma type with worse prognosis than ordinary non-NE NSCLC, while others disagree. Biologically this category is very interesting as NSCLCs and SCLCs are ultimately thought to be derived from the same cell. The regulation of NE differentiation is poorly understood. The proneural achaete-scute homolog 1 (ASCL1), a lineage-dependent oncogene may be a key factor for NE differentiation. On the other hand, it was recently shown that the deletion of the tumor suppressor genes Rb1 and p53 specifically in NE cells, a subpopulation of cells in normal airways, or in alveolar non-NE type2 pneumocytes of mice resulted in SCLCs closely mimicking human disease. These results provide experimental evidence for the close relationship of NE and non-NE epithelial cells in carcinogenesis.
Abstract IA18: Molecular mechanisms regulating synergistic effect of immunotherapy and chemotherapy of cancer
Authors: Gabrilovich Dmitry , Ramakrishnan Rupal , Huang Chun
Published: March 2013

Despite recent advances in the development of new chemotherapeutic drugs and improvements in radiation therapy, conventional cancer therapy often falls short of the goal of controlling tumor progression. Therapeutic cancer vaccines and adoptive T-cell transfer are long considered as very attractive therapeutic options in the treatment of cancer. However, clinical trials of different cancer vaccines performed in recent years demonstrated rather weak clinical efficacy. It has become apparent that therapeutic cancer vaccines given as a single agent may not produce substantial clinical benefits and combination with conventional methods of treatment will be necessary. The use of conventional cancer chemotherapy in combination with cancer vaccines was previously not considered as very attractive due to the potent immunosuppressive effect usually associated with chemotherapy. This paradigm was challenged in recent years by serendipitous observations made in a number of phase I/II clinical trials that reported high rates of objective clinical responses when cancer vaccines were combined with chemotherapy. The paradox is that the types of chemotherapy that are used in lung cancer are known to suppress the immune system. We have recently demonstrated that chemotherapy and cytotoxic T cells (CTL) have a synergistic cytotoxic effect that may be mediated by mannose-6-phosphate receptor (MPR). We have shown that when cancer vaccines or adoptive T-cell transfer are used as a single modality, only limited numbers of T cells are able to penetrate the tumor parenchyma. CTLs exert their cytotoxic effect only against tumor cells that express specific antigen, since it requires a direct cell-cell contact, and release of perforin and granzymes (GrzB). If chemotherapy is administered immediately after immunotherapy, it causes disruption of tumor stroma that allows for better penetration of antigen-specific T cells. Chemotherapy via autophagy causes substantial increase in MPR expression on tumor cells. Small number of activated CTLs interacting with tumor cells expressing tumor antigen can release GrzB that can penetrate into neighboring tumor cells without requirement for cell-cell contact. Therefore large number of tumor cells including those that do not express specific antigen would be susceptible to the effect of CTLs. This may explain substantial enhancement of antitumor effect of combined treatment. It is likely that this effect would not be long-lasting since CTL activity will be eliminated by chemotherapy and the immune suppressive tumor microenvironment. However, it may provide a sufficient window to achieve a significant antitumor effect. Since memory T cells are more resistant to chemotherapy than effector T cells, it is possible that subsequent immunization would be able to boost antitumor immunity and thus provide a longer lasting effect of combined therapy.
Abstract IA2: Network models in oncogene-addicted lung cancer
Authors: Haura Eric B. , Yoshida Takeshi , Fang Bin , Stukalov Alexey , Eschrich Steven , Welsh Eric , Colinge Jacques , Koomen John , Superti-Furga Giulio , Li Jiannong , Rix Uwe , Zhang Guolin , Kim Jae-Young , Bennett Keiryn , Song Lanxi , Chen Ann , Bai Yun
Published: March 2013

Lung cancer is a devastating worldwide disease yet enthusiasm exists for treatment of subsets of the disease with molecularly targeted agents. Mutations in the epidermal growth factor receptor (EGFR) or translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) define two unique subsets of lung cancer characterized by sensitivity to tyrosine kinase inhibitors (TKI). Despite striking results with TKI, not all patients respond, the drugs are non-curative, and resistance is universal. Mutations in KRAS also define a group of patients awaiting therapeutic opportunities. We are characterizing signaling networks using tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions (PPI) and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. In PC9 cells with mutated EGFR, we characterized a physical EGFR network consisting of 266 proteins by integrating both TAP and pTyr MS data. In H3122 cells harboring EML4-ALK, we identified a PPI network consisting of 113 proteins and using pTyr MS identified changes in tyrosine phosphorylation in 120 proteins (58 decreased, 62 increased) following exposure to ALK tyrosine kinase inhibitor. Functional proteins are being discovered from these networks using siRNA and inhibitor screens. In parallel studies, we have exploited the use of chemical proteomics to discern targets of promiscuous kinase inhibitors and enable optimal combination approaches. In this way, we view network mapping linked to chemical proteomics as one approach to discern novel drug combination studies for in vivo validation and ultimately translation to early phase clinical trials. To translate PPI based mass spectrometry studies to clinic, we are developing in situ assays that identify and quantify PPI using proximal ligation assays (PLA). Pilot studies identify EGFR:Grb2 interactions in formalin fixed human lung cancers. These approaches have the potential to enable ‘network medicine’ by identifying novel combination approaches as well as through identifying subtypes of cancers through network views of cancer.
Abstract IA22: miRNA and lung cancer: Early detection in high-risk subjects
Authors: Sozzi Gabriella , Boeri Mattia , Verri Carla , Roz Luca , Suatoni Paola , Croce Carlo M. , Pastorino Ugo
Published: March 2013

Background: Lung cancer remains the major cause of cancer mortality in the world. In addition to primary prevention, earlier detection and more targeted treatments, tailored on the biological characteristics of the tumor and its microenvironment, could significantly reduce morbidity and mortality for this disease. The real efficacy of lung cancer screening by spiral-computed tomography (CT) in heavy smokers is still to be defined since, in spite of a proved capacity to detect small asymptomatic nodules, the frequency of false positive CTs as well as unnecessary treatments is very high if compared to the limited mortality reductions. The development of biomarkers able to identify tumors in a pre-clinical phase and to track the different aggressiveness of lung tumors is of paramount importance. microRNAs (miRNAs) represent a recently identified class of regulatory molecules and several studies showed that miRNA are involved in lung tumor development and progression and also circulate in plasma and serum of lung cancer patients.

Materials and Methods: We analyzed miRNA profiles of lung tumors, normal lung tissues and plasma samples from cases with variable prognosis identified in two independent spiral-CT screening trials where multiple plasma samples, collected from one to four years before radiological detection of the disease were available.

Results: We found miRNA expression profiles associated with aggressiveness of the disease and poor survival in tumors and also in normal lung tissues of the patients, thus proving the critical influence of a smoking-related lung microenvironment on tumor progression. Specific microRNA signatures were identified in plasma samples collected up to two years before spiral-CT detection of the disease, thus able to catch the earlier biological phases of disease development. We also defined a plasma signature that discriminates subjects according to aggressiveness of their future tumors, and in particular the occurrence of early metastatic but spiral-CT invisible lung tumors or small spiral-CT detected lesions with aggressive potential. Of interest plasma miRNas involved in the signatures of lung cancer risk and of aggressive disease more closely reflected miRNAs expressed in the normal lung rather than those characterizing the tumor samples of patients, supporting the concept that normal lung microenvironment has a critical influence on tumor development and aggressiveness.

Conclusion: These results open up the prospective of using plasma miRNAs as non invasive lung cancer biomarkers.
Abstract IA23: A systems approach to the discovery of lung cancer biomarkers
Authors: Hanash Samir
Published: March 2013

We have implemented a systems approach for the discovery of lung cancer biomarkers. Applications include risk assessment, early detection, and predicting response to treatment. The approach which includes genomic, proteomic, metabolomic, and immune profiling integrates data from human biospecimens, a large number of lung cancer cell lines grown under various conditions and several genetically engineered mouse models of lung cancer. An important component of the human studies consists of profiling of pre-diagnostic plasmas available through longitudinal cohort studies, particularly useful for the discovery and validation of blood based risk and early detection markers. Findings include molecular signatures and networks for lung cancer sub-types. Mouse model studies yielded an NKX2.1 signature relevant to early detection, an EGFR network signature for EGFR driven lung cancer and a neuroendocrine signature for small cell lung cancer which were validated in human samples. Integration of proteome and transcriptomic profiling of lung cancer cell line yielded several signatures and a marker panel that predict survival for early stage lung cancer. Mining of the large and continuously expanding sets of data currently being collected benefits from an organized collaborative effort.
Abstract IA4: Defining the mechanisms of tumorigenesis by mutant EGFR using mouse models
Authors: Politi Katerina A.
Published: March 2013

Mutations in exons encoding the epidermal growth factor receptor (EGFR) are found in 10–15% of lung adenocarcinomas. The two most common mutations: a point mutation in exon 21 that leads to substitution of an arginine for a leucine at position 858 and small in-frame deletions in exon 19 are associated with sensitivity to the specific tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Approximately 70% of tumors harboring EGFR mutations respond to treatment with these drugs, however 30% of these tumors exhibit primary resistance to treatment with these agents. Moreover, responses are transient and, on average, within a year of initiating treatment patients who initially respond eventually develop TKI-inhibitor-resistant disease. Drug resistance, in most cases, is due to a secondary mutation in EGFR (EGFR T790M). Other less frequent mechanisms of resistance include MET amplification, PIK3CA mutations and transformation to small cell lung cancer. However, the mechanism of resistance is still unknown in approximately 30% of cases. More effective treatment of patients with EGFR mutant lung cancer requires a better understanding of the mechanisms of primary and acquired resistance to EGFR TKIs and the development of strategies to overcome this resistance.

To study these problems in vivo, we developed tetracycline-inducible transgenic mouse models of EGFR mutant lung cancer. Expression of lung cancer associated EGFR mutants gives rise to lung adenocarcinomas with bronchioloalveolar carcinoma features that are dependent upon the continuous activity of mutant EGFR for survival. Thus, treatment of the mice with erlotinib leads to tumor regression and long-term treatment with the drug gives rise to drug-resistant tumors that harbor some of the alterations observed in human TKI-resistant tumors such as the T790M mutation. Current efforts to use these mouse models to: 1) study mechanisms of primary and acquired resistance to EGFR TKIs and 2) evaluate new therapies to overcome resistance to first generation TKIs will be discussed.
Abstract IA7: Somatic genome alterations in human lung cancers
Authors: Meyerson Matthew
Published: March 2013

Cancer is a disease of the genome. High-throughput genome analysis tools now enable the detection of somatic alterations in cancer cells including point mutations, copy number alterations, translocations, and infections. Our approaches include next-generation sequencing of cancer genomes, exomes, and transcriptomes as well as single nucleotide polymorphisms (SNP) array analysis of copy number.

As part of “The Cancer Genome Atlas” or TCGA project of the National Institutes of Health, which aims to characterize the genomes of 10,000 human cancers, we are performing genomic analysis of lung carcinomas. In this presentation, I will discuss analysis of the genomes of squamous cell lung carcinomas. New results regarding mutations, genomic structure, and classification will be presented.